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15: DNA Technologies - Biology

15: DNA Technologies - Biology


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Learning Objectives

  1. Suggest molecular techniques to design experiments (e.g., how would you use cDNA or a PCR product to clone a gene).
  2. Determine when to make or use a cDNA library or a genomic library
  3. Outline an experiment to purify rRNA from eukaryotic cells.
  4. Outline an experiment to isolate a cDNA for a human protein and clone it so you can manufacture insulin for the treatment of disease.
  5. Explain why you might want to clone and express a human growth hormone gene.
  6. List components needed to make a cDNA library using purified poly(A) RNA.
  7. List the components needed to make a genomic library from isolated genomic DNA.
  8. Compare PCR and genomic cloning as strategies for isolating a gene.
  9. Outline a strategy for using fly DNA to obtain copies of a human DNA sequence.
  10. Ask a question that requires screening a genomic library to obtain a gene you want to study
  11. Ask a question that requires using a microarray to obtain a gene you want to study
  • 15.1: Overview
    We start this chapter by looking at technologies that led to genetic engineering. The ability of make recombinant DNA is such a seminal technology that just realizing it could be done and then doing it in a test tube for the first time earned Paul berg a half-share in the 1980 Nobel Prize in Chemistry (the other half was shared by Walter Gilbert and Frederick Sanger for studies that enabled efficient DNA sequencing).
  • 15.2: Make and Screen a cDNA Library
    The first step in making a cDNA library is to isolate cellular mRNA. This mRNA extract should represent all of the transcripts in the cells at the time of isolation, or the cell’s transcriptome. This term is used by analogy to genome. However, a genome is all of the genetic information of an organism. In contrast, a transcriptome (usually eukaryotic) reflects all of the genes expressed in a given cell type at a moment in time.
  • 15.3: DNA Sequencing
    RNA sequencing came first, when Robert Holley sequenced a tRNA in 1965. The direct sequencing of tRNAs was possible because tRNAs are small, short nucleic acids, and because many of the bases in tRNAs are chemically modified after transcription. An early method for DNA sequencing developed by Walter Gilbert and colleagues involved DNA fragmentation, sequencing of the small fragments of DNA, and then aligning the overlapping sequences of the short fragments to assemble longer sequences.
  • 15.4: Genomic Libraries
    A genomic library might be a tube full of recombinant bacteriophage. Each phage DNA molecule contains a fragmentary insert of cellular DNA from a foreign organism. The library is made to contain a representation of all of possible fragments of that genome. The need for vectors like bacteriophage that can accommodate long inserts becomes obvious from the following bit of math.
  • 15.5: The Polymerase Chain Reaction (PCR)
    The polymerase chain reaction (PCR) can amplify a region of DNA from any source, even from a single cell’s worth of DNA or from fragments of DNA obtained from a fossil. This amplification usually takes just a few hours, generating millions of copies of the desired target DNA sequence. The effect is to purify the DNA from surrounding sequences in a single reaction!
  • 15.6: Genomic Approaches- The DNA Microarray
    Traditionally, when cellular levels of a protein were known to change in response to a chemical effector, molecular studies focused on control of the transcription of its gene. These studies often revealed that the control of gene expression was at the level of transcription, turning a gene on or off through interactions of transcription factors with DNA.
  • 15.7: Ome-Sweet Ome
    Early molecular technologies, including the ones described in this chapter, were applied to understanding the structure, function and regulation of specific genes. Some of the more recent technologies (e.g., microarrays) are well adapted to holistic approaches to understanding cell function. Terms we have already seen (genome, epigenome, transcriptome) were coined in an effort to define the different objects of study whose underlying network of molecular interactions can more accurately explain
  • 15.8: From Genetic Engineering and Genetic Modification
    By enabling us to focus on how genes and their regulation have evolved, these genomic, transcriptomic and proteomic technologies have vastly increased our knowledge of how cells work at a molecular level. We continue to add to our knowledge of disease process and in at least a few cases, how we can treat disease.
  • 15.9: Key Words and Terms

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Principle of Recombinant DNA Technology (4 Steps)

Read this article to learn about the principle of recombinant DNA technology. The principle of recombinant DNA technology involved four steps.

The four steps are: (1) Gene Cloning and Development of Recombinant DNA (2) Transfer of Vector into the Host (3) Selection of Transformed Cells and (4) Transcription and Translation of Inserted Gene.

Knowledge about cell and its functioning has increased to a great magnitude during 20th century. Science and genetics provided basic inputs and now we have gathered information about the structure of master molecule, DNA, its replication and control of gene expression.

Developments in last three decades were very rapid and gene sequencing, gene cloning and gene transfer in eukaryotes and prokaryotes (and vice versa) have been achieved. This has become possible because genetic code is assumed to be universal.

Genetic engineering may or may not have recombinant DNA (rDNA) preparation step and with the advancement of gene transfer technology, artificially DNA can be transferred to different hosts (host cells) without any vector and host organism can be engineered to carry desired properties. The genetic manipulations are used to produce individuals having a new combination of inherited properties.

Such manipulations may be of two kinds:

(1) Cellular manipulation involving culturing of cells (e.g., haploid cells) and hybridization of somatic cells (protoplast fusion), and

(2) Molecular manipulation, involving construction of artificial rDNA molecules, their insertion into a vector and their establishment in a host cell or organism.

The latter approach has been called “recombinant DNA (rDNA) technology”. Therefore, use of term “genetic engineering” and “rDNA technology” is overlapping. However, all the manipulations involving use of constructed gene or constructed gene transfer are termed as rDNA technology.

The different steps of recombinant technology include (Fig. 14.7):

a. Gene cloning and development of recombinant DNA:

The foreign DNA (gene of interest) from the source is enzymatically cleaved and ligated (joined) to other DNA molecule i.e. cloning vector (plasmid, phagemid etc.) to form recombinant DNA.

b. Transfer of vector into the host:

This cloning vector with recombinant DNA is transferred into and maintained within a host cell. The introduction of rDNA into a bacterial host cell is called transformation.

c. Selection of transformed cells (host):

Those host cells that take up the rDNA are identified and selected from the pool.

d. Transcription and translation of inserted gene:

If required, an rDNA construct can be prepared to ensure that the protein product that is encoded by the cloned DNA sequence is produced by the host cell.

1. Gene Cloning and Development of Recombinant DNA:

Any gene to be cloned must be inserted in a cloning vector (plasmid). A foreign gene (DNA fragment) introduced (by transformation) into a bacterium cell will not be replicated with bacterium. The reason for this is that the enzyme DNA polymerase, which is responsible for copying DNA, does not initiate the process at random. It is initiated at selected sites known as “origin of replication”.

Generally, small fragments of DNA do not possess an origin of replication. Using rDNA technology, it is possible to insert the gene into a ‘cloning vector’, which in turn will make copies of the fragment (inserted DNA). A cloning vector is simply a DNA molecule possessing an ‘origin of replication’ and which can replicate in the host cell of choice. Most commonly ‘plasmids’, extra chromosomal, autonomously replicating, circular DNA molecules, are used as vectors. Sometimes, viruses are used as vector for gene insertion into micro-organisms, but they are better vectors for animal cells.

Cutting and insertion of desired foreign gene into the plasmid require special enzymes known as restriction endonucleases or restriction enzymes. These enzymes cut large DNA molecules into shorter fragments by cleavage at specific nucleotide sequences called ‘recognition sites’. Therefore, restriction endo-nucleases are highly specific deoxy-ribonucleases (DNAse).

Both vector DNA and foreign DNA to be inserted is cut by the same restriction enzyme, generating complementary ends. Thus, ends of foreign DNA make perfect match with cut ends of vector and join to make again a circular molecule. This process can be understood in detail by taking the example of pBR 322 plasmid. It is commonly used cloning vector (Fig. 14.2).

Firstly, purified, closed, circular pBR322 molecules are cut with a restriction enzyme that lies within either of the antibiotic resistance genes and cleaves the plasmid DNA only once to create single, linear, sticky- ended DNA molecules. These linear molecules are combined with prepared target DNA from a source organism. This DNA is cut with the same restriction enzyme, which generates the same sticky ends as those on plasmid DNA. The DNA mixture is treated with T4 ligase in the presence of ATR Under these conditions, a number of different ligated combinations are produced, including the original closed circular plasmid DNA.

To reduce the amount of this particular unwanted ligation product, the cleaved plasmid DNA preparation is treated with the enzyme alkaline phosphatase to remove the 5′-phosphate groups from the linearized plasmid DNA. Due to this, T4 DNA ligase cannot join the ends of the dephosphorylated linear plasmid DNA.

However, the two phosphodiester bonds that are formed by T4 DNA ligase after the ligation and circularization of alkaline phosphate-treated plasmid DNA with restriction endonuclease digested source DNA (which provides the phosphate groups), are sufficient to hold both molecules together, despite the presence of two nicks (Fig. 14.8). After transformation, these nicks are sealed by the host cell DNA ligase. In addition, fragments from the source DNA are also joined to each other by T4 DNA ligase.

2. Transfer of Vector into the Host:

The next step in a recombinant DNA experiment requires the uptake by E. coli of the rDNA. The process of introducing purified DNA into a bacterial cell is called transformation. This is carried out by treating cells with calcium chloride and high temperature. A few transformed cells are obtained by this method.

Extra chromosomal DNA that lacks an origin of replication cannot be maintained within a bacterial cell. Thus, uptake of non-plasmid DNA is of no significance in a recombinant DNA experiment. Suitable strain of E. coli is used which lacks capabilities to destroy plasmid DNA or carrying out exchanges between DNA molecules (Fig. 14.9).

3. Selection of Transformed Cells:

After transformation, it is necessary to identify the cells that contain plasmid-cloned DNA constructs. In pBR 322 in which target DNA was inserted onto the BamHI site, recombinant bacteria (bacteria with recombinant plasmid) are selected.

All cells are grown successively on media containing antibiotic, ampicillin or tetracycline and cells showing the recombinant DNA (depending upon the restriction enzyme site and loss of particular antibiotic resistance due to disruption of the gene). Selected recombinant bacteria are grown in bioreactor to obtain the gene product (Fig. 14.10).

Other methods for selection of recombinant bacteria are:

1. If synthesized gene is used, selection is easy as compared to DNA fragments used from genomic library of an organism, which requires selection for a suitable characters.

2. By nucleic acid hybridization technique.

All the steps are required to be considered carefully in making a recombinant bacterium. Prokaryotic organism E. coli is the most frequently used bacterium for recombinant technology.

4. Transcription and Translation of Inserted Gene:

Transcription of DNA into mRNA is mediated through the enzyme RNA polymerase, which recognizes the binding site on DNA called promoter. The process of mRNA synthesis is terminated by a termination signal (terminator codon). This means, only gene lying between promoter and terminator will be transcribed. Gene isolated in certain ways such as cDNA cloning or artificial synthesis, do not have their own promoter, therefore they must be inserted into a vector close to promoter site.

Even if a cloned gene carries its own promoter, this promoter may not function in the new host cell. In such circumstances, the original promoter has to be replaced. The position of cloned insulin gene with lac promoter in the vector is given in the figure 14.11. This gene is transcribed in presence of lactose because lac operon functions in presence of lactose and transcribe the gene attached downstream to attach it. Similarly, positions of ribosome binding site (rbs) and termination codon are shown in the figure 14.11 in respect to the insulin gene in the vector.

In the cell, transcription takes place inside the nucleus or close to the nucleoid in bacteria by the action of the RNA polymerase on DNA. This process can also be mimicked outside the cell, i.e. cloned DNA can be mixed with RNA polymerase and the four nucleotide in a tube and under appropriate conditions, RNA transcripts can be formed as it does inside the cell. This is known as in vitro transcription. The cellular RNA polymerase, whether it is bacterial or from higher organisms, is an extremely complicated enzyme, containing several subunits.

It is very difficult to purify in an active form. On the other hand, several bacteriophages encode their own RNA polymerases, which are much simpler enzymes, are easy to purify and transcribe genes at a high efficiency. This is because they have evolved to only recognize rate. This phenomenon has been utilized in designing in vitro transcription systems.

The bacteriophage RNA polymerases commonly used for this purpose include those from bacteriophage T3 and T7 which infect E. coli and SP6, which infects Salmonella typhimurim. The promoter sequences recognized by each of these RNA polymerases are different, but can be as small as 21bp in length. Thus, these promoters are designed to be part of the special cloning for in vitro transcription, just upstream of the cloning sites for the DNA fragments to be transcribed.

The cloned DNA, placed downstream of the above promoters, is then incubated with the purified RNA polymerase from the bacteriophage, along with the precursor ribonucleotide triphosphate, which synthesizes transcripts specific for the cloned DNA.

In vitro derived transcripts are used extensively as probes for the detection of specific nucleic acid fragments both in Southern as well as in Northern hybridization.

Translation of mRNA into proteins is a complex process which involves interaction of the mRNA with the ribosomes. For translation to take place the mRNA must carry a ribosome binding site in front (upstream) of the gene to be translated. Ribosome binds to this site and move along the mRNA and initiates protein synthesis at the first AUG codon, it encounters.

This process is stopped by stop codon (UAA, UAG, UGA). In case the cloned gene lacks a rbs, then it is necessary to use a vector with promoter and rbs and the gene is inserted downstream to both these (promoter and rbs).

Translation in a cell is carried out by the ribosomes, which synthesize polypeptides by decoding the information carried by mRNA. In addition, amino-acyl tRNAs and other proteinaceous accessory factors are also utilized. Biochemically the process of translation is not yet fully characterized, which means that we do not know all the requirements for polypeptide synthesis and very few of the components required for the process of translation have actually been purified.

Despite having the above drawbacks, translation of a given mRNA can still be performed outside the cell, if the mRNA is incubated in a mixture of components which supports translation. This is termed in vitro translation. The above components are generally isolated from E. coli cells (S-30 fraction), plants (wheat germ) or animals (rabbit reticulocytes). Because of the universality of the genetic code a given mRNA gets translated to the same extent and with the same efficiency irrespective of the in vitro translation system used.

Since there are already many types of protein molecules in the translation mix, new protein synthesis is usually monitoring by adding radioactive amino acids in the translation mix. Thus, all newly synthesized proteins get radioactively labeled and can be easily detected by autoradiography. In many instances, a combined in vitro transcription and translation system is used which can produce the encoded polypeptide directly from a given cloned gene. Such systems are now commercially available.

Bacterial and viral genes have simple structure as all the genetic information in the mRNA between the initiation and stop codons is translated into protein. Many genes of eukaryotic organisms, including the human insulin gene, have a more complex structure. They are consist of coding regions (or exons), which contribute to the final protein sequence, and non- coding regions (or introns), which are not translated into proteins.

In eukaryotes, genes containing introns are transcribed into mRNA in the usual manner, but then the corresponding intron sequences are spliced out. As bacteria cannot spliced out introns, they cannot be used directly to express many genes from mammals or other eukaryotes. This is done by ribozymes-RNA molecules having catalytic activity.

Insulin (a dipeptide) formation by processing of mRNA (removal of introns) and translation of exons is well studied. There are two ways to overcome this problem.

1. By use of re verse transcriptase, a cDNA copy of the processed mRNA is prepared and this cDNA (gene) is used for insertion in the vector.

2. The gene for protein may be synthesized in test tube, which lacks introns.

Posttranscriptional modification in the gene:

A number of proteins undergo posttranscriptional modifications. Proteins that are destined to be transported out of cell are synthesized with extra 15-30 amino acids at the amino terminal (N- terminus). These extra amino acids are referred to as a signal sequence. The common feature of these sequences is that they have a central core of hydrophobic amino acids flanked by polar or hydrophilic residues. During passage through the membrane the signal sequence is cleaved off, making the protein active.


Recombinant DNA Technology (With Diagram)

In this article we will discuss about Recombinant DNA Technology:- 1.Steps in Recombinant DNA Technology 2. Tools for Recombinant DNA Technology 3. Techniques Used In Recombinant DNA Technology 4. Applications of Recombinant DNA Technology.

Steps in Recombinant DNA Technology:

Basic steps involved in rec DNA technology (or genetic engineering) are given below (Fig. 1):

i. Selection and isolation of DNA insert

ii. Selection of suitable cloning vector

iii. Introduction of DNA-insert into vector to form rec DNA molecule

iv. rec DNA molecule is introduced into a suitable host.

v. Selection of transformed host cells.

vi. Expression and multiplication of DNA-insert in the host.

(i) Selection and isolation of DNA insert:

First step in rec DNA technology is the selection of a DNA segment of interest which is to be cloned. This desired DNA segment is then isolated enzymatically. This DNA segment of interest is termed as DNA insert or foreign DNA or target DNA or cloned DNA.

(ii) Selection of suitable cloning vector:

A cloning vector is a self-replicating DNA molecule, into which the DNA insert is to be integrated. A suitable cloning vector is selected in the next step of rec DNA technology. Most commonly used vectors are plasmids and bacteriophages.

(iii) Introduction of DNA-insert into vector to form recDNA molecule:

The target DNA or the DNA insert which has been extracted and cleaved enzymatically by the selective restriction endonuclease enzymes [in step (i)] are now ligated (joined) by the enzyme ligase to vector DNA to form a rec DNA molecule which is often called as cloning-vector-insert DNA construct.

(iv) rec DNA molecule is introduced into a suitable host:

Suitable host cells are selected and the rec DNA molecule so formed [in step (iii)] is introduced into these host cells. This process of entry of rec DNA into the host cell is called transformation. Usually selected hosts are bacterial cells like E. coli, however yeast, fungi may also be utilized.

(v) Selection of transformed host cells:

Transformed cells (or recombinant cells) are those host cells which have taken up the recDNA molecule. In this step the transformed cells are separated from the non-transformed cells by using various methods making use of marker genes.

(vi) Expression and Multiplication of DNA insert in the host:

Finally, it is to be ensured that the foreign DNA inserted into the vector DNA is expressing the desired character in the host cells. Also, the transformed host cells are multiplied to obtain sufficient number of copies. If needed, such genes may also be transferred and expressed into another organism.

Tools for Recombinant DNA Technology:

1WDNA technology utilizes a number of biological tools to achieve its objectives, most important of them being the enzymes.

Important biological tools for rec DNA technology are:

a. Restriction Endonucleases

(D) DNA insert or foreign DNA

(E) Linker and adaptor sequences.

An account of all these biological tools of genetic engineering is given below:

(A) ENZYMES:

A number of specific enzymes are utilized to achieve the objectives of rec DNA technology.

The enzymology of genetic engineering includes the following types of enzymes:

(a) Restriction Endonuclease:

These enzymes serve as important tools to cut DNA molecules at specific sites, which is the basic need for rec DNA technology.

These are the enzymes that produce internal cuts (cleavage) in the strands of DNA, only within or near some specific sites called recognition sites/recognition sequences/ restriction sites 01 target sites. Such recognition sequences are specific for each restriction enzyme. Restriction endonuclease enzymes are the first necessity for rec DNA technology.

The presence of restriction enzymes was first of all reported by W. Arber in the year 1962. He found that when the DNA of a phage was introduced into a host bacterium, it was fragmented into small pieces. This led him to postulate the presence of restriction enzymes. The first true restriction endonuclease was isolated in 1970s from the bacterium E. coli by Meselson and Yuan.

Another important breakthrough was the discovery of restriction enzyme Hind-II in 1970s by Kelly, Smith and Nathans. They isolated it from -the bacterium Haemophilus influenza. In the year 1978, the Nobel Prize for Physiology and Medicine was given to Smith, Arber and Nathans for the discovery of endonucleases.

Types of Restriction Endonucleases:

There are 3 main categories of restriction endonuclease enzymes:

Type-I Restriction Endonucleases

Type-II Restriction Endonucleases

Type-III Restriction Endonucleases

Type-I Restriction Endonucleases:

These are the complex type of endonucleases which cleave only one strand of DNA. These enzymes have the recognition sequences of about 15 bp length (Table 1).

They require Mg++ ions and ATP for their functioning. Such types of restriction endonucleases cleave the DNA about 1000 bp away from the 5′ end of the sequence ‘TCA’ located within the recognition site. Important examples of Type-I restriction endonuclease enzyme are EcoK, EcoB, etc.

Type-II Restriction Endonucleases:

These are most important endonucleases for gene cloning and hence for rec DNA technology. These enzymes are most stable. They show cleavage only at specific sites and therefore they produce the DNA fragments of a defined length. These enzymes show cleavage in both the strands of DNA, immediately outs.de then- recognition sequences. They require Mg ++ ions for their functioning.

Such enzymes are advantageous because they don’t require ATP for cleavage and they cause cleavage in both strands of DNA. Only Type II Restriction Endonucleases are used tor gene cloning due to their suitability.

The recognition sequences for Type-II Restriction Endonuclease enzymes are in the form of palindromic sequences with rotational symmetry, i.e., the base sequence .n the first half of one strand of DNA is the mirror image of the second half of other strand of that DNA double helix (Fig. 2). Important examples of Type-II Restriction endonucleases include Hinfl, EcoRI, PvuII, Alul, Haelll etc.

Type-III Restriction Endonucleases:

These are not used for gene cloning. They are the intermediate enzymes between Type-I and Type-II restriction endonuclease. They require Mg ++ ions and ATP for cleavage and they cleave the DNA at well-defined sites in the immediate vicinity of recognition sequences, e.g. Hinf III, etc.

Nature of cleavage by Restriction Endonucleases:

The nature of cleavage produced by a restriction endonuclease is of considerable importance.

They cut the DNA molecule in two ways:

i. Many restriction endonucleases cleave both strands of DNA simply at the same point within the recognition sequence. As a result of this type of cleavage, the DNA fragments with blunt ends are generated. PvuII, Haelll, Alul are the examples of restriction endonucleases producing blunt ends. Blunt ends may also be referred to as flush ends.

ii. In the other style of cleavage by the restriction endonucleases, the two strands of DNA are cut at two different points. Such cuts are termed as staggered cuts and this results into the generation of protruding ends i.e., one strand of the double helix extends a few bases beyond the other strand. Such ends are, called cohesive or sticky ends.

Such ends have the property to pair readily with each other when pairing conditions are provided. Another feature of the restriction endonucleases producing such sticky ends is that two or more of such enzymes with different recognition sequences may generate the same sticky ends.

Exonuclease is an enzyme that removes nucleotides from the ends of a nucleic acid molecule. An exonuclease removes nucleotide from the 5′ or 3′ end of a DNA molecule. An exonuclease never produces internal cuts in DNA.

In rec DNA technology, various types of exonucleases are employed like Exonuclease Bal31, E. coli exonuclease III, Lambda exonuclease, etc.

Exonculease Bal31 are employed for making the DNA fragment with blunt ends shorter from both its ends.

E coli Exonuclease III is utilized for 3’end modifications because it has the capability to remove nucleotides from 3′-OH end of DNA.

Lambda exonuclease is used to modify 5′ ends of DNA as it removes the nucleotides from 5′ terminus of a linear DNA molecule.

The function of these enzymes is to join two fragments of DNA by synthesizing the phosphodiester bond. They function to repair the single stranded nicks in DNA double helix and in rec DNA technology they are employed for sealing the nicks between adjacent nucleotides. This enzyme is also termed as molecular glue.

These are the enzymes which synthesize a new complementary DNA strand of an existing DNA or RNA template. A few important types of DNA polymerases are used routinely in genetic engineering. One such enzyme is DNA polymerase ! which , prepared from E coli. The Klenow fragment of DNA polymerase-I .s employed to make the protruding ends double-stranded by extension of the shorter strand.

Another type of DNA polymerase used in genetic engineering is Taq DNA polymerase which is used in PCR (Polymerase Chain Reaction).

Reverse transcriptase is also an important type of DNA polymerase enzyme for genetic engineering. It uses RNA as a template for synthesizing a new DNA strand called as cDNA a e complementary DNA). Its main use is in the formation of cDNA libraries. Apart from all these above mentioned enzymes, a few other enzymes also mark their importance in genetic engineering.

A brief description of these is given below:

(a) Terminal deoxynucleotidyl transferase enzyme:

It adds single stranded sequences to 3′-terminus of the DNA molecule. One or more deoxynbonucleotides (dATP, dGTP, dl IP, dCTP) are added onto the 3′-end of the blunt-ended fragments.

(b) Alkaline Phosphatase Enzyme:

It functions to remove the phosphate group from the 5′-end of a DNA molecule.

(c) Polynucleotide Kinase Enzyme:

It has an effect reverse to that of Alkaline Phosphatase, i.e. it functions to add phosphate group to the 5′-terminus of a DNA molecule

(B) Cloning Vectors:

It is another important natural tool which geneticists use in rec DNA technology. The cloning vector is the DNA molecule capable of replication in a host organism, into which the target DNA is introduced producing the rec DNA molecule.

A cloning vector may also be termed as a cloning vehicle or earner DNA or simply as a vector or a vehicle a great variety of cloning vectors are present for use with E. coli is the host organism.

However under certain circumstances it becomes desirable to use different host for cloning experiments. So, various cloning vectors have been developed based on other bacteria like Bacillus, Pseudomonas, Agrobacterium, etc. and on different eukaryotic organisms like yeast and other fungi.

The cloning vector which has only a single site for cutting by a particular restriction endormclease is Considered as a good cloning vector. Different types of DNA molecules may be used as cloning vehicles such as they may be plasmids, bacteriophages, cosmids, phasmids or artificial chromosomes.

(C) Host Organism:

A good host organism is an essential tool tor genetic engineering. Most widely used host for rec DNA technology is the bacterium E. coli. because cloning and isolation of DNA inserts is very easy in this host. A good host organism is the one winch easy to transform and in which the replication of rec DNA is easier. There should not be any interfering element against the replication of rec DNA in the host cells

(D) DNA Insert Or Foreign DNA:

The desired DNA segment which is to be cloned is called as DNA insert or foreign DNA or target DNA. The selection of a suitable target DNA is the very first step of rec DNA technology. The target DNA (gene) may be of viral, plant, animal or bacterial origin.

Following points must be kept in mind while selecting the foreign DNA:

1. CD It can be easily extracted from source.

2. It can be easily introduced into the vector.

3. The genes should be beneficial for commercial or research point of view.

A number of foreign genes are being cloned for benefit of human beings. Some of these DNA inserts are the genes responsible for the production of insulin, interferon’s, lymphotoxins various growth factors, interleukins, etc.

(E) Linker and Adaptor Sequences:

Linkers and adaptors are the DNA molecules which help in the modifications of cut ends of DNA fragments. These can be joined to the cut ends and hence produce modifications as desired.

Both are short, chemically synthesized, double stranded DNA sequences. Linkers have (within them) one or more restriction endonuclease sites and adaptors have one or both sticky ends. Different types of linkers and adaptors are used for different purposes.

Linkers contain target sites for the action of one or more restriction enzymes. They can be ligated to the blunt ends of foreign DNA or vector DNA (Fig. 5a). Then they undergo a treatment with a specific restriction endonuclease to produce cohesive ends of DNA fragments EcoRI-linker is a common example of frequently used linkers.

Adaptors are the chemically Synthesized molecules which have pre-formed cohesive ends (Fig. 5b). Adaptors are employed for end modification in cases where the recognition site for restriction endonuclease enzyme is present within the foreign DNA.

The foreign DNA is ligated with adaptor on both ends. This new molecule, so formed, is then phosphorylated at the 5′-terminii. Finally foreign DNA modified with adaptors is integrated into the vector DNA to form the recombinant DNA molecule.

Techniques Used In Recombinant DNA Technology:

A number of techniques are used for various purposes during different steps of rec DNA technology.

Such techniques serve for the fulfilment of different requirements or to obtain proper information for drawing an exact inference during genetic engineering. Some of these important techniques are gel electrophoresis, blotting techniques, dot-blot hybridization, DNA sequencing, artificial gene synthesis, polymerase chain reaction, colony hybridization, etc.

Gel Electrophoresis:

It is the technique of separation of charged molecules (in aqueous phase) under the influence of an electrical field so that they move on the gel towards the electrode of opposite charge i.e., cations move towards the negative electrode and anions move towards the positive electrode.

The genomic DNA is extracted from the desired host and is then fragmented using restriction endonucleases.

For separation of these cut fragments and isolation of desired DNA fragment, the technique of gel electrophoresis is employed. Gel electrophoresis may be of horizontal or vertical type. Usually agarose gel is used for separation of large segments of DNA while the polyacrylamide gel is used for the separation of small DNA fragments which are only a few base pairs long.

Gel electrophoresis employs a buffer system, a medium which is a gel and a source of direct current (Fig. 6). Samples having DNA fragments are applied on the gel and current is passed through the system for an appropriate time. Different DNA fragments move up to different distances on the gel depending on their charge to mass ratio.

The heavier fragments move a little, while the lighter DNA fragments move up to a larger distance. Following the migration of the molecules, the gel is treated with selective stains to show the location of separated molecules in the form of bands.

Very large DNA molecules or chromosomes cannot be separated even by Agarose Gel electrophoresis. For separation of such very large DNA molecules (sometimes representing whole chromosomes), a new technique is used which is known as Pulse Field Gel Electrophoresis (PFGE).

Blotting Techniques:

Visualization of a specific DNA (or RNA or protein) fragment out of many molecules requires a technique called blot transfer. In this technique, the separated bands are transferred onto a nitrocellulose membrane from the gel.

Mainly there are three types of blot transfer procedures:

Southern Blotting, Northern Blotting and Western blotting.

Southern blotting is named after the person who devised this technique, viz. E.M. Southern (1975). The other names began as laboratory jargon but they are now accepted terms.

Technically, blotting may be defined as the transfer of macromolecules from the gel onto the surface of an immobilizing membrane like nitrocellulose membrane. It is to note here that during such transfer, the relative positions of bands (of macromolecules) are same on the membrane as they occurred on the gel.

The membranes which may.be used in blotting are nitrocellulose membrane, nylon membrane, carboxymethyl membrane, diazobenzyl-oxymethyl (DBM) membranes, etc.

Southern blotting is used for the transfer of DNA from gel onto the membrane while Northern and Western blotting are used for the transfer of RNA and protein bands respectively. One other blotting technique is south-western blotting which examines the protein-DNA interactions.

A schematic representation of southern blotting technique is given in the fig. 7. In this technique first of all, the sample DNA is digested with restriction enzymes to obtain fragments of different lengths. These differently sized DNA segments are then passed through Agarose Gel Electrophoresis for their separation based on their lengths.

The gel so obtained with different bands of DNA fragments is placed on top of buffer saturated filter papers which act as a filter paper wick. Above gel is put a nitrocellulose filter and over nitrocellulose filter are placed many dry filter paper sheets. With the movement of buffer towards the dry filter papers, the DNA bands are also moved upwards and hence they get bound to the nitrocellulose filter membrane.

Now, the nitrocellulose filter is removed and baked in vacuum. DNA fragments on the nitrocellulose filter are hybridized with single stranded radioactively labeled probes. Washing is done to remove unbound probes and finally the DNA bands with radioactivity are visualized by autoradiography.

In Northern Blotting, RNA molecules are blot transferred from the gel onto a chemically reactive paper. Western blotting is used for proteins and its working is based on the specificity of antibody-antigen reaction. In this technique the hybridization of bound proteins is done with radioactively labeled antibodies.

Dot Blot Hybridization:

The procedure of this technique is almost the same as blotting, but the only difference is that the DNA fragments are not separated by electrophoresis, instead they are directly applied as a dot on the nitrocellulose membrane.

Then radioactively labeled DNA probes having the complementary base sequences to the DNA of interest are applied on this membrane to allow its hybridization. The position of this hybridization is then detected by autoradiography method.

DNA Sequencing:

The segments of specific DNA molecules obtained by recombinant DNA technology can be analysed for determining their nucleotide sequence.

The methods commonly used for DNA sequencing are:

i. Enzymatic method or Sanger’s Dideoxy method.

ii. Chemical method or Maxam-Gilbert Method.

(i) Enzymatic method of DNA sequencing is also called as Sanger-Coulson method of sequencing of DNA molecules. This method involves the use of single stranded DNA as a template for DNA synthesis.

The dideoxynucleotide triphosphates (ddNTPs like ddCTP, ddGTP, ddATP, ddTTP) are incorporated in the growing chain and they terminate the chain synthesis because they are unable to form a phosphodiester bond with next deoxy-nucleotide triphosphate.

For sequencing, the reaction mixture is taken in four separate test tubes. In each test tube is added one particular ddNTP. As a result, different sizes of newly synthesized DNA strands are obtained in each test tube which are terminated by a particular ddNTP. These segments are then separated by electrophoresis and then the DNA sequences are obtained by reading the bands on autoradiogram from bottom to the top of gel.

(ii) Chemical method of DNA sequencing involves the degradation of DNA by using chemicals, rather than synthesis of new DNA. In this type of sequencing, the DNA sample is labeled radioactively at 3′ ends and separated into single strands. Sample is then divided into four test tubes, each treated with a specific chemical reagent which degrades only at specific nucleotide base like G or C or ‘A and G’ or ‘C and T’.

As a result of this partial chemical cleavage, a number of differently sized fragments are obtained in each test tube. These fragments are separated by gel electrophoresis and then observed under autoradiography to interpret the nucleotide sequence of sample DNA. Chemical method is not used very Commonly because it is a slow and labour intensive process.

(iii) Automatic DNA sequencing methods have been developed by improvements in dideoxy-method. A number of automatic DNA sequencing machines have also been invented which are capable of sequencing thousands of nucleotides within few hours.

Such methods involve the tagging of fluorescent dyes to ddNTPs, slab gel sequencing systems, capillary gel sequencing systems and PCR-based DNA sequencing techniques. Such techniques are faster and more reliable.

Artificial Gene Synthesis:

This technique may also be called as oligonucleotide synthesis. It is one of those techniques which have been adopted for the synthesis of desired gene or DNA fragment. Gene synthesis is now a routine laboratory procedure to be utilized in the rec DNA technology.

First success in the approach of artificial gene synthesis was achieved by Dr. Har Govind Khorana and his co-workers in 1970 when they synthesized the artificial gene for a t-RNA in vitro which had potential for functioning within a living cell.

Major approaches available for the artificial synthesis of genes are:

Enzymatic synthesis of Gene:

When details of base sequence of concerned gene are available, the polynucleotide of that same base sequence can be synthesized by enzymatic method. In this method the bacterial enzyme Polynucleotide phosphorylase is utilized. This method is easy to perform and does not require any template.

Chemical synthesis of Gene:

Once the base sequence of a gene is deducted, this gene can be synthesized by a purely chemical method as used by Khorana and his co-workers for the synthesis of gene for yeast alanyl t-RNA. This method utilizes different chemical reagents for various steps of the process.

There are mainly three distinct methods, which are phosphodiester phosphotriester, and phosphite-triester methods. These methods differ in their strategies for protecting the hydroxyl group of the phosphate residues.

If the detailed sequence of the concerned gene is unknown then the artificial gene is synthesized in the form of cDNA i.e. complementary DNA from the mRNA of that gene. In this method, the enzyme employed is RNA directed DNA polymerase.

PCR (Polymerase Chain Reaction):

PCR is a technique for the amplification (or cloning) of a target sequence of DNA. It is sometimes also referred to as in vitro gene cloning (without expression of that gene). PCR is an important technique in molecular biology and it was discovered by Kary Mullis in 1985 (Fig. 8). It is carried out in vitro and by it, upto billion copies of the target DNA sequence can be obtained from a single copy within few hours only.

Outline of PCR:

PCR is a technique which results in selective amplification of a selected DNA molecule. One limitation of PCR is that the border region sequences of the DNA (to be amplified) must be known in order to select the appropriate primers which anneal (attach) at its 3′ ends. Primer annealing is important due to the fact that enzyme DNA polymerases require double stranded (ds) primer regions for initiating the DNA synthesis.

The whole reaction of PCR takes place in a tube called eppendorf tube. Scientists are using PCR in a number of disciplines due to the advantages like it is a quick, simple and extremely accurate technique. Major limitation of PCR is that due to its extreme sensitivity it may produce erroneous results caused by several inhibitors or contaminating DNA segments present in the sample DNA preparation.

Main Requirements of PCR:

a. Two nucleotide primers which are complementary to 3′ ends of target DNA strands

c. A heat stable DNA polymerase e.g. Taq polymerase.

d. Deoxy adenosine triphosphate (dATP)

e. Deoxy thymidine triphosphate (dTTP)

f. Deoxy cytidine triphosphate (dCTP)

g. Deoxy guanosine triphosphate (dGTP)

h. A thermal cycler in which PCR is carried out

Steps in PCR:

A generalized PCR-protocol involves following steps (however, the temperature-time profile may vary according to the requirements):

(a) Mix target DNA sequence, excess of primers, dATP, dTTP, dCTP, dGTP and Taq polymerase in the reaction mixture in eppendorf tube. Place this tube in thermal cycler.

(b) Reaction mixture is given high temperature of about 90-98°C for few seconds to denature the DNA. As a result the double stranded DNA becomes single stranded.

(c) Temperature is changed to about 55°C for 20 seconds so that primers are annealed at 3′ ends of DNA.

(d) Now the temperature is maintained at 72°C for 30 seconds which facilitates the functioning of Taq polymerase thus synthesizing the complementary strand of DNA.

(e) Hence, one cycle of PCR is completed here resulting in the formation of two ds DNA molecules from one ds DNA.

(f) Same cycle is repeated till the required number of DNA copies are obtained.

Main Types of PCR:

Uses of PCR:

1. For amplification of DNA

3. To diagnose genetic disorders

4. To produce in vitro mutations

5. For preparing DNA for sequencing

6. To analyse genetic defects in single cells from human embryos.

7. To identify virus & bacteria in infectious diseases.

8. For characterization of genotypes.

Colony Hybridization Technique:

This technique is used in genetic engineering for the identification of transformed bacterial cells (i.e. cells which contain foreign DNA). After transformation of cells with a specific DNA, it is likely that only some of those cells may have foreign DNA. For further procedure, firstly it is important to screen such cells which are having foreign DNA.

This screening is done by using the technique of colony hybridization in case of bacterial cells (Fig. 9). A similar technique namely Plaque Hybridization is utilized for screening of transformed bacteriophages.

Basic principle of this technique lies in the in-situ hybridization of transformed bacterial cells with a radioactive probe sequence. Due to the specificity of probe, it enables rapid identification of one colony (through radioactivity) even amongst many thousands of colonies.

The transformed bacterial cells are first of all plated on a suitable agar plate which is termed as the master plate. Colonies are grown in the master plate. These colonies on the master plate are replica-plated onto a nitrocellulose or nylon membrane by placing it gently over the master plate. This replica-plate carrying the colonies is removed and treated with alkaline reagent to lyse the bacteria.

DNA of those bacterial cells is denatured. Proteins on the membrane are digested. Finally the membrane is washed to remove all other molecules, leaving behind only the denatured DNA bound to it, in the form of DNA print of the colonies.

This DNA print is then hybridized with a radioactively labeled RNA/DNA probe. Membrane is washed to remove any unbound probe and then autoradiography is done to detect radioactivity. The positions of the DNA prints showing up in autoradiograph are then compared with the master plate to identify the transformed colony.

Applications of Recombinant DNA Technology:

Genetic engineering or rec DNA technology has enormous and wide-spread applications in all the fields of biological sciences.

Some important applications of rec DNA technology are enlisted below:

(1) Production of Transgenic Plants:

By utilizing the tools and techniques of genetic engineering it is possible to produce transgenic plants or the genetically modified plants. Many transgenic plants have been developed with better qualities like resistance to herbicides, insects or viruses or with expression of male sterility, etc.

Also they allow the production of commercially important biochemical, pharmaceutical compounds, etc. Genetic engineering is capable of introducing the improved post-harvest characteristics in plants also. Transgenic plants also aid in the study of the functions of genes in plant species.

(2) Production of Transgenic Animals:

By the use of rec DNA technology, desired genes can be inserted into the animal so as to produce the transgenic animal. The method of rec DNA technology aids the animal breeders to increase the speed and range of selective breeding in case of animals. It helps for the production of better farm animals so as to ensure more commercial benefits.

Another commercially important use of transgenic animals is the production of certain proteins and pharmaceutical compounds. Transgenic animals also contribute for studying the gene functions in different animal species. Biotechnologists have successfully produced transgenic pigs, sheep, rats and cattle.

(3) Production of Hormones:

By the advent of techniques of rec DNA technology, bacterial cells like E.coli are utilized for the production of different fine chemicals like insulin, somatostatin, somatotropin and p-endorphin. Human Insulin Hormone i.e., Humulin is the first therapeutic product which was produced by the application of rec DNA technology.

The genes of interest are incorporated into the bacterial cells which are then cloned. Such clones are capable of producing a fair amount of hormones like insulin which have great commercial importance.

(4) Production of Vaccines:

Vaccines are the chemical preparations containing a pathogen in attenuated (or weakened) or inactive state that may be given to human beings or animals to confer immunity to infection. A number of vaccines have been synthesized biologically through rec DNA technology.

These vaccines are effective against numerous serious diseases caused by bacteria, viruses or protozoa. These include vaccines for polio, malaria, cholera, hepatitis, rabies, smallpox, etc. The generation of DNA vaccines has revolutionized the approach of treatment of infectious diseases. DNA-vaccine is the preparation that contains a gene encoding an immunogenic protein from the concerned pathogen.

(5) Biosynthesis of Interferon:

Interferon’s are the glycoproteins which are produced in very minute amounts by the virus-infected cells. Interferon’s have antiviral and even anti-cancerous properties. By recDNA technology method, the gene of human fibroblasts (which produce interferon’s in human beings) is inserted into the bacterial plasmid.

These genetically engineered bacteria are cloned and cultured so that the gene is expressed and the interferon’s are produced in fairly high quantities. This interferon, so produced, is then extracted and purified.

(6) Production of Antibiotics:

Antibiotics produced by microorganisms are very effective against different viral, bacterial or protozoan diseases. Some important antibiotics are tetracyclin, penicillin, streptomycin, novobiocin, bacitracin, etc.

recDNA technology helps in increasing the production of antibiotics by improving the microbial strains through modification of genetic characteristics.

(7) Production of Commercially Important Chemicals:

Various commercially important chemicals can be produced more efficiently by utilizing the methods of rec DNA technology. A few of them are the alcohols and alcoholic beverages obtained through fermentation organic acids like citric acid, acetic acid, etc. and vitamins produced by microorganisms.

(8) Application in Enzyme Engineering:

As we know that the enzymes are encoded by genes, so if there are changes in a gene then definitely the enzyme structure also changes. Enzyme engineering utilizes the same fact and can be explained as the modification of an enzyme structure by inducing alterations in the genes which encode for that particular enzyme.

(9) Prevention and Diagnosis of Diseases:

Genetic engineering methods and techniques have greatly solved the problem of conventional methods for diagnosis of diseases. It also provides methods for the. prevention of a number of diseases like AIDS, cholera, etc. Monoclonal antibodies are useful tools for disease diagnosis. Monoclonal antibodies are produced by using the technique called hybridoma technology.

The monoclonal antibodies bear specificity against a specific antigen. These are used in the diagnosis of diseases due to their specificity. Genetic engineering allows the production of hybridoma which is a cell obtained by the fusion of a lymphocyte cell capable of producing antibodies and a single myeloma cell (tumour cell).

(10) Gene Therapy:

Gene therapy is undoubtedly the most beneficial area of genetic engineering for human beings. It involves delivery of specific genes into human body to correct the diseases. Thus it is the treatment of diseases by transfer and expression of a gene into the patients’ cells so as to ensure the restoration of a normal cellular activity.

On the basis of types of cells into which the functional genes are introduced, the gene therapy may be classified as somatic gene therapy and germ line gene therapy. Gene therapy is done either by using in vivo strategy (also called as patient therapy) or by using the ex vivo strategy.

(11) Practical Applications of Genetic Engineering:

recDNA technology has an immense scope in Research and Experimental studies.

a. Localizing specific genes.

b. Sequencing of DNA or genes.

c. Study of mechanism of gene regulation.

d. Molecular analysis of various diseases.

e. Study of” mutations in DNA, etc.

(12) Applications in forensic science:

The applications of rec DNA technology (or genetic engineering) in forensic sciences largely depend on the technique called DNA profiling or DNA fingerprinting. It enables us to identify any person by analysing his hair roots Wood stains, serum, etc. DNA fingerprinting also helps to solve the problems of parentage and to identify the criminals.

(13) Biofuel Production:

Biofuels are derived from biomass and these are renewable and cost effective. Genetic engineering plays an essentially important role in a beneficial and large scale production of biofuels like biogas. bio hydrogen biodiesel bio-ethanol., etc. Genetic engineering helps to improve organisms for obtaining higher product yields and product tolerance.

Genetically stable high producing microorganisms are being developed by using modern recDNA techniques, which aid in an efficient production of bioenergy.

The energy crop plants are those plants which use solar energy in a better way for production of biomass. Genetic improvements of these energy crop plants greatly help for quick and high Product on of biomass which in turn reduces the biofuel production cost. The fermenting microbes which are utilized for biogas production are improved at the genetic level for achieving better result.

(14) Environment Protection:

Genetic engineering makes its contributions to the environment protection in various ways. Most important to mention are the new approaches utilized for waste treatments and bioremediation Environment protection means the conservation of resources and hence to limit the degradation of environment.

Major approach in environment protection is the use of recDNA technology for degradation of toxic pollutants which harm the environment. Different microbes used for sewage treatment, waste water treatment, industrial effluent treatment and for bioremediation are greatly improved by genetic engineering practices and thus present better results.

The plant species can also be developed by using various gene transfer techniques for acquiring better options for phytoremediation. Biological deodorization is a newer technology that involves the decomposition of bad stalling ingredients by microorganisms Genetic -peering play an equally sincere attention towards the improvisation and betterment of such deodorizing microbes.

Transgenic Plants:

A genetically modified plant, consisting in its genome, one or more inserted genes of an unrelated plant is termed as a transgenic plant and those inserted genes are called as transgenic. The development of transgenic plant is possible by using recDNA technology, gene delivery strategies and the tissue culture techniques.

Production of transgenic plants involves two main steps, that are: transformation of the target plant cells and then regeneration of transformed cells into whole plans In the transformation step, foreign gene of interest is introduced into the target plant cells.

This can be done by following any of the gene delivery systems available like AMGT (Agrobacterium mediated gene transfer), using plant viruses as vectors or by direct gene delivery system i.e., electroporation, microinjection, particle-gun method, etc. At present, the particle gun method and AMGT are the most favourable methods of gene delivery into plant cells.

Use of Marker Genes:

After the transformation step is over, it becomes essential to identify the transformed cells. Here, the role of marker genes is marked. The detection of integration of foreign genes into the plant genome requires the use of marker gene that either allow the transformed cells to be selected (selectable marker gene) or that encodes an activity which can be assayed or scored (scorable marker gene).

The use of a dominant selectable marker gene serves as a direct means of obtaining only transformed cells in culture because the non-transformed cells die on the selective medium. Therefore selectable marker genes are the frequently used marker genes. Usually, these selectable marker genes are introduced along with the foreign gene into the plant cells.

Although these marker genes are of immense utility in differentiating transformed cells from the non-transformed cells, but they pose some problems too. There is a threat of accidental transfer of resistance genes (used as selectable markers) to the pathogenic soil-bacteria which may cause disaster.

Secondly, the presence of a selectable marker gene makes it difficult to insert additional foreign genes into the transgenic plant as the same selectable markers gene cannot be used twice. To avoid these problems, a number of advanced strategies have been developed for the removal of marker genes and for the production of marker-free transgenic plants.

Production of transgenic crops has become a crucial part of plant breeding and it has immensely uplifted the possibilities of crop improvement programmes all over the world Numerous transgenic plants of different species have already been in cultivation and some are undergoing field trials.

They are beneficial because they contain improved agronomic traits which are commercially efficient. Transgenic crop plants have many beneficial traits like resistance against different pathogens pests, abiotic stresses, and improved nutritional quality higher yield, better phenotypes, etc. The first transgenic plant was generated in early 1980s when diverse foreign genes were introduced into tobacco plants.

Flavr-Savr tomato marketed in USA in 1994 by Calgene Co. was the first transgenic variety to reach the market. These transgenic tomatoes retain their freshness for long periods.

Freedom II squash marketed by Agrow Seed Co. resist the infection by viral diseases. High lauric rapeseed is an approved genetically engineered plant which produced rapeseeds rich in laurate (fatty-acid) which is useful in making soaps, detergents and shampoos.

Roundup Ready soya been developed by. Monosanto Co. is intended for making animal feed and not tor human use. Food and Drug Administration (FDA) of U.S.A. has so far approved a number of transgenic crops of plants species like rapeseeds, cotton, tomato, maize, sugar beet. papaya, soybean, etc., which have transgenes inserted for producing improved traits.

Some improved traits generally present in transgenic plants are one or more from the following (Table 2):

(i) Resistance to biotic stresses like:

d. Fungal Disease Resistance

e. Bacterial Disease Resistance

(ii) Herbicide Resistance:

(iii) Resistance against abiotic stresses like:

(iv) Modified quality of starch, edible-oils, proteins obtained from crop plants.

(v) Improved Nitrogen fixing capacity

(vi) Delayed ripening for improved storage and longer shelf-life.

(vii) Seedless fruits for better commercial values.

(viii) Improved colour, fragrance and longer life of commercial flowers.

Beyond possessing one or more of the above traits, transgenic plants are also utilized as bioreactors for manufacture of pharmaceutical chemicals and other commercial chemicals. These are also applied for studying the regulation of gene expressions under different conditions of factors like light and temperature (Table 2).

Transgenic research is being carried over in India also with a view to strengthen the plant biotechnology in country. Department of Biotechnology (DBT) makes funds for the promotion of transgenic research in India.

A number of institutes like Central Potato Research Institute (CPRI), Shimla Indian Agricultural Research Institute (IARI), New Delhi Central Rice Research Institute (CRRI), Cuttack Delhi University, Punjab University, Ludhiana, etc. have made significant development in transgenic research producing a number of transgenic crop plants mainly in the species like rice, cotton, rapeseed and tobacco.

Molecular Farming:

This term describes the use of genetically modified plants for the production of scientifically, medically and/or industrially important biomolecules.

The concept behind molecular farming involves the growing and harvesting of plants with novel traits (i.e., transgenic plants) for producing biomolecules rather than food, feed and fibre. Potential biomolecules which can be produced through molecular farming include the medical products like pharmaceuticals (drugs), vaccines, diagnostic products, industrial chemicals, biodegradable plastics, biologies, etc.

Molecular farming has emerged as a promising industry having its base in plant biotechnology. It has attained a great importance in the field of pharmaceutical and industrial production because it ensures the cost-effective production of safe and functional products, which are expensive and difficult to be produced by any other means.

Molecular farming is actually an application of genetic engineering. It uses the genetically modified plants as ‘biological factories’ to produce recombinant protein products for a variety of uses.

In plant molecular farming, first of all the plants to be used as the ‘expression system’ are chosen. The organism into which the foreign gene is inserted for expression of the desired new product in molecular farming is called as expression system.

A foreign gene associated with the production of desired biomolecule is then integrated into plants’ genome. Such genetically engineered i.e., transgenic plants are then grown on agricultural scale providing them with water, sunlight and essential nutrients. During their growth, these transgenic plants synthesize the useful biomolecules which get accumulated in the plant tissues.

These plants are then harvested and the desired product is extracted and purified from the plants.

Some important examples of products that have been developed experimentally through plant molecular farming are interleukin in tobacco, edible vaccines, various enzymes for use in food processing, enzymes for treatment of human diseases, bio plastics from biodegradable molecules in corn, functional antibodies, hormones, blood proteins, trypsin and gastric lipase in corn, etc.

Molecular farming also involves the use of transgenic bacteria, plant viruses, yeasts, animal cell culture and transgenic animals as the expression systems for production of the desirable novel compounds.

But, as the plants offer numerous advantages over animals and animal cultures therefore, the molecular farming involving plants (i.e., plant molecular farming) is the most talked about and practically useful technique. A more closely related term is ‘pharming’ which is mostly applied for the use of ‘transgenic animals’ for the production of pharmaceuticals.

DNA Fingerprinting:

DNA fingerprinting is also called as DNA profiling. This technique was discovered in the year 1986 by British geneticist Alec Jeffrey’s of Leicester University. DNA fingerprinting aids in identification of individuals at the genetic level. It is a well-known fact that every living organism can be differentiated from the other only due to the sequence of nucleotides in the chromosome.

DNA profiling technology characterizes the segments of DNA which helps in the identification of individuals. Its basic requirement is the availability of samples like blood stains, semen, urine, tears, saliva, sweat, hair roots, etc.

For DNA fingerprinting, first of all, the DNA isolated from sample are digested with the help of suitable restriction enzymes.

This digested DNA is then subjected to electrophoresis and southern hybridization which involves its hybridization with a specific probe representing the highly variable region of the organism’s genome. As a result of this, polymorphism is generated. Due to this polymorphism it is very rare that two persons may have same pattern in DNA fingerprints.

This technique of DNA fingerprinting has revolutionized the field of forensic medicine as it is very beneficial for identification of criminals like murderers or rapists. This also helps in solving parentage disputes by identifying the real biological father of the child by analysing the DNA fingerprints of child and suspected father.

In cases where the samples of blood stain, semen etc. has partially degraded DNA the technique of PCR can be applied for amplification of DNA from sample. This enables better characterization of DNA which would not be possible otherwise. In India, facilities for DNA fingerprinting are available at CDFD (Centre for DNA Fingerprinting and Diagnostics), Hyderabad.

Mini-satellites which are tandem repeats of short sequences are used as probes while preparing DNA fingerprints of humans. In case of plants, such probes are not present, therefore, usually RFLPs (Restriction Fragment Length Polymorphism) or simple sequence repeats like (CT) or (AC)n etc. are used as probes for DNA fingerprinting for varietal identification.

GEMs:

GEM stands for Genetically Engineered Microbes. Those microorganisms which are modified through the use of genetic engineering techniques to fulfil specific needs are called as GEMs They are utilized for performing functions which would not be possible through the use of their natural counterparts.

For modifying microbes, foreign genes are introduced in o to genome using the recDNA technology, so as to obtain desired functioning of those microbes GEMs are of great use in different fields specially in industries Amongst different microbe.

The genetically engineered bacteria have found enormous utility in every field. Transfer and expression of beneficial genes into microorganisms has opened a new era for exploiting the microbial bioprocesses for attainment of better commercial outputs.

Some applications of GEMs in different areas are discussed below:

(i) GEMs specially bacterial strains are helpful for crop protection from diseases or abiotic stresses. Development of a virulent strains and antibiotic-producing strains of microbes are commonly used methods for crop protection.

(ii) Genetically engineered bacteria have also been applied for better crop production by enhancing their nitrogen fixation capacity. This is done by transferring of efficient nif genes into the bacterial genome. Rhizobium melilottii is a successful example in this case.

(iii) GEMs are better sources used for enzyme productions on commercial scales.

(iv) GEMs can also be applied for achieving enhanced production and quality of SCP and other compounds used as food and feed.

(v) A number of commercially important chemicals like amino acids, organic acids, ethanol antibiotics, etc. can be efficiently produced by utilizing GEMs. For example, genetically engineered strains of bacteria Bacillus amyloliquefaciens have been in use for large scale production of amino acids.

(vi) GEMs are very useful for abatement of environmental pollution. They have an immense potential for bioremediation of contaminants. Most prominent example for this « superbug which is an oil-eating bacteria Pseudomonas putida (patented in 1980) developed by an India born American scientist Dr. Ananda Mohan Chakraborty.

Splicing Genes:

It is an important step in gene cloning/genetic engineering. It is actually a gene manipulation where one DNA molecule is attached to another. The most important tools for splicing genes are the restriction endonuclease enzymes which cut DNA at specific sites and Produce specific DNA fragments which can be joined to some. Other DNA are techniques available for breaking a DNA molecule into shorter fragments.

Two such important techniques for splicing genes are the fragmentation of DNA by cleaving with restriction enzymes or by the synthesis of complementary DNA. The DNA fragments so isolated or synthesized are subsequently separated and then joined together if the ends are Complementary.

Gene Cloning:

Gene cloning is also referred to as DNA cloning or molecular cloning. In simple words it is the introduction of recombinant DNA molecule into a host cell which is then multiplied to produce the clones of rec DNA. However, often this term i.e., gene cloning is used as a synonym to rec DNA technology or genetic engineering.

Taq Polymerase:

It is a DNA polymerase enzyme which has an important role in PCR (Polymerase Chain Reaction).

It is a DNA polymerase-I type of enzyme which is isolated from the bacteria Thermus aquaticus which lives in hot springs. Taq polymerase enzyme is a thermo stable enzyme and it can withstand even the high temperature used for denaturation of DNA in PCR I hat is why, it is the suitable enzyme for polymerisation of DNA during PCR.

Human Genome Project:

Human Genome project (HGP) is an ambitious plan which is administered by National Institute of Health and Department of Energy, U.S.A. The idea behind HGP is to map and sequence all the genes found in human genome.

The main goal of the HGP is to obtain a complete knowledge of the structure, function, organisation and sequence of human DNA Thus the ultimate aim of HGP is to know the sequence of bases of each gene of a human and to apply this knowledge for benefits of mankind. The development of rec DNA technology specially the use of restriction enzymes has provided an extra boost to Human Genome Project.

Risks of Genetic Engineering:

It is true that biotechnology and genetic engineering have immense applications in almost all the areas related to the betterment of humanity.

But there are certain risks/harms related to it also It has been found that biotechnological processes may also have adverse effects in several areas. A destructive mind can use the biotechnological tools and techniques like genetic engineering for production of new arms race or biological warfare, etc.

Producing new and superior breeds of plants and animals can pose a danger to biodiversity, as only superior breeds would be used and others would be excluded. It is not wrong to say that if recDNA technology is not handled with a caution, it may prove to be disastrous.

There are a few confirmed cases showing the negative aspects of genetic engineering, these are listed below:

i. Genetically engineered human growth hormone (HGH) was found to cause leukemia in children on consumption, so, it was banned for sale in U.S.

ii. U.S. scientists reported the production of a super- AIDS virus which was formed when the ordinary AIDS virus was combined with the mice virus. It is believed to be more hazardous and can be transmitted even by air.

iii. A genetically engineered soybean manufactured by a British company was banned as it caused allergies in some persons on consumption.

iv. The transgenic maize manufactured by a Swiss company Ciba-Geigy was denied permission for sale because there was a fear that the antibiotic resistance gene present in it might go into the bacteria.

Several risks associated to the genetic engineering can be summarized as follows:

1. Hazardous toxins can be produced by genetic engineering of several organism like botulinum toxin, neurotoxins, aflatoxins etc.. can be used as biological weapons.

2. Advancements in biotechnology has also aided the terrorists to produce potent biological warfare agents. The genetically engineered microbes causing severe diseases like E.coli, Haemophilus influenza, etc., can be used for the same.

3. Large scale release of genetically modified plants may disturb the ecological and environmental equilibrium.

4. Introduction of superior genetically engineered varieties are replacing the wild type varieties and are causing a considerable loss to biodiversity.

5. During gene transfer process, the antibiotic resistance marker genes might get introduced to the bacteria which are pathogenic to humans. This may pose a great difficulty to get rid of this bacteria.

6. Gene drug preparation and Gene therapy approaches involve the introduction of genes into the target cells. There is a fear that incorrect integration of gene into target cells may cause problems by inactivating the essential genes.

7. Careless handling of tools of genetic engineering may result into the escape of organisms carrying recDNA molecule from the laboratory into the natural environment. This would be extremely harmful if it is involving a pathogenic gene.

8. Bacillus thuringiensis, baculoviruses, etc. are modified genetically to produce potent pesticides. If such GMOs attack the non-specific targets, they would result into disastrous consequences.

9. During the process of genetic modification the insertion of foreign gene into incorrect site in the host genome may result into the progeny with deformities.

10. GEM may disturb the ecosystem in which it is released by its rapid rate multiplication which may affect the native microbes of that ecosystem.

11. There is a likelihood of the transgene (like insect resistance, herbicide resistance, etc.) to be transferred from GMP to a related sexually compatible weed species. In such case the weed would become more persistent and it would be difficult to control it.

12. Some transgenic plants may pose threats to the human health by production of toxic and/or allergic metabolites. When consumed, such plant products cause allergy and/or infection to the human consumers.

From the above discussion it is clear that the field of biotechnology especially the genetic engineering is a double edged sword. The advances in genetic engineering are of immense help for humanity but if mishandled, their prospects are quite frightening.


Decoding the microbiome

Elhanan Borenstein Computational systems biologist at Tel Aviv University, Israel.

Over the past decade, methods for sequencing the genetic content of microbial communities have probed the composition of the human microbiome. More recently, scientists have tried to learn what the microbiome is doing by integrating information about genes, transcripts, proteins and metabolites. Metabolites are especially interesting: they could offer the closest understanding of how the microbiome affects our health, because many host–microbiome interactions occur through the metabolites that bacteria generate and consume.

Priorities for the next 10 years of human microbiome research

There has been an explosion of micro-biome–metabolome studies looking at, for instance, a set of stool samples — identifying the species present in each sample and their abundances through metagenomic sequencing, and using mass spectrometry and other technologies to measure the concentrations of different metabolites. By combining these two profiles, the hope is to understand which member of the microbiome is doing what, and thus whether specific microbes determine the level of certain metabolites.

But these data are complex and multi-dimensional, and there might be a whole web of interactions, involving multiple species and pathways, which ultimately produce a set of metabolites. Scientists have published computational methods to link microbiome and metabolome data and to learn these quirks and patterns. Such methods range from simple correlation-based analyses to complex machine-learning approaches that use existing microbiome–metabolome data sets to predict the metabolome in new microbial communities, or to recover microbe–metabolite relationships.

Our lab takes a different strategy. Rather than apply statistical methods to find microbe–metabolite associations, we build mechanistic models of how we think a specific microbial composition affects the metabolome, and use these as part of the analyses themselves. In effect, we are asking: on the basis of genomic and metabolic information, what do we know about each microbe’s ability to produce or take up specific metabolites? We can then predict the potential of a given collection of microbes to produce or degrade specific metabolites, and compare those predictions with actual metabolomic data. We showed that this approach avoids the pitfalls of simple correlation-based analyses 4 , and will release a new version of the analysis framework in the coming months.

Such studies could improve micro-biome-based therapies by identifying, for example, specific microbes responsible for producing too much of a harmful metabolite or too little of a beneficial one.

Software code can be used to build models that simulate tumour development. Credit: Getty


Inexpensive biology kits offer hands-on experience with DNA

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To help students gain a better grasp of biological concepts, MIT and Northwestern University researchers have designed educational kits that can be used to perform experiments with DNA, to produce glowing proteins, scents, or other easily observed phenomena.

Biology teachers could use the BioBits kits to demonstrate key concepts such as how DNA is translated into proteins, or students could use them to design their own synthetic biology circuits, the researchers say.

“Our vision is that these kits will serve as a creative outlet for young individuals, and show them that biology can be a design platform,” says James Collins, the Termeer Professor of Medical Engineering and Science in MIT’s Institute for Medical Engineering and Science (IMES) and Department of Biological Engineering. “The time is right for creating educational kits that could be utilized in classrooms or in the home, to introduce young folks as well as adults who want to be retrained in biotech, to the technologies that underpin synthetic biology and biotechnology.”

The new kits contain no living cells but instead consist of freeze-dried cellular components, which makes them inexpensive, shelf-stable, and accessible to any classroom, even in schools with minimal resources.

“Synthetic biology is a technology for the 21st century, and these ‘just add water’ kits are poised to transform synthetic biology education. Indeed, BioBits kits are user-friendly, engage the senses in a fun and exciting way, and reduce biosafety concerns,” says Michael Jewett, the Charles Deering McCormick Professor of Teaching Excellence, an associate professor of chemical and biological engineering, and co-director of the Center for Synthetic Biology at Northwestern University, who led the research team with Collins.

The researchers describe the two kits, BioBits Bright and BioBits Explorer, in two papers appearing in Science Advances on Aug. 1. The lead authors of both papers are Ally Huang, an MIT graduate student Peter Nguyen, a postdoc at Harvard University’s Wyss Institute for Biologically Inspired Engineering and Jessica Stark, a Northwestern University graduate student.

Hands-on biology

In recent years, Collins’ lab has been working on technology to extract and freeze-dry the molecular machinery needed to translate DNA into proteins. They developed freeze-dried pellets, which contain dozens of enzymes and other molecules extracted from cells, and can be stored for an extended period of time at room temperature. Upon the addition of water and DNA, the pellets begin producing proteins encoded by the DNA.

The Collins and Jewett labs recently began to adapt this technology to educational biology kits, in hopes of bringing hands-on, laboratory experiences to high school students, as well as younger students.

“I fell in love with biology in high school, but I never really truly understood the biological concepts until college, when I started working in a research lab and actually doing all the real experiments,” says Huang, who took on the project after joining Collins’ lab a few years ago. “The intent of this project was to find a way to bring these laboratory experiments into a nonlaboratory setting in an easy-to-do and cheap way.”

The researchers set out to create the equivalent of the toy chemistry kit, which allows users to perform their own simple chemical reactions at home.

“One of the best gifts I got as a kid was a chemistry kit,” Collins says. “I did all the prescribed reactions and then went off-script and created my own reactions, some of which were probably not recommended. But I had a tremendous time, and, like many faculty here, was inspired, in part, to consider a career in science because of those kits.”

Similar kits are available to help children build their own simple electronic or robotic systems, but right now, the researchers say, there is no cost-effective equivalent for biology. One reason for that is that most biology experiments involve living cells, which require expensive equipment to keep them alive and can also pose safety risks. The MIT and Northwestern researchers were able to overcome that obstacle with their freeze-dried cellular components.

“The goal was to create a kit where the teacher could open the box and hand out all the components to the kids, without any prep time,” Huang says. “You add the water that contains your DNA to these freeze-dried pellets, and just by doing that the kids can produce a variety of different proteins, and visualize or sense different outputs from these proteins.”

The BioBits Bright kit is based on fluorescent proteins. The kit includes tubes with freeze-dried pellets containing all of the cellular components needed to translate DNA into proteins, as well as DNA that encodes fluorescent proteins of several different colors. Students can add DNA to the pellets, put the tubes into an inexpensive incubator the researchers designed, and then image them using a $15 device that the researchers also developed.

This kind of experimentation, which allows students to vary the amount of DNA added, length of incubation, and temperature of the reaction, helps students to grasp firsthand the “central dogma” of biology: how information encoded by genes flows from DNA to RNA to proteins. The kit can be produced for less than $100 for a classroom of 30 students, making it feasible for use in schools with limited budgets.

In the BioBits Explorer kit, the researchers included DNA that encodes proteins with outputs other than fluorescence, helping to teach additional biological concepts such as reaction catalysis. One DNA sequence included in the kit codes for an enzyme that converts isoamyl alcohol into banana oil, producing a distinctive scent. Another DNA sequence produces an enzyme that can catalyze the formation of hydrogels. The kit also allows students to extract DNA from a fruit such as a banana or kiwi and then test it with a sensor that can distinguish between DNA sequences found in different types of fruit.

Mix and match

In addition to classroom experiments, the researchers believe these kits could be useful for school science clubs where students could “mix and match the components and try to come up with new reactions, or experiment to find what new combinations of outputs they could make,” Huang says.

In trial runs in the Chicago public schools, which began last year, the researchers found that students ranging in age from elementary school to high school were able to successfully perform their own experiments using the kits.

“Seeing the students’ and teachers’ results, which showed that a first-time user could run the BioBits Bright labs successfully, was when it started to become real,” Stark says. “That data gives us evidence that these kits have the potential to significantly expand the kinds of hands-on biology activities that are possible in classrooms or other non-lab settings.”

The team is now building new prototypes of the BioBits Bright kit that will be tested in high schools in Boston, Cambridge, and Chicago this fall. The researchers have launched a website to help enable the creation of an open source community that would allow teachers to add their own supporting curriculum, and scientists to add new components to the kits.

“Eventually, we hope to form a larger community of scientists and educators who are interested in continuing to translate cutting-edge science into hands-on educational experiences,” Stark says.

The researchers hope that the kits will not only help students grasp the connections between what they learn from their biology textbook and real-life biological events, but also stimulate their interest in careers in biology or other science, technology, engineering, and mathematics (STEM) fields.

“We want the BioBits kits to help students see themselves as scientists and hope that these open-access kits might inspire the next generation of students to pursue STEM education,” Jewett says.

The research was funded, in part, by the Army Research Office, the National Science Foundation, the Air Force Research Laboratory Center of Excellence, the Defense Threat Reduction Agency, the David and Lucile Packard Foundation, the Camille Dreyfus Teacher-Scholar Program, and the Department of Energy.


Methodology

The following criteria were used in compiling this list of Top 15 DNA Analyst Professors. Not all criteria below applied to each and every professor, but many of the listed professors may have:

    Undertaken interesting research:

Some of these professors have become involved in research or projects that have promulgated new technologies or strategies in DNA analysis.

All of the professors on this list have received doctoral-level degrees. Many have expertise that is grounded in biology or other scientific fields, but may also have gained DNA analysis experience from previous professional employment.

Many of these professors are involved in forensic science programs at the student level, or may provide training to police, detectives and technician personnel.

Many of these professors have co-authored academic articles about new DNA analysis techniques, or the processes that are resulting in unusual or interesting DNA findings.

  • Interviews
  • Professors
  • Resources
  • How to Become
  • Follow the Money

DNA Technology in Forensic Science (1992)

Characterization, or ''typing," of deoxyribonucleic acid (DNA) for purposes of criminal investigation can be thought of as an extension of the forensic typing of blood that has been common for more than 50 years it is actually an extension from the typing of proteins that are coded for by DNA to the typing of DNA itself. Genetically determined variation in proteins is the basis of blood groups, tissue types, and serum protein types. Developments in molecular genetics have made it possible to study the person-to-person differences in parts of DNA that are not involved in coding for proteins, and it is primarily these differences that are used in forensic applications of DNA typing to personal identification. DNA typing can be a powerful adjunct to forensic science. The method was first used in casework in 1985 in the United Kingdom and first used in the United States by commercial laboratories in late 1986 and by the Federal Bureau of Investigation (FBI) in 1988.

DNA typing has great potential benefits for criminal and civil justice however, because of the possibilities for its misuse or abuse, important questions have been raised about reliability, validity, and confidentiality. By the summer of 1989, the scientific, legal, and forensic communities were calling for an examination of the issues by the National Research Council of the National Academy of Sciences. As a response, the Committee on DNA Technology in Forensic Science was formed its first meeting was held in January 1990. The committee was to address the general applicability and appropriateness of the use of DNA technology in forensic science, the need to develop standards for data collection and analysis, aspects of the

technology, management of DNA typing data, and legal, societal, and ethical issues surrounding DNA typing. The techniques of DNA typing are fruits of the revolution in molecular biology that is yielding an explosion of information about human genetics. The highly personal and sensitive information that can be generated by DNA typing requires strict confidentiality and careful attention to the security of data.

DNA, the active substance of the genes, carries the coded messages of heredity in every living thing: animals, plants, bacteria, and other microorganisms. In humans, the code-carrying DNA occurs in all cells that have a nucleus, including white blood cells, sperm, cells surrounding hair roots, and cells in saliva. These would be the cells of greatest interest in forensic studies.

Human genes are carried in 23 pairs of chromosomes, long threadlike or rodlike structures that are a person's archive of heredity. Those 23 pairs, the total genetic makeup of a person, are referred to as the human "diploid genome." The chemistry of DNA embodies the universal code in which the messages of heredity are transmitted. The genetic code itself is spelled out in strings of nucleotides of four types, commonly represented by the letters A, C, G, and T (standing for the bases adenine, cytosine, guanine, and thymine), which in various combinations of three nucleotides spell out the

FIGURE 1 Diagram of the double-helical structure of DNA in a chromosome. The line shown in the chromosome is expanded to show the DNA structure.

codes for the amino acids that constitute the building blocks of proteins. A gene, the basic unit of heredity, is a sequence of about 1,000 to over 2 million nucleotides. The human genome, the total genetic makeup of a person, is estimated to contain 50,000-100,000 genes.

The total number of nucleotides in a set of 23 chromosomes&mdashone from each pair, the "haploid genome"&mdashis about 3 billion. Much of the DNA, the part that separates genes from one another, is noncoding. Variation in the genes, the coding parts, are usually reflected in variations in the proteins that they encode, which can be recognized as "normal variation" in blood type or in the presence of such diseases as cystic fibrosis and phenylketonuria but variations in the noncoding parts of DNA have been most useful for DNA typing.

Except for identical twins, the DNA of a person is for practical purposes unique. That is because one chromosome of each pair comes from the father and one from the mother which chromosome of a given pair of a parent's chromosomes that parent contributes to the child is independent of which chromosome of another pair that parent gives to that child. Thus, the different combinations of chromosomes that one parent can give to one child is 2 23 , and the number of different combinations of paired chromosomes a child can receive from both parents is 2 46 .

The substitution of even one nucleotide in the sequence of DNA is a variation that can be detected. For example, a variation in DNA consisting of the substitution of one nucleotide for another (such as the substitution of a C for a T) can often be recognized by a change in the points at which certain biological catalysts called "restriction enzymes" cut the DNA. Such an enzyme cuts DNA whenever it encounters a specific sequence of nucleotides that is peculiar to the enzyme. For example, the enzyme HaeI cuts DNA wherever it encounters the sequence AGGCCA. A restriction enzyme will cut a sample of DNA into fragments whose lengths depend on the location of the cutting sites recognized by the enzyme. Assemblies of fragments of different lengths are called "restriction fragment length polymorphisms" (RFLPs), and RFLPs constitute one of the most important tools for analyzing and identifying samples of DNA.

An important technique used in such analyses is the "Southern blot," developed by Edwin Southern in 1975. A sample of DNA is cut with a restriction enzyme, and the fragments are separated from one another by electrophoresis (i.e., they are separated by an electrical field). The fragments of particular interest are then identified with a labeled probe, a short segment of single-stranded DNA containing a radioactive atom, which hybridizes (fuses) to the fragments of interest because its DNA sequence is complementary to those of the fragments (A pairing with T, C pairing with G). Each electrophoretic band represents a separate fragment of DNA, and a given person will have no more than two fragments derived from a partic-

ular place in his or her DNA&mdashone representing each of the genes that are present on the two chromosomes of a given pair. The forms of a given gene are referred to as alleles. A person who received the same allele from the mother and the father is said to be "homozygous" for that allele a person who received different alleles from the mother and the father is said to be "heterozygous." Many RFLP systems are based on change in a single nucleotide. They are said to be "diallelic," because there are only two common alternative forms. And there are only three genotypes: two kinds of homozygous genotypes and a heterozygous genotype. Another form of RFLP is generated by the presence of variable number tandem repeats (VNTRs). VNTRs are sequences, sometimes as small as two different nucleotides (such as C and A), that are repeated in the DNA. When such a structure is subjected to cutting with restriction enzymes, fragments of varied length are obtained.

It was variation of the VNTR type to which Alex Jeffreys in the United Kingdom first applied the designation "DNA fingerprinting." He used probes that recognized not one locus, but multiple loci, and "DNA fingerprinting" has come to refer particularly to multilocus, multiallele systems. A locus is a specific site of a gene on a chromosome. In the United States, in particular, single-locus probes are preferred, because their results are easier to interpret. "DNA typing" is the preferred term, because "DNA fingerprinting'' is associated with multilocus systems. Discriminating power for personal identification is achieved by using several&mdashusually at least four&mdashsingle-locus, multiallelic systems.

The entire procedure for analyzing DNA with the RFLP method is diagrammed in Figure 2.

After the bands (alleles) are visualized, those in the evidence sample and the suspect sample are compared. If the bands match in the two samples, for all three or four enzyme-probe combinations, the question is: What is the probability that such a match would have occurred between the suspect and a person drawn at random from the same population as the suspect?

Answering that question requires calculation of the frequency in the population of each of the gene variants (alleles) that have been found, and the calculation requires a databank where one can find the frequency of each allele in the population. On the basis of some assumptions, so-called Hardy-Weinberg ratios can be calculated. For a two-allele system, the ratios are indicated by the expressions p2 and q2 for the frequency of the two homozygotes and 2pq for heterozygotes, p and q being the frequencies of the two alleles and p + q being equal to 1. Suppose that a person is heterozygous at a locus where the frequencies of the two alleles in the population are 0.3 and 0.7. The frequency of that heterozygous genotype in the population would be 2 × 0.3 × 0.7 = 0.42. Suppose, further, that at three

FIGURE 2 Schematic representation of Southern blotting of single-locus, multiallelic VNTR. In example shown here, DNA from four persons is tested. All have different patterns. Three are heterozygous and one homozygous, for a total of seven different alleles. From L. T. Kirby, "DNA Fingerprinting: An Introduction," Stockton Press, New York, 1990. Copyright © 1990 by Stockton Press. Reprinted with permission of W.H. Freeman and Company.

other loci the person being typed has genotypes with population frequencies of 0.01, 0.32, and 0.02. The frequency of the combined genotype in the population is 0.42 × 0.01 × 0.32 × 0.02 or 0.000027, or approximately 1 in 37,000.

That example illustrates what is called the product rule, or multiplication rule. Its use assumes that the alleles at a given locus are inherited independently of each other. It also assumes that there are no subpopulations in which a particular allele at one locus would have a preferential probability of being associated with a particular allele at a second locus.

Techniques for analyzing DNA are changing rapidly. One key technique introduced in the last few years is the polymerase chain reaction (PCR), which allows a million or more copies of a short region of DNA to be easily made. For DNA typing, one amplifies (copies) a genetically informative sequence, usually 100-2,000 nucleotides long, and detects the genotype in the amplified product. Because many copies are made, DNA

typing can rely on methods of detection that do not use radioactive substances. Furthermore, the technique of PCR amplification permits the use of very small samples of tissue or body fluids&mdashtheoretically even a single nucleated cell.

The PCR process (Figure 3) is simple indeed, it is analogous to the process by which cells replicate their DNA. It can be used in conjunction with various methods for detecting person-to-person differences in DNA.

It must be emphasized that new methods and technology for demonstrating individuality in each person's DNA are being developed. The present methods explained here will probably be superseded by others that are more efficient, error-free, automatable, and cost-effective. Care should be taken to ensure that DNA typing techniques used for forensic purposes do not become "locked in" prematurely, lest society and the criminal justice system be unable to benefit fully from advances in science and technology.

TECHNICAL CONSIDERATIONS

The forensic use of DNA typing is an outgrowth of its medical diagnostic use&mdashanalysis of disease-causing genes based on comparison of a patient's DNA with that of family members to study inheritance patterns of genes or comparison with reference standards to detect mutations. To understand the challenges involved in such technology transfer, it is instructive to compare forensic DNA typing with DNA diagnostics.

DNA diagnostics usually involves clean tissue samples from known sources. Its procedures can usually be repeated to resolve ambiguities. It involves comparison of discrete alternatives (e.g., which of two alleles did a child inherit from a parent?) and thus includes built-in consistency checks against artifacts. It requires no knowledge of the distribution of patterns in the general population.

Forensic DNA typing often involves samples that are degraded, contaminated, or from multiple unknown sources. Its procedures sometimes cannot be repeated, because there is too little sample. It often involves matching of samples from a wide range of alternatives in the population and thus lacks built-in consistency checks. Except in cases where the DNA evidence excludes a suspect, assessing the significance of a result requires statistical analysis of population frequencies.

Each method of DNA typing has its own advantages and limitations, and each is at a different state of technical development. However, the use of each method involves three steps:

Laboratory analysis of samples to determine their genetic-marker types at multiple sites of potential variation.

Comparison of the genetic-marker types of the samples to determine

Polymerase chain reaction (PCR). Courtesy, Perkin-Elmer Cetus Instruments.

whether the types match and thus whether the samples could have come from the same source.

If the types match, statistical analysis of the population frequencies of the types to determine the probability that a match would have been observed by chance in a comparison of samples from different persons.

Before any particular DNA typing method is used for forensic purposes, precise and scientifically reliable procedures for performing all three steps must be established. It is meaningless to speak of the reliability of DNA typing in general&mdashi.e., without specifying a particular method.

Despite the challenges of forensic DNA typing, it is possible to develop reliable forensic DNA typing systems, provided that adequate scientific care is taken to define and characterize the methods.

Recommendations

Any new DNA typing method (or a substantial variation of an existing method) must be rigorously characterized in both research and forensic settings, to determine the circumstances under which it will yield reliable results.

DNA analysis in forensic science should be governed by the highest standards of scientific rigor, including the following requirements:

Each DNA typing procedure must be completely described in a detailed, written laboratory protocol.

Each DNA typing procedure requires objective and quantitative rules for identifying the pattern of a sample.

Each DNA typing procedure requires a precise and objective matching rule for declaring whether two samples match.

Potential artifacts should be identified by empirical testing, and scientific controls should be designed to serve as internal checks to test for the occurrence of artifacts.

The limits of each DNA typing procedure should be understood, especially when the DNA sample is small, is a mixture of DNA from multiple sources, or is contaminated with interfering substances.

Empirical characterization of a DNA typing procedure must be published in appropriate scientific journals.

Before a new DNA typing procedure can be used, it must have not only a solid scientific foundation, but also a solid base of experience.

The committee strongly recommends the establishment of a National Committee on Forensic DNA Typing (NCFDT) under the auspices of an

appropriate government agency or agencies to provide expert advice primarily on scientific and technical issues concerning forensic DNA typing.

Novel forms of variation in the genome that have the potential for increased power of discrimination between persons are being discovered. Furthermore, new ways to demonstrate variations in the genome are being developed. The current techniques are likely to be superseded by others that provide unambiguous individual identification and have such advantages as automatability and economy. Each new method should be evaluated by the NCFDT for use in the forensic setting, applying appropriate criteria to ensure that society derives maximal benefit from DNA typing technology.

STATISTICAL BASIS FOR INTERPRETATION

Because any two human genomes differ at about 3 million sites, no two persons (barring identical twins) have the same DNA sequence. Unique identification with DNA typing is therefore possible, in principle, provided that enough sites of variation are examined. However, the DNA typing systems used today examine only a few sites of variation and have only limited resolution for measuring the variability at each site. There is a chance that two persons have DNA patterns (i.e., genetic types) that match at the small number of sites examined. Nevertheless, even with today's technology, which uses 3-5 loci, a match between two DNA patterns can be considered strong evidence that the two samples came from the same source. Interpreting a DNA typing analysis requires a valid scientific method for estimating the probability that a random person by chance matches the forensic sample at the sites of DNA variation examined. To say that two patterns match, without providing any scientifically valid estimate (or, at least, an upper bound) of the frequency with which such matches might occur by chance, is meaningless. The committee recommends approaches for making sound estimates that are independent of the race or ethnic group of the subject.

A standard way to estimate frequency is to count occurrences in a random sample of the appropriate population and then use classical statistical formulas to place upper and lower confidence limits on the estimate. (Because forensic science should avoid placing undue weight on incriminating evidence, an upper confidence limit of the frequency should be used in court.) If a particular DNA pattern occurred in 1 of 100 samples, the estimated frequency would be 1%, with an upper confidence limit of 4.7%. If the pattern occurred in 0 of 100 samples, the estimated frequency would be 0%, with an upper confidence limit of 3%. (The upper bound cited is the traditional 95% confidence limit, whose use implies that the true value has only a 5% chance of exceeding the upper bound.) Such estimates produced

by straightforward counting have the virtue that they do not depend on theoretical assumptions, but simply on the samples having been randomly drawn from the appropriate population. However, such estimates do not take advantage of the full potential of the genetic approach.

In contrast, population frequencies often quoted for DNA typing analyses are based not on actual counting, but on theoretical models based on the principles of population genetics. Each matching allele is assumed to provide statistically independent evidence, and the frequencies of the individual alleles are multiplied together to calculate a frequency of the complete DNA pattern. Although a databank might contain only 500 people, multiplying the frequencies of enough separate events might result in an estimated frequency of their all occurring in a given person of 1 in a billion. Of course, the scientific validity of the multiplication rule depends on whether the events (i.e., the matches at each allele) are actually statistically independent.

Because it is impossible or impractical to draw a large enough population to test directly calculated frequencies of any particular DNA profile much below 1 in 1,000, there is not a sufficient body of empirical data on which to base a claim that such frequency calculations are reliable or valid. The assumption of independence must be strictly scrutinized and estimation procedures appropriately adjusted if possible. (The rarity of all the genotypes represented in the databank can be demonstrated by pairwise comparisons, however. Thus, in a recently reported analysis of the FBI databank, no exactly matching pairs were found in five-locus DNA profiles, and the closest match was a single three-locus match among 7.6 million pairwise comparisons.)

The multiplication rule has been routinely applied to blood-group frequencies in the forensic setting. However, that situation is substantially different. Because conventional genetic markers are only modestly polymorphic (with the exception of human leukocyte antigen, HLA, which usually cannot be typed in forensic specimens), the multilocus genotype frequencies are often about 1 in 100. Such estimates have been tested by simple empirical counting. Pairwise comparisons of allele frequencies have not revealed any correlation across loci. Hence, the multiplication rule does not appear to lead to the risk of extrapolating beyond the available data for conventional markers. But highly polymorphic DNA markers exceed the informative power of protein markers and so multiplication of their estimated frequencies leads to estimates that are far less than the reciprocal of the size of the databanks, i.e., 1/N, N being the number of entries in the databank.

The multiplication rule is based on the assumption that the population does not contain subpopulations with distinct allele frequencies&mdashthat each person's alleles constitute statistically independent random selections from

a common gene pool. Under that assumption, the procedure for calculating the population frequency of a genotype is straightforward:

Count the frequency of alleles. For each allele in the genotype, examine a random sample of the population and count the proportion of matching alleles&mdashthat is, alleles that would be declared to match according to the rule that is used for declaring matches in a forensic context.

Calculate the frequency of the genotype at each locus. The frequency of a homozygous genotype a1/a1 is calculated to be pa1 2 , where pa1 denotes the frequency of allele a1. The frequency of a heterozygous genotype a1/a2 is calculated to be 2pa1pa2, where pa1 and pa2 denote the frequencies of alleles a1 and a2. In both cases, the genotype frequency is calculated by multiplying the two allele frequencies, on the assumption that there is no statistical correlation between the allele inherited from one's father and the allele inherited from one's mother. When there is no correlation between the two parental alleles, the locus is said to be in Hardy-Weinberg equilibrium.

Calculate the frequency of the complete multilocus genotype by multiplying the genotype frequencies at all the loci. As in the previous step, this calculation assumes that there is no correlation between the genotypes at the individual loci the absence of such correlation is called linkage equilibrium. Suppose, for example, that a person has genotype a1/a2, b1/b2, c1/c1. If a random sample of the appropriate population shows that the frequencies of alleles a1, a2, b1, b2, and c1 are approximately 0.1, 0.2, 0.3, 0.1, and 0.2, respectively, then the population frequency of the genotype would be estimated to be [2(0.1)(0.2)][2(0.3)(0.1)][(0.2)(0.2)] = 0.000096, or about 1 in 10,417.

The validity of the multiplication rule depends on the assumption of absence of population substructure. Population substructure violates the assumption of statistical independence of alleles. In a population that contains groups each with different allele frequencies, the presence of one allele in a person's genotype can alter the statistical expectation of the other alleles in the genotype. For example, a person who has one allele that is common among Italians is more likely to be of Italian descent and is thus more likely to carry additional alleles that are common among Italians. The true genotype frequency is thus higher than would be predicted by applying the multiplication rule using the average frequency in the entire population.

To illustrate the problem with a hypothetical example, suppose that a particular allele at a VNTR locus has a 1% frequency in the general population, but a 20% frequency in a specific subgroup. The frequency of homozygotes for the allele would be calculated to be 1 in 10,000 according to the allele frequency determined by sampling the general population, but would actually be 1 in 25 for the subgroup. That is a hypothetical and

extreme example, but illustrates the potential effect of demography on gene frequency estimation.

The key question underlying the use of the multiplication rule&mdashi.e., whether actual populations have significant substructure for the loci used for forensic typing&mdashhas provoked considerable debate among population geneticists. Some have expressed serious concern about the possibility of significant substructure. They maintain that census categories&mdashsuch as North American Caucasians, blacks, Hispanics, Asians, and Native Americans&mdashare not homogeneous groups, but rather that each group is an admixture of subgroups with somewhat different allele frequencies. Allele frequencies have not yet been homogenized, because people tend to mate within their subgroups.

Those population geneticists also point out that, for any particular genetic marker, the actual degree of subpopulation differentiation cannot be predicted in advance, but must be determined empirically. Furthermore, they doubt that the presence of substructure can be detected by the application of statistical tests to data from large mixed populations. Population differentiation must be assessed through direct studies of allele frequencies in ethnic groups.

Other population geneticists, while recognizing the possibility or likelihood of population substructure, conclude that the evidence to date suggests only a minimal effect on estimates of genotype frequencies. Recent empirical studies concerning VNTR loci detected no deviation from independence within or across loci. Moreover, as pointed out earlier, pairwise comparisons of all five-locus DNA profiles in the FBI database showed no exact matches the closest match was a single three-locus match among 7.6 million pairwise comparisons. Those studies are interpreted as indicating that multiplication of gene frequencies across loci does not lead to major inaccuracies in the calculation of genotype frequency&mdashat least not for the specific polymorphic loci examined.

Although mindful of those opposing views, the committee has chosen to assume for the sake of discussion that population substructure may exist and to provide a method for estimating population frequencies in a manner that would adequately account for it. Our decision is based on four considerations:

It is possible to provide conservative estimates of population frequency, without giving up the inherent power of DNA typing.

It is appropriate to prefer somewhat conservative numbers for forensic DNA typing, especially because the statistical power lost in this way can often be recovered through typing of additional loci, where required.

It is important to have a general approach that is applicable to any loci used for forensic typing. Recent empirical studies pertain only to the population genetics of the VNTR loci in current use. However, we expect

forensic DNA typing to undergo much change over the next decade&mdashincluding the introduction of different types of DNA polymorphisms, some of which might have different properties from the standpoint of population genetics.

It is desirable to provide a method for calculating population frequencies that is independent of the ethnic group of the subject.

The committee is aware of the need to account for possible population substructure, and it recommends the use of the ceiling principle. The multiplication rule will yield conservative estimates even for a substructured population, provided that the allele frequencies used in the calculation exceed the allele frequencies in any of the population subgroups. The ceiling principle involves two steps: (1) for each allele at each locus, determine a ceiling frequency that is an upper bound of the allele frequency that is independent of the ethnic background of a subject and (2) to calculate a genotype frequency, apply the multiplication rule according to the ceiling allele frequencies.

To determine ceiling frequencies, the committee strongly recommends the following approach: (1) Draw random samples of 100 persons from each of 15-20 populations that represent groups relatively homogeneous genetically. (2) Take as the ceiling frequency the largest frequency in any of those populations or 5%, whichever is larger.

Use of the ceiling principle yields the same frequency of a given genotype, regardless of the suspect's ethnic background, because the reported frequency represents a maximum for any possible ethnic heritage. Accordingly, the ethnic background of the individual suspect should be ignored in estimating the likelihood of a random match. The calculation is fair to suspects, because the estimated probabilities are likely to be conservative in their incriminating power.

Some legal commentators have pointed out that frequencies should be based on the population of possible perpetrators, rather than on the population to which a particular suspect belongs. Although that argument is formally correct, practicalities often preclude use of that approach. Furthermore, the ceiling principle eliminates the need for investigating the perpetrator population, because it yields an upper bound to the frequency that would be obtained by that approach.

Although the ceiling principle is a conservative approach, we feel that it is appropriate. DNA typing is unique, in that the forensic analyst has an essentially unlimited ability to adduce additional evidence: whatever power is sacrificed by requiring conservative estimates can be regained by examining additional loci. (Although there might be some cases in which the DNA sample is insufficient to permit typing additional loci with RFLPs, this limitation is likely to disappear with the eventual use of PCR.)

That no evidence of population substructure is demonstrable with the

markers tested so far cannot be taken to mean that such does not exist for other markers. Preservation of population DNA samples in the form of immortalized cell lines will ensure that DNA is available for determining population frequencies of any DNA pattern as new and better techniques become available, without the necessity of collecting fresh samples. It will also provide samples for standardization of methods across laboratories.

Because of the similarity in DNA patterns between relatives, databanks of DNA of convicted criminals have the ability to point not just to individuals but to entire families&mdashincluding relatives who have committed no crime. Clearly, this raises serious issues of privacy and fairness. It is inappropriate, for reasons of privacy, to search databanks of DNA from convicted criminals in such a fashion. Such uses should be prevented both by limitations of the software for searching and by statutory guarantees of privacy.

The genetic correlation among relatives means that the probability that a forensic sample will match a relative of the person who left it is considerably greater than the probability that it will match a random person.

Especially for a technology with high discriminatory power, such as DNA typing, laboratory error rates must be continually estimated in blind proficiency testing and must be disclosed to juries.

Recommendations

As a basis for the interpretation of the statistical significance of DNA typing results, the committee recommends that blood samples be obtained from 100 randomly selected persons in each of 15-20 relatively homogeneous populations that the DNA in lymphocytes from these blood samples be used to determine the frequencies of alleles currently tested in forensic applications and that the lymphocytes be ''immortalized" and preserved as a reference standard for determination of allele frequencies in tests applied in different laboratories or developed in the future. The collection of samples and their study should be overseen by a National Committee on Forensic DNA Typing.

The ceiling principle should be used in applying the multiplication rule for estimating the frequency of particular DNA profiles. For each allele in a person's DNA pattern, the highest allele frequency found in any of the 15-20 populations or 5% (whichever is larger) should be used.

In the interval (which should be short) while the reference blood samples are being collected, the significance of the findings of multilocus DNA typing should be presented in two ways: (1) If no match is found with any sample in a total databank of N persons (as will usually be the case), that should be stated, thus indicating the rarity of a random match. (2) In applying the multiplication rule, the 95% upper confidence limit of the frequency of each allele should be calculated for separate U.S. "racial"

groups and the highest of these values or 10% (whichever is the larger) should be used. Data on at least three major "races" (e.g., Caucasians, blacks. Hispanics, Asians, and Native Americans) should be analyzed.

Any population databank used to support DNA typing should be openly available for scientific inspection by parties to a legal case and by the scientific community.

Laboratory error rates should be measured with appropriate proficiency tests and should play a role in the interpretation of results of forensic DNA typing.

STANDARDS

Critics and supporters of the forensic uses of DNA typing agree that there is a lack of standardization of practices and a lack of uniformly accepted methods for quality assurance. The deficiencies are due largely to the rapid emergence of DNA typing and its introduction in the United States through the private sector.

As the technology developed in the United States, private laboratories using widely differing methods (single-locus RFLP, multilocus RFLP, and PCR) began to offer their services to law-enforcement agencies. During the same period, the FBI was developing its own RFLP method, with a different restriction enzyme and different single-locus probes. The FBI's method has become the one most widely used in public forensic-science laboratories. Each method has its own advantages and disadvantages, databanks, molecular-weight markers, match criteria, and reporting methods.

Regardless of the causes, practices in DNA typing vary, and so do the educational backgrounds, training, and experience of the scientists and technicians who perform the tests, the internal and external proficiency testing conducted, the interpretation of results, and approaches to quality assurance.

It is not uncommon for an emerging technology to go without regulation until its importance and applicability are established. Indeed, the development of DNA typing technology has occurred without regulation of laboratories and their practices, public or private. The committee recognizes that standardization of practices in forensic laboratories in general is more problematic than in other laboratory settings stated succinctly, forensic scientists have little or no control over the nature, condition, form, or amount of sample with which they must work. But it is now clear that DNA typing methods are a most powerful adjunct to forensic science for personal identification and have immense benefit to the public&mdashso powerful, so complex, and so important that some degree of standardization of laboratory procedures is necessary to assure the courts of high-quality results. DNA typing is capable, in principle, of an extremely low inherent rate of false results, so the risk of error will come from poor laboratory

practice or poor sample handling and labeling and, because DNA typing is technical, a jury requires the assurance of laboratory competence in test results.

At issue, then, is how to achieve standardization of DNA typing laboratories in such a manner as to assure the courts and the public that results of DNA typing by a given laboratory are reliable, reproducible, and accurate.

Quality assurance can best be described as a documented system of activities or processes for the effective monitoring and verification of the quality of a work product (in this case, laboratory results). A comprehensive quality-assurance program must include elements that address education, training, and certification of personnel specification and calibration of equipment and reagents documentation and validation of analytical methods use of appropriate standards and controls sample handling procedures proficiency testing data interpretation and reporting internal and external audits of all the above and corrective actions to address deficiencies and weight their importance for laboratory competence.

Recommendations

Although standardization of forensic practice is difficult because of the nature of the samples, DNA typing is such a powerful and complex technology that some degree of standardization is necessary to ensure high standards.

Each forensic-science laboratory engaged in DNA typing must have a formal, detailed quality-assurance and quality-control program to monitor work, on both an individual and a laboratory-wide basis.

The Technical Working Group on DNA Analysis and Methods (TWGDAM) guidelines for a quality-assurance program for DNA RFLP analysis are an excellent starting point for a quality-assurance program, which should be supplemented by the additional technical recommendations of this committee.

The TWGDAM group should continue to function, playing a role complementary to that of the National Committee on Forensic DNA Typing (NCFDT). To increase its effectiveness, TWGDAM should include additional technical experts from outside the forensic community who are not closely tied to any forensic laboratory.

Quality-assurance programs in individual laboratories alone are insufficient to ensure high standards. External mechanisms are needed, to ensure adherence to the practices of quality assurance. Potential mechanisms include individual certification, laboratory accreditation, and state or federal regulation.

One of the best guarantees of high quality is the presence of an active

professional-organization committee that is able to enforce standards. Although professional societies in forensic science have historically not played an active role, the American Society of Crime Laboratory Directors (ASCLD) and the American Society of Crime Laboratory Directors-Laboratory Accreditation Board (ASCLD-LAB) recently have shown substantial interest in enforcing quality by expanding the ASCLD-LAB accreditation program to include mandatory proficiency testing. ASCLD-LAB must demonstrate that it will actively discharge this role.

Because private professional organizations lack the regulatory authority to require accreditation, further means are needed to ensure compliance with appropriate standards.

Courts should require that laboratories providing DNA typing evidence have proper accreditation for each DNA typing method used. Any laboratory that is not formally accredited and that provides evidence to the courts&mdashe.g., a nonforensic laboratory repeating the analysis of a forensic laboratory&mdashshould be expected to demonstrate that it is operating at the same level of standards as accredited laboratories.

Establishing mandatory accreditation should be a responsibility of the Department of Health and Human Services (DHHS), in consultation with the Department of Justice (DOJ). DHHS is the appropriate agency, because it has extensive experience in the regulation of clinical laboratories through programs under the Clinical Laboratory Improvement Act and has extensive expertise in molecular genetics through the National Institutes of Health. DOJ must be involved, because the task is important for law enforcement.

The National Institute of Justice (NIJ) does not appear to receive adequate funds to support proper education, training, and research in the field of forensic DNA typing. The level of funding should be re-evaluated and increased appropriately.

DATABANKS AND PRIVACY OF INFORMATION

DNA typing in the criminal-justice system has so far been used primarily for direct comparison of DNA profiles of evidence samples with profiles of samples from suspects. However, that application constitutes only the tip of the iceberg of potential law-enforcement applications. If DNA profiles of samples from a population were stored in computer databanks (databases), DNA typing could be applied in crimes without suspects. Investigators could compare DNA profiles of biological evidence samples with profiles in a databank to search for suspects.

In many respects, the situation is analogous to that of latent finger-prints. Originally, latent fingerprints were used for comparing crime-scene evidence with suspects. With the development of the Automated Fingerprint Identification Systems (AFIS) in the last decade, the investigative use

of fingerprints has dramatically expanded. Forensic scientists can enter an unidentified latent-fingerprint pattern into an automated system and within minutes compare it with millions of person's patterns contained in a computer file. In its short history, automated fingerprint analysis has been credited with solving tens of thousands of crimes.

The computer technology required for an automated fingerprint identification system is sophisticated and complex. Fingerprints are complicated geometric patterns, and the computer must store, recognize, and search for complex and variable patterns of ridges and minutiae in the millions of prints on file. Several commercially available but expensive computer systems are in use around the world. In contrast, the computer technology required for DNA databanks is relatively simple. Because DNA profiles can be reduced to a list of genetic types (hence, a list of numbers), DNA profile repositories can use relatively simple and inexpensive software and hardware. Consequently, computer requirements should not pose a serious problem in the development of DNA profile databanks.

Confidentiality and security of DNA-related information are especially important and difficult issues, because we are in the midst of two extraordinary technological revolutions that show no signs of abating: in molecular biology, which is yielding an explosion of information about human genetics, and in computer technology, which is moving toward national and international networks connecting growing information resources.

Even simple information about identity requires confidentiality. Just as fingerprint files can be misused, DNA profile information could be misused to search and correlate criminal-record databanks or medical-record databanks. Computer storage of information increases the possibilities for misuse. For example, addresses, telephone numbers, social security numbers, credit ratings, range of incomes, demographic categories, and information on hobbies are currently available for many of our citizens in various distributed computerized data sources. Such data can be obtained directly through access to specific sources, such as credit-rating services, or through statistical disclosure, which refers to the ability of a user to derive an estimate of a desired statistic or feature from a databank or a collection of databanks. Disclosure can be achieved through one query or a series of queries to one or more databanks. With DNA information, queries might be directed at obtaining numerical estimates of values or at deducing the state of an attribute of an individual through a series of Boolean (yes-no) queries to multiple distributed databanks.

Several private laboratories already offer a DNA-banking service (sample storage in freezers) to physicians, genetic counselors, and, in some cases, anyone who pays for the service. Typically, such information as name, address, birth date, diagnosis, family history, physician's name and address, and genetic counselor's name and address is stored with samples.

That information is useful for local, independent bookkeeping and record management. But it is also ripe for statistical or correlative disclosure. Just the existence in a databank of a sample from a person, independent of any DNA-related information, may be prejudicial to the person. In some laboratories, the donor cannot legally prevent outsiders' access to the samples, but can request its withdrawal. A request for withdrawal might take a month or more to process. In most cases, only physicians with signed permission of the donor have access to samples, but typically no safeguards are taken to verify individual requests independently. That is not to say that the laboratories intend to violate donors' rights they are simply offering a service for which there is a recognized market and attempting to provide services as well as they can.

Recommendations

In the future, if pilot studies confirm its value, a national DNA profile databank should be created that contains information on felons convicted of particular violent crimes. Among crimes with high rates of recidivism, the case is strongest for rape, because perpetrators typically leave biological evidence (semen) that could allow them to be identified. Rape is the crime for which the databank will be of primary use. The case is somewhat weaker for violent offenders who are most likely to commit homicide as a recidivist offense, because killers leave biological evidence only in a minority of cases.

The databank should also contain DNA profiles of unidentified persons made from biological samples found at crime scenes. These would be samples known to be of human origin, but not matched with any known persons.

Databanks containing DNA profiles of members of the general population (as exist for ordinary fingerprints for identification purposes) are not appropriate, for reasons of both privacy and economics.

DNA profile databanks should be accessible only to legally authorized persons and should be stored in a secure information resource.

Legal policy concerning access and use of both DNA samples and DNA databank information should be established before widespread proliferation of samples and information repositories. Interim protection and sanctions against misuse and abuse of information derived from DNA typing should be established immediately. Policies should explicitly define authorized uses and should provide for criminal penalties for abuses.

Although the committee endorses the concept of a limited national DNA profile databank, it doubts that existing RFLP-based technology provides an appropriate wise long-term foundation for such a databank. We expect current methods to be replaced soon with techniques that are sim-

pler, easier to automate, and less expensive&mdashbut incompatible with existing DNA profiles. Accordingly, the committee does not recommend establishing a comprehensive DNA profile databank yet.

For the short term, we recommend the establishment of pilot projects that involve prototype databanks based on RFLP technology and consisting primarily of profiles of violent sex offenders. Such pilot projects could be worthwhile for identifying problems and issues in the creation of databanks. However, in the intermediate term, more efficient methods will replace the current one, and the forensic community should not allow itself to become locked into an outdated method.

State and federal laboratories, which have a long tradition and much experience in the management of other types of basic evidence, should be given primary responsibility, authority, and additional resources to handle forensic DNA testing and all the associated sample-handling and data-handling requirements.

Private-sector firms should not be discouraged from continuing to prepare and analyze DNA samples for specific cases or for databank samples, but they must be held accountable for misuse and abuse to the same extent as government-funded laboratories and government authorities.

DNA INFORMATION IN THE LEGAL SYSTEM

To produce biological evidence that is admissible in court in criminal cases, forensic investigators must be well trained in the collection and handling of biological samples for DNA analysis. They should take care to minimize the risk of contamination and ensure that possible sources of DNA are well preserved and properly identified. As in any forensic work, they must attend to the essentials of preserving specimens, labeling, and the chain of custody and must observe constitutional and statutory requirements that regulate the collection and handling of samples. The Fourth Amendment provides much of the legal framework for the gathering of DNA samples from suspects or private places, and court orders are sometimes needed in this connection.

In civil (noncriminal) cases&mdashsuch as paternity, custody, and proof-of-death cases&mdashthe standards for admissibility must also be high, because DNA evidence might be dispositive. The relevant federal rules (Rules 403 and 702-706) and most state rules of evidence do not distinguish between civil and criminal cases in determining the admissibility of scientific data. In a civil case, however, if the results of a DNA analysis are not conclusive, it will usually be possible to obtain new samples for study.

The advent of DNA typing technology raises two key issues for judges: determining admissibility and explaining to jurors the appropriate standards for weighing evidence. A host of subsidiary questions with respect to how

expert evidence should be handled before and during a trial to ensure prompt and effective adjudication apply to all evidence and all experts.

In the United States, there are two main tests for admissibility of scientific information through experts. One is the Frye test, enunciated in Frye v. United States. The other is a "helpfulness" standard found in the Federal Rules of Evidence and many of its state counterparts. In addition, several states have recently enacted laws that essentially mandate the admission of DNA typing evidence.

The test for the admissibility of novel scientific evidence enunciated in Frye v. United States is still probably the most frequently invoked test in American case law. A majority of states profess adherence to the Frye rule, although a growing minority have adopted variations on the helpfulness standard suggested by the Federal Rules of Evidence.

Frye predicates the admissibility of novel scientific evidence on its general acceptance in a particular scientific field: "While courts will go a long way in admitting expert testimony deduced from a well-recognized scientific principle or discovery, the thing from which the deduction is made must be sufficiently established to have gained general acceptance in the particular field in which it belongs." Thus, admissibility depends on the quality of the science underlying the evidence, as determined by scientists themselves. Theoretically, the court's role in this preliminary determination is narrow: it should conduct a hearing to determine whether the scientific theory underlying the evidence is generally accepted in the relevant scientific community and whether the specific techniques used are reliable for their intended purpose.

In practice, the court is much more involved. The court must determine the scientific fields from which experts should be drawn. Complexities arise with DNA typing, because the full typing process rests on theories and findings that pertain to various scientific fields. For example, the underlying theory of detecting polymorphisms is accepted by human geneticists and molecular biologists, but population geneticists and other statisticians might differ as to the appropriate method for determining the population frequency of a genotype in the general population or in a particular geographic, ethnic, or other group. The courts often let experts on a process, such as DNA typing, testify to the various scientific theories and assumptions on which the process rests, even though the experts' knowledge of some of the underlying theories is likely to be at best that of a generalist, rather than a specialist.

The Frye test sometimes prevents scientific evidence from being presented to a jury unless it has sufficient history to be accepted by some subspecialty of science. Under Frye, potentially helpful evidence may be excluded until consensus has developed. By 1991, DNA evidence had been considered in hundreds of Frye hearings involving felony prosecutions in

more than 40 states. The overwhelming majority of trial courts ruled that such evidence was admissible, but there have been some important exceptions.

In determining admissibility according to the helpfulness standard under the Federal Rules of Evidence, without specifically repudiating the Frye rule, a court can adopt a more flexible approach. Rule 702 states that, "if scientific, technical or other specialized knowledge will assist the trier of fact to understand the evidence or to determine a fact in issue, a witness qualified as an expert by knowledge, skill, experience, training, or education, may testify thereto in the form of an opinion or otherwise."

Rule 702 should be read with Rule 403, which requires the court to determine the admissibility of evidence by balancing its probative force against its potential for misapplication by the jury. In determining admissibility, the court should consider the soundness and reliability of the process or technique used in generating evidence the possibility that admitting the evidence would overwhelm, confuse, or mislead the jury and the proffered connection between the scientific research or test result to be presented and particular disputed factual issues in the case.

The federal rule, as interpreted by some courts, encompasses Frye by making general acceptance of scientific principles by experts a factor, and in some cases a decisive factor, in determining probative force. A court can also consider the qualifications of experts testifying about the new scientific principle, the use to which the technique based on the principle has been put, the technique's potential for error, the existence of specialized literature discussing the technique, and its novelty.

With the helpfulness approach, the court should also consider factors that might prejudice the jury. One of the most serious concerns about scientific evidence, novel or not, is that it possesses an aura of infallibility that could overwhelm a jury's critical faculties. The likelihood that the jury would abdicate its role as critical fact-finder is believed by some to be greater if the science underlying an expert's conclusion is beyond its intellectual grasp. The jury might feel compelled to accept or reject a conclusion absolutely or to ignore evidence altogether. However, some experience indicates that jurors tend not to be overwhelmed by scientific proof and that they prefer experiential data based on traditional forms of evidence. Moreover, the presence of opposing experts might prevent a jury from being unduly impressed with one expert or the other. Conversely, the absence of an opposing expert might cause a jury to give too much weight to expert testimony, on the grounds that, if the science were truly controversial, it would have heard the opposing view. Nevertheless, if the scientific evidence is valid, the solution to those possible problems is not to exclude the evidence, but to ensure through instructions and testimony that the jury is equipped to consider rationally whatever evidence is presented.

In determining admissibility with the helpfulness approach, the court should consider a number of factors in addition to reliability. First is the significance of the issue to which the evidence is directed. If the issue is tangential to the case, the court should be more reluctant to allow a time-consuming presentation of scientific evidence that might itself confuse the jury. Second, the availability and sufficiency of other evidence might make expert testimony about DNA superfluous. And third, the court should be mindful of the need to instruct and advise the jury so as to eliminate the risk of prejudice.

Recommendations

Courts should take judicial notice of three scientific underpinnings of DNA typing:

The study of DNA polymorphisms can, in principle, provide a reliable method for comparing samples.

Each person's DNA is unique (except that of identical twins), although the actual discriminatory power of any particular DNA test will depend on the sites of DNA variation examined.

The current laboratory procedure for detecting DNA variation (specifically, single-locus probes analyzed on Southern blots without evidence of band shifting) is fundamentally sound, although the validity of any particular implementation of the basic procedure will depend on proper characterization of the reproducibility of the system (e.g., measurement variation) and inclusion of all necessary scientific controls.

The adequacy of the method used to acquire and analyze samples in a given case bears on the admissibility of the evidence and should, unless stipulated by opposing parties, be adjudicated case by case. In this adjudication, the accreditation and certification status of the laboratory performing the analysis should be taken into account.

Because of the potential power of DNA evidence, authorities should make funds available to pay for expert witnesses, and the appropriate parties must be informed of the use of DNA evidence as soon as possible.

DNA samples (and evidence likely to contain DNA) should be preserved whenever that is possible.

All data and laboratory records generated by analysis of DNA samples should be made freely available to all parties. Such access is essential for evaluating the analysis.

Protective orders should be used only to protect the privacy of individuals.

DNA TYPING AND SOCIETY

The introduction of any new technology is likely to raise concerns about its impact on society. Financial costs, potential harm to the interests of individuals, and threats to liberty or privacy are only a few of the worries typically voiced when a new technology is on the horizon. DNA typing technology has the potential for uncovering and revealing a great deal of information that most people consider to be intensely private. Examples might be the presence of genes involved in known genetic disorders or genes that have been linked to a heightened risk of particular major diseases in some populations.

Although DNA technology involves new scientific techniques for identifying or excluding people, the techniques are extensions and analogues of techniques long used in forensic science, such as serological and fingerprint examinations. Ethical questions can be raised about other aspects of this new technology, but the committee does not see it as violating a fundamental ethical principle.

A new practice or technology can be subjected to further ethical analysis by using two leading ethical perspectives. The first examines the action or practice in terms of the rights of people who are affected the second explores the potential positive and negative consequences (nonmonetary costs and benefits) of the action or practice, in an attempt to determine whether the potential good consequences outweigh the bad.

Two main questions can be asked about moral rights: Does the use of DNA technology give rise to any new rights not already recognized? Does the use of DNA technology enhance, endanger, or diminish the rights of anyone who becomes involved in legal proceedings? In answer to the first question, it is hard to think of any new rights not already recognized that come into play with the introduction of DNA technology into forensic science. The answer to the second question requires a specification of the classes of people whose rights might be affected and what those rights might be.

Concerns about intrusions into privacy and breaches of confidentiality regarding the use of DNA technology in such enterprises as gene mapping are frequently voiced, and they are legitimate ethical worries. The concerns are pertinent to the role of DNA technology in forensic science, as well as to its widespread use for other purposes and in other social contexts. A potential problem related to the confidentiality of any information obtained is the safeguarding of the information and the prevention of its unauthorized release or dissemination that can also be classified under the heading of abuse and misuse, as well as seen as a violation of individual rights in the forensic context.

Another factor to be weighed in a consequentialist ethical analysis is

whose interests are to count and whether some people's interests should be given greater weight than others'. For example, there are the interests of the accused, the interests of victims of crime or their families in apprehending and convicting perpetrators, and the interests of society. Whether the interests of society in seeing that justice is done should count as much as the interests of the accused or the victim is open to question.

A major issue is the preservation of confidentiality of information obtained with DNA technology in the forensic context. When databanks are established in such a way that state and federal law-enforcement authorities can gain access to DNA profiles, not only of persons convicted of violent crimes but of others as well, there is a serious potential for abuse of confidential information. The victims of many crimes in urban areas are relatives or neighbors of the perpetrators, and these victims might themselves be former or future perpetrators. There is greater likelihood that DNA information on minority-group members, such as blacks and Hispanics, will be stored or accessed. However, it is important to note that use of the ceiling principle removes the necessity to categorize criminals (or defendants in general) by ethnic group for the purposes of DNA testing and storage of information in databanks.

The introduction of a powerful new technology is likely to set up expectations that might be unwarranted or unrealistic in practice. Various expectations regarding DNA typing technology are likely to be raised in the minds of jurors and others in the forensic setting. For example, public perception of the accuracy and efficacy of DNA typing might well put pressure on prosecutors to obtain DNA evidence whenever appropriate samples are available. As the use of the technology becomes widely publicized, juries will come to expect it, just as they now expect fingerprint evidence.

Two aspects of DNA typing technology contribute to the likelihood of its raising inappropriate expectations in the minds of jurors. The first is a jury's perception of an extraordinarily high probability of enabling a definitive identification of a criminal suspect the second is the scientific complexity of the technology, which results in laypersons' inadequate understanding of its capabilities and failings. Taken together, those two aspects can lead to a jury's ignoring other forensic evidence that it should be considering.

As large felon databanks are created, the forensic community could well place more reliance on DNA evidence, and a possible consequence is the underplaying of other forensic evidence. Unwarranted expectations about the power of DNA technology might result in the neglect of relevant evidence.

The need for international cooperation in law enforcement calls for appropriate scientific and technical exchange among nations. As in other areas of science and technology, dissemination of information about DNA

typing and training programs for personnel likely to use the technology should be encouraged. It is desirable that all nations that will collaborate in law-enforcement activities have similar standards and practices, so efforts should be furthered to exchange scientific knowledge and expertise regarding DNA technology in forensic science.

Recommendations

In the forensic context as in the medical setting, DNA information is personal, and a person's privacy and need for confidentiality should be respected. The release of DNA information on a criminal population without the subjects' permission for purposes other than law enforcement should be considered a misuse of the information, and legal sanctions should be established to deter the unauthorized dissemination or procurement of DNA information that was obtained for forensic purposes.

Prosecutors and defense counsel should not oversell DNA evidence. Presentations that suggest to a judge or jury that DNA typing is infallible are rarely justified and should be avoided.

Mechanisms should be established to ensure accountability of laboratories and personnel involved in DNA typing and to make appropriate public scrutiny possible.

Organizations that conduct accreditation or regulation of DNA technology for forensic purposes should not be subject to the influence of private companies, public laboratories, or other organizations actually engaged in laboratory work.

Private laboratories used for testing should not be permitted to withhold information from defendants on the grounds that trade secrets are involved.

The same standards and peer-review processes used to evaluate advances in biomedical science and technology should be used to evaluate forensic DNA methods and techniques.

Efforts at international cooperation should be furthered, in order to ensure uniform international standards and the fullest possible exchange of scientific knowledge and technical expertise.


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