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Is one DNA molecule same as one chromosome

Is one DNA molecule same as one chromosome


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Is one DNA molecule = one chromosome or is one DNA molecule = all the chromosomes, ie, all the genetic material in our cells? I have googled it but I am not getting clear answers ?


Considering the encyclopedic definition of a molecule…

Molecules are held together by shared electron pairs, or covalent bonds.

… one DNA molecule is one DNA strand.

Put another way, one DNA molecule is a polymer of nucleotides connected by phosphodiester bonds. This means that one intact double-stranded chromosome is composed of two molecules of DNA interacting by hydrogen bonds.

In practice, however, one piece of double-stranded DNA is commonly called a DNA molecule.

All of the chromosomes in a cell constitute a single genome.

To further add to the confusion, identical chromatids may be said to compose a single chromosome, such that one replicated chromosome composed of two chromatids may comprise four DNA molecules, by the definition outlined above.


Note, the same encyclopedia lists ionic bonds as being on the same "continuum" as covalent bonds…

Ionic and covalent bonding therefore can be regarded as constituting a continuum rather than as alternatives.

… so atoms held together in ionic interactions are also considered molecules, by this definition.


Senior Seminar Medical Ethics in the 21st Century

INSTRUCTOR: Dr. Henry Jakubowski

Structure of DNA

DNA is a polymer, consisting of monomers call nucleotides. The monomer contains a simple sugar (deoxyribose), a phosphate group, and a cyclic organic group that is a base (not an acid). Only four bases are used in DNA, which we will abbreviate, for simplicity, as A, G, C and T. The polymer consists of a sugar - phosphate - sugar - phosphate backbone, with 1 base attached to each sugar molecule. DNA can exist as single (with one sugar-phosphate backbone), double-stranded (with two sugar-phosphate backbones which bind to each other through their bases) , or mixed forms. It is actually a misnomer to call dsDNA a molecule, since it really consisted of two different, complementary strands held together by IMF's. However, most people talk about a molecule of dsDNA, and so will I. dsDNA varies in length (number of sugar-phosphate units connected), base composition (how many of each set of bases) and sequence (the order of the bases in the backbone. the links links below will help you understand the properties of DNA.

Structure of a chromosome

Most people have seen pictures of chromosomes viewed through microscopes. Check out this amazing picture of a chromosome taken form Scientific American, September, 1995.

Chromosomes consist of one dsDNA molecule. Each somatic (body) cell of your body has 23 pairs of chromosomes, one member of each pair contributed by your mother and the other by your father. (In germ cells - eggs and sperm - there are 23 individual chromosomes, not chromosome pairs.) One pair are the sex chromosomes, which can come in two forms, X and Y. A pair of X's gives a female, and an XY results in a male.

The human genome has about 3 billion base pairs of DNA. Therefore, on average, each single chromosome of a pair has about 150 million base pairs, which consists of one molecule of DNA and lots of proteins bound to it. dsDNA is a highly charged molecule, and can be viewed, to a first approximation, as a long rod-like molecule with a large negative. charge. This very large molecule must somehow be packed into a small nucleus. The packing problem is solved by coiling DNA and packing it with proteins, which usually have a net positive charge. The chromosomes are usually dispersed within the nucleus and are not visible with an ordinary microscope. When the cell is ready to divide, the DNA in the chromosomes replicates, and the chromosomes condense in a fashion that they are visible (when stained) using an ordinary microscope. At this point the chromosomes can be stained with a variety of stains (hence the name chromosomes), some of which bind differentially to different chromosomes. The different chromosomes can hence be distinguished by their size, shape, and dye-binding properties.

The standard picture of a chromosome with which you are familiar, including the one shown above, is actually one chromosome of a pair that has just replicated!. One of the chromosomes will stay will the mother cell, and the other will go to the daughter cell. These two chromosomes which are aligned and appear joined at their centers are called sister chromatids. These large DNA/protein complexes must be further packaged in the nucleus, as shown in the "Carl Saganesque" reducing view of the chromosome, a double stranded DNA molecule winds around a core of proteins.

Fun DNA Facts to Know and Tell

  • Largest known continuous DNA sequence (yeast chromosome 3): 350 x 10 6 BP ?
  • E. Coli Genome: 4.6 x 10 6 BP (4.6 million BP)
  • Yeast Genome: 16 x 10 6 BP
  • Smallest human chromosome (Y) 50 x 10 6 BP
  • Worm: 100 x 10 6 BP
  • Fruit Fly: 160 x 10 6 BP
  • Largest human chromosome (1) 250 x 10 6 BP
  • Entire human genome 3 x 10 9 (3 billion) BP
  • Mouse Genome: 3 x 10 9 BP
  • Length of uncoiled dsDNA in a human cell: approx 2 meters
  • Number of human cells: about 100 trillion
  • Number of times DNA from all human cells, if stretched out, could reach to sun and back: about 700
  • If compiled in books, the data would fill an estimated 200 volumes the size of a Manhattan telephone book (at 1000 pages each), and reading it would require 26 years working around the clock (Fig.14). The fruit fly genome would be 10 books, yeast 1 book, E. Coli 300 pages, and yeast chromosome 3 would be 14 pages.
  • Any two individuals differ in about three million - 3 x 10 6 bases (0.1%). The population is now about 6 x 10 9 (6 billion).A catalog of all sequence differences would require 15 x 10 15 entries. This catalog may be needed to find the rarest or most complex disease genes.

Central Dogma of Biology:

DNA is the carrier of genetic information in organisms. What does that mean? Large molecules in organism can have many functions: they can provide structure, act as catalyst for chemical reactions, serve to sense changes in their environment (leading to immune responses to foreign invaders and to neural responses to stimuli such as light, heat, sound, touch, etc) and provide motilty. DNA really does none of these things. Rather you can view it as an information storage system. The information must be decode to allow the construction of other large molecules. The other molecules are usually proteins, another class of large polymers in the body. Chromosomes are located in the nucleus of a cell. DNA must be duplicated in a process called replication before a cell divides. The replication of DNA allows each daughter cell to contain a full complement of chromosomes.

Animation of Replication: requires Hypercosm plugin (available when select link)

The actual information in the DNA of chromosomes is decoded in a process called transcription through the formation of another nucleic acid, ribonucleic acid or RNA. The RNA, made by the enzyme RNA polymerase, is complementary to one strand of the DNA. RNA differs from DNA in that RNA contains a ribose, not deoxyribose, sugar in its backbone. In addition, RNA lacks the base T. It is replaced, instead, with the base U, which is complementary to A (as T is complementary to A in DNA). The RNA formed acts as a messenger, which passes from the nucleus into the cytoplasm of the cell. In fact, this type of RNA is often called messenger RNA, mRNA. The information from the DNA, now in the form of a linear RNA sequence, is decoded in a process called translation, to form a protein, another biological polymer. The monomer in a protein is called an amino acid, a completely different kind of molecule than a nucleotide. There are twenty different naturally occurring amino acids that differ in one of the 4 groups connected to the central carbon. In an amino acid, the central (alpha) carbon has an amine group (RNH2), a carboxylic acid group (RCOOH), and H, and an R group attached to it. With four different groups attached to the central carbon, all amino acis (except Glycine) are chiral and exists in enantiomers or mirror image forms. Only one of the mirror image is found in proteins.

20 Naturally Ocuring Amino Acids - Molecular Models: Notice the common blue and red groups in al amino acids. Notice the different "R" groups pointing down in each figure.

The monomers come together to form a long chain called a protein. The linear sequence of a protein can be depicted in many ways, as shown below.

In contrast to the complementarity of DNA and RNA (1 base in RNA complementary to 1 base in DNA), there is not a 1:1 correspondence between a base (part of the monomeric unit of RNA) in RNA to the monomer in a protein. After much work it was discovered that a contiguous linear sequence of 3 nucleotides in RNA is decoded by the molecular machinery of the cytoplasm with the result that 1 amino acid is added to the growing protein. Hence a triplet of nucleotides in DNA and RNA have the information for 1 amino acid in a protein. That there was not a 1:1 correspondence between nucleotides in nucleic acids and amino acids in proteins was evident long ago since there are only 4 different DNA monomers (with A, T, G, and C) and 4 different RNA monomers (with A, U, G, and C) but there are 20 different amino acid monomers that compose proteins.

Now, it turns out that not all the information in the DNA sequence of a organism encodes for a protein. In fact only about 2% of the 3 billion base pairs seem to be transcribed into RNA which can be translated into protein. The function of the rest of the DNA is at present uncertain. How does the molecular machinery of the cell know which part of the DNA encodes for proteins. It turns out that there are unique DNA sequences at the beginning and end of the part of the DNA sequence that codes for a protein. Proceed down the DNA of a chromosome and suddenly you come to those signals, which are recognized by the cells machinery. A complementary RNA is made from that section, and the complementary RNA is then decoded into a single protein. Continue further down the DNA sequence and another such coding sequence is found, which can be transcribed into a mRNA, which then can be translated into another unique protein. In all there are about 30-40 thousand such sections of DNA in all the chromosomes that encode the information for 30-40 thousand unique proteins. These unique coding sections of DNA that ultimately are transcribed into unique mRNA which are translated into unique proteins are called genes. For issue of simplicity, we conclude that one gene has the information for one protein. Each of the protein differ from each other in both length, and the specific sequence of amino acids in the protein. The DNA is indeed the blueprint of the cell. What determines the actual characteristics of the cells are the actual proteins that are made by the cell.

Not only must DNA be transcribed into DNA, but the genetic information in the DNA must be replicated before a given cell divides, so that the daughter cells both contain the same genetic information. In replication, the dsDNA separate, and an enzyme, DNA polymerase, makes complimentary copies of each strand. The two resulting dsDNA strands separate to different daughter cells during division. The process where by DNA is replicated when cells divide, and is transcribed into RNA which is translated into protein is called the Central Dogma of Biology. (disregard tRNA, rRNA, and snRNA in the preceding web link)

As mentioned above, each amino acid is specified by a particular combination of three nucleotides in RNA. The three bases are called a codon. The Genetic Code consists of a chart which shows what triplet RNA sequence or codon in mRNA codes for which of the 20 amino acids. One of the codon codes for no amino acids and serves to stop the synthesis of the protein from the mRNA sequence. The genetic code is shown below:

Determining the protein sequence from a DNA sequence.

For a given gene, only one strand of the DNA serves as the template for transcription. An example is shown below. The bottom (blue) strand in this example is the template strand, which is also called the minus (-) strand,or the sense strand. It is this strand that serves as a template for the mRNA synthesis. The enzyme RNA polymerase sythesizes an mRNA in the 5' to 3' direction complementary to this template strand. The opposite DNA strand (red) is called the c oding strand, the nontemplate strand, the plus (+) strand, or the antisense strand.

The easiest way to find the corresponding mRNA sequence (shown in green below) is to read the c oding, nontemplate , plus (+) , or antisense strand directly in the 5' to 3' direction substituting U for T. Find the triplet in the coding strand, change any T's to U's, and read from the Genetic Code the corresponding amino acid that would be incorporated into the growing protein.

In contrast to the linear polymers of DNA and RNA, proteins (linear polymers of amino acids) fold in 3D space to form structures of unique shapes. Each unique protein sequence (of a given length and sequence of amino acids) folds to a unique 3D shape. Hence there are about 30-40 thousand proteins of different shapes in humans. Not only do proteins have unique shapes, but they also have unique nooks and crannies and pockets which allow them to bind other molecules. Binding of other molecules to proteins or DNA initiates or terminates the function of the protein or nucleic, much like an on/off switch. The example below show different protein structures, some of which have small molecules or large molecules (like DNA) bound to them. Some common motiffs are found within the 3D structure of the protein. The include alpha helices and beta sheets. These are held together by H-bonds between the slightly positive H on the N in the protein backbone and a slightly negative O further away on the protein backbone. In the Chime models below, use the mouse controls to rotate the molecule. (Also shift L-mouse click will change the size of the molecule). Click on the command in the right hand frame to change the rendering of the proteins. The cartoon view allows a simple way to interpret the overall structure of the main chain.

Myoglobin - an oxygen binding protein

(a protein enzyme that detoxifies the body of toxic oxygen byproducts and which high level of the protein have been associated with longer life spans.)

If the DNA sequence in a coding region becomes changed, the resulting mRNA will also be changed, which will lead to changes in the protein sequence. These changes might have no effect and be silent, if the change in the protein does not affect the folding of the protein or its binding to another important molecule. However, if the changes affects either the folding or the binding region, the protein may not be able to peform its usual function. If the function was to put on break on cell division, the result might lead the cell to become a cancerous. Likewise, if the normal protein had a role in causing the cell to die after its intended life span, the cell with the mutant protein might not die and more likely become a tumor. The opposite scenarios could happen leading the cell to a premature death.

Point mutations: From bad luck and nucleotide analogs

Large mutations: Deletions, insertions, duplications, and inversions

Toa first approximation, we decided that genes are continuous segments of DNA that are transcribed into a single continuous strand of RNA which is translated into a single protein. The human genome appears to have about 40,000 genes, which then would imply that 40,000 proteins could be made. But consider this. We have an immune system that can recognize almost any foreign molecules thrown at it. One immune response is the production of antibodies which are proteins that recognize and bind to foreign molecules. How many different antibody molecules can we make. Hopefully the number is greater than 40,000, but how our limited genome encode for all of them as well as the other proteins required. One solution to the problem would be if more than one protein can arise from one DNA gene sequence. This is a common occurrence in nonbacterial cell. How does this happen?

One mechanisms arises from the fact that a single "gene" is divided into coding (exons) and nocoding intervening (intron) sequences. The entire gene, including introns, is transcribed into RNA. The the coding parts of the RNA must be sliced together, with removal of the noncoding RNA sequences, to give the mature RNA. If different exons in a given "gene" are spliced together to form different "mature" RNA molecules, then different proteins could arise from the same initial "gene" sequence.

Genes and Disease

DNA: Mastery of a Language

The four letter alphabet (A, G, C, and T) that makes up DNA represents a language that when transcribed and translated leads to the myriad of proteins that make us who we are as a species and as individuals. Let's continue with the metaphor that DNA is a language. To master that language, as with any other language, we need to be able to read, write, copy, and edit that language. If you were using a word processor to find one line in a hundred page document, or one article from one book out of the Library of Congress or of the Manhattan phone book, you would also need a way to search the large print base available. You might want to compare two different copies of files to see if they differ from each other. From this online discussion, you will learn how modern scientists read, write, copy, edit, search, and compare the language of the genome. These abilities, acquired over the last twenty years, have revolutionized our understanding of life and have given us the potential to alter, for good or evil, life itself.

MANAGING DNA

DNA in human chromosomes exists as one long double stranded molecule. It is too long to physically study and manipulate in the lab. Using a battery of enzymes, the DNA of chromosomes can be chemically cleaved into smaller fragments which are more readily manipulable. (Similar techniques are used to sequence proteins, which require overlapping polypeptide fragments to be made.) After the fragments have been made, they must be separated from each other in order to study them. DNA fragments can be separated on the basis of some structural feature that differentiates the fragments from each other. The best way to separate the fragments from each other is to base the separation on the actual size of the fragment by separating the molecules based on their charge using a technique called electrophoresis on an agarose or polyacrylamide gel.

A carbohydrate extract called agarose is made from algae. Water is added to the extract, which is then heated. The carbohydrate extract dissolves in the water to form a viscous solution. The agarose solution is poured into a mold (like warm jello) and is allowed to solidify. A plastic comb with wide teeth was placed in the agarose when it was still liquid. When the agarose is solid, the comb can be removed, leaving in its place little wells. A solution of DNA fragments can be placed in the wells. The agarose slab with sample is covered with a buffer solution and electrodes placed at each end of the slab. The negative electrode is placed near the well end of the agarose slab while the positive electrode is placed at the other end. If a voltage is applied across the agarose slab, the negatively charged DNA fragments will move through the agarose gel toward the positive electrode. This migration of charged molecules in solution toward an oppositely charged electrode is called electrophoresis. Pretend you are one of the fragments. To you the gel looks like a tangle cobweb. You sneak your way through the openings in the web as you move straight forward to the positive electrode. The larger the fragment, the slower you move because it is hard to get through the tangled web. Conversely, the shorter the fragment, the faster you move. Using this technique and its many modifications, oligonucleotides differing by just one nucleotides can be separated from each other. As you read the rest of this tutorial, you will come to the profound realization that all of these techniques are based in some way on the simple principle that small oligonucleotides interact with DNA through intermolecular forces!

To determine the sequence of a single stranded piece of DNA, the complementary strand is synthesized using an enzyme, DNA polymerase. The enzyme is added to the DNA along with the 4 monomers that are used to make DNA with A's, C's, G's, and T's. The monomers are called dATP, dCTP, dGTP, dTTP. In addition, small amounts of dideoxyATP (ddATP) are added to one reaction tube. Likewise, small amounts of dd CTP, ddGTP, and ddTTP are added to three other reaction tubes. In the tube with dATP and small amounts of ddATP, the dATP and ddATP attach randomly to the growing 3' end of the complementary stranded. If ddATP is added no further nucleotides can be added after since its 3' end has an H and not a OH. That's why they call it dideoxy. The new chain is terminated.. If dATP is added, the chain will continue to grow until another A needs to be added. Hence a whole series of discreet fragments of DNA chains will be made, all terminated when ddATP was added. The same scenario occurs for the other 3 tubes, which contain dCTP and ddCTP, dTTP and ddTTP, and dGTP and ddGTP respectively. All the fragments made in each tube will be placed in separate lanes for electrophoresis, where the fragments will separate by size. More modern methods allow the use of differ fluorescent tags to be added to synthesized fragments ending in G, T, A, or C.

The Human Genome Project (a public consortium) and the private initative by Celera, have recenty announced that they have completed a working draft of the entire sequence of the human genome. The Human Genome Project is sequencing DNA collected from a number of volunteers whose identity will remain secret to protect their privacy.Completing the DNA sequence of the human genome will provide scientists with a tool for genetics akin to the Periodic Table of the Elements. Instead of 100 chemical elements, though, the genome contains about 30-40 thousand elements, the human genes. Once you have the table of the elements, you can begin to figure out how the genes function, how genes interact, and how they contribute to common disorders. Other projects are determining the entire sequences of many infectious bacteria, and from higher organisims including fruit flys, round worms, mice, and chimpanzees, our closest genetic relative (98.6% identical).

Oligonucleotide can be synthesized on a solid bead. By adding one nucleotide at a time, the sequence and length of the oligonucleotide can be controlled.

Several methods exists for copying a sequence of DNA millions of times. Most methods make use of plasmids (which are found in bacteria) and viruses (which can infect any cell). The DNA of the plasmid or virus is engineered to contain a copy of a specific DNA sequence of interest. The plasmid or virus is then reintroduced into the cell where amplification occurs.

Initially, a DNA containing a gene is cut at specific places with an enzyme called a restriction endonuclease, or restriction enzyme for short. The enzyme, which you can think of a molecular scissors, doesn't cleave DNA any old place, but rather at "restricted" places in the sequence. The restriction endonuclease must cleave both strands of dsDNA. It can cut the strands cleanly to leave blunt ends, or in a staggered fashion, to leave small tails of ssDNA. Multiple such sites exist at random in the genome. The gene of interest must be flanked on either side by such a sequence. The same enzyme is used to cleave the plasmid or virus DNA.

The foreign fragment of DNA can then be added to the plasmid or viral DNA as shown to make a recombinant DNA molecule. This technique of DNA cloning is the basis for the entire field of recombinant DNA technology.

Animation of Gene Splicing - Requires Hypercosm Plugin (available when select link)

The plasmid can be added to bacteria, which take it up in a process called transformation. The plasmid can be replicated in the bacteria which will copy the DNA fragment of interest. A similar method can be used to copy DNA in which the foreign fragment is recombined with the DNA of bacteriophage , a virus which infects bacteria like E. Coli. The recombinant DNA can be packaged into actual viruses, as shown below. When the virus infects the bacteria, it instructs the cells to make millions of new viruses, hence copying the foreign fragment of interest.

Sometimes, "cloning" or copying a fragment of DNA is not what an investigator really wants. One such possible method exists in which you start with the actual mRNA for a protein of interest. In this technique, a dsDNA copy is made from a ss-mRNA molecule. Such dsDNA is call cDNA, for complementary or copy DNA. This can then be cloned into a plasmid or bacteriophage vector and amplified as described above.

In the mid 80's a new method was developed to copy (amplify) DNA in a test tube. It doesn't require a plasmid or a virus. It just requires a DNA fragment, some primers (small polynucleotides complementary to sections of DNA on each strand and straddling the section of DNA to be amplified. Just add to this mixture dATP, dCTP, dGTP, dTTP, and a heat stable DNA polymerase from the organism Thermophilus aquaticus (which lives in hot springs), and off you go. The mixture is first heated to a temperature which will cause the dsDNA strands to separate. The temperature is cooled allowing the primers to bind to the ssDNA. The heat stable Taq polymerase (from Thermophilus aquaticus) polymerizes DNA from the primers. The temperature is raised again, allowing dsDNA strand separation. On cooling the primers anneal again to the original and newly synthesized DNA from the last cycle and synthesis of DNA occurs again. This cycle is repeated as shown in the diagram. This chain reaction is called the polymerase chain reaction (PCR). The target DNA synthesized is amplified a million times in 20 cycles, or a billion times in 30 cycles, which can be done in a few hours.

Using recombinant DNA technology, the gene that encodes the protein can be altered at one or more nucleotide, in a way which would either change one or more amino acids, or add or delete one or more amino acids. This technique, called site-specific mutagenesis, is used extensively by protein chemist to detemine the importance of a given amino acid in the folding, structure, and activity of a protein. The techniques is described in the diagram below

Where on a chromosome is the gene that codes for a given protein? One way to find the gene is to synthesize a small oligonucleotide "probe" which is complementary to part of the actual DNA sequence of the gene (determined from previous experiments). Attach a fluorescent molecule to the DNA probe. Then take a cell preparation in which the chromosomes can be seen under the microscope. To the cell add base which unwinds the double stranded DNA helix, add the fluorescent probe to the cell, and allow double stranded DNA to reform. The fluorescent probe will bind to the chromosome at the site of the gene to which the DNA is complementary, as shown below.

An example of a DNA-RNA complex is shown below.

The DNA sequence of each individual must be different from every other individual in the world (with the exception of identical twins). The difference must be less than the difference between a human and a chimp, which are 98.5 % identical. Let us say that each of have DNA sequences that are 99.9 % identical as compared to some "normal human". Given that we have about 43 billion base pairs of DNA, that means we are all different in about 0.001 x 3,000,000,000 which is about 3 million base pairs different. This means that on the average we have one nucleotide difference for each 1000 base pairs of DNA. Some of these are in genes, but most are probably in between DNA, and many have been shown to be clustered in areas of highly repetitive DNA sequences at the ends of chromosomes (called the teleomers) and in the middle (called the centromers).

Now remember that their are restriction enzyme sites interspersed randomly along the DNA as well. If some of the differences in the DNA among individuals occurs within the sequences where the DNA is cleaved by restriction enzymes, then in some individuals a particular enzyme won't cleave at the usual site, but at a more distal site. Hence, the size of the restriction enzyme fragments should differ for each person. Each persons DNA, when cut by a battery of restriction enzymes, should give rise to a unique set of DNA fragments of sizes unique to that individual. Each persons DNA has a unique Restriction Fragment Length Polymorphism (RFLP). How could you detect such polymorphism?

You already know how to cut sample DNA with restriction enzymes, and then separate the fragments on an agarose gel. An additional step is required, however, since thousands of fragments could appear on the gel, which would be observed as one large continuous smear. If however, each fragment could be reacted with a set of small, labeled DNA probes which are complementary to certain highly polymorphic sections of DNA (like teleomeric DNA) and then visualized, only a few sets of discrete bands would be observed in the agarose gel. These discrete bands would be different from the DNA bands seen in another individual's gene treated the same way. This technique is called Southern Blotting and works as shown below. DNA fragments are electrophoresed in an agarose gel. The ds DNA fragments are unwound by heating, and then a piece of nitrocellulose filter paper is placed on top of the gel. The DNA from the gel transfers to the filter paper. Then a small radioactive oligonucleotide probe, complementary to a polymorphic site on the DNA, is added to the paper. It binds only to the fragment containing DNA complementary to the probe. The filter paper is dried, and a piece of x-ray film is placed over the sheet. Also run on the gel, and transferred to the sheet, are a set of radioactive fragments (which are not complementary to the probe), which serve as a set of markers to ensure that the gel electrophoresis and transfer to the filter paper was correct. This technique is shown on the next page, along with a RFLP analysis from a particular family.

When this technique is used in forensic cases (such as the OJ Simpson trial) or in paternity cases, it is called DNA fingerprinting. With present techniques, investigators can state unequivocally that the odds of a particular pattern not belong to a suspect are in the range of one million to one. The x-ray film shown below is a copy of real forensic evidence obtained from a rape case. Shown are the Southern blot results from suspect 1, suspect 2, the victim, and the forensic evidence. Analyze the data.

SNPs are single bases in the DNA that differ in at least 1% of the population. They occur about every 1250 bases. Recent work show they are mostly in non protein-coding regions of the genome (in regions of DNA between the genes and in introns) Venter has shown that "more than 99% of human SNPs are not associated with biology. Only 2000 SNPs change an amino acid sequence of a regulatory site. That is a few thousand out of 2-3 million SNPs cataloged so far." If they occur in gene, they may not lead to change in the amino acid sequence of a resulting protein. (Example: CCA in mRNA to CCG both give the amino acid Pro in the protein). All the emerging DNA analyses suggest that Homo Sapiens arose form one common ancestor inn Africa about 200,000 years ago and people migrated out of Africa around 125,000 years ago. Observed difference in people emerged just recently. There is, in fact, no genetically pure population (in contrast to Arian mythology). .


What is a Chromatid?

Chromatid is one of the two similar copies of DNA that makes up a single chromosome. One chromosome has two chromatids joined by a centromere. During the cell division (meiosis and mitosis), they are separated from each other they are then called sister chromatids since they are identical to each other.

Figure 02: Chromatid and Chromosome

To be more specific, a chromosome is like an X shape structure when viewed under a microscope. Divide the X by half and it would result in two identical parts > and <. A single > or < is what you call a chromatid. The center point of contact is the centromere and the whole X is the chromosome.


A1. The structure of DNA

DNA can exist as single, double-stranded, or mixed forms. It is actually a misnomer to call dsDNA a molecule, since it really consisted of two different, complementary strands held together by IMF's. However, most people talk about a molecule of dsDNA, and so will I. In analogy to protein structure, dsDNA has a linear sequence (primary structure), secondary structure (right handed double helix), and tertiary/quaternary structure (it is folded and packed in the cell).

Alternative DNA structures

The links above show the classic dsDNA helix, in which DNA is in the B form. Other forms of DNA exists, including A-DNA and Z-DNA. Single-stranded sections of DNA can, through intramolecular base-pairing, form stem-loop hairpin structures and quadruplex structures (found at the ends of chromosomes (telomeres).


Sequence organization was revealing by heating DNA to a single-stranded state then allowing the DNA to reanneal by cooling.
This revealed several types of repetitive DNA.
Multiple "copies" of similar functional repetitive sequences can be described as dispersed gene families (globin genes, actins, tubulins).
Non-functional copies of genes are known as pseudogenes.
Tandem gene family arrays are made up of multiple copies of the same gene all next to each other (such as histones).
The nucleolar organizer, which is cytologically distinct, is a tandem array of genes that encode ribosomal RNA.
Noncoding functional sequences, such as the short tandem repeats that act to maintain the telomeres at the ends of a linear chromosome.

There are a number of sequences with no known function include
1) Highly repetitive centromeric DNA including satellite DNA.
2) Variable number tandem repeats (VNTRs) or minisatellite DNA which provide the differences in DNA used in DNA fingerprinting.
3) Microsatellites, regions of dinucleotide repeats

Transposed sequences are "jumping genes" that are dispersed throughtout the genome.
Transposons move as DNA elements and retrotransposons move via an RNA intermediate which is reverse transcribed and reinerted into the genome.
Examples of a retrotransposons include the 1-to 5 kilobase Long interspersed elements (LINES) and the much smaller (>200 basepairs) short interspersed elements (SINES)
Such as the human Alu sequences.
The presence of these various elements provides a great deal of variety to the spacing and locations of genes in the genomes of organisms.


11.1 Structure And Function of Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) is a molecule composed of two chains that coil around each other to form a double helix carrying genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses. DNA and ribonucleic acid (RNA) are nucleic acids alongside proteins, lipids and complex carbohydrates (polysaccharides), nucleic acids are one of the four major types of macromolecules that are essential for all known forms of life.

Figure 11.2: The structure of the DNA double helix. A section of DNA. The bases lie horizontally between the two spiraling strands. The atoms in the structure are colour-coded by element (based on atomic coordinates of PDB 1bna rendered with open source molecular visualization tool PyMol.)

The two DNA strands are also known as polynucleotides as they are composed of simpler monomeric units called nucleotides. Each nucleotide is composed of one of four nitrogen-containing nucleobases (cytosine [C], guanine [G], adenine [A] or thymine [T]), a sugar called deoxyribose, and a phosphate group. The nucleotides are joined to one another in a chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone. The nitrogenous bases of the two separate polynucleotide strands are bound together, according to base pairing rules (A with T and C with G), with hydrogen bonds to make double-stranded DNA. The complementary nitrogenous bases are divided into two groups, pyrimidines and purines. In DNA, the pyrimidines are thymine and cytosine the purines are adenine and guanine.

Figure 11.3: The structure of the four nucleotides and their base pairing in the DNA double helix. The atoms in the structure are colour-coded by element (based on atomic coordinates of PDB 1bna rendered with open source molecular visualization tool PyMol.)

Both strands of DNA store biological information. This information is replicated as and when the two strands separate. A large part of DNA (more than 98% for humans) is non-coding, meaning that these sections do not serve as patterns for protein sequences. The two strands of DNA run in opposite directions to each other and are thus antiparallel. Attached to each sugar is one of four types of nucleobases (informally, bases). It is the sequence of these four nucleobases along the backbone that encodes genetic information. RNA strands are created using DNA strands as a template in a process called transcription, where DNA bases are exchanged for their corresponding bases except in the case of thymine (T), which RNA substitutes for uracil (U). Under the genetic code, these RNA strands specify the sequence of amino acids within proteins in a process called translation.

Within eukaryotic cells, DNA is organized into long structures called chromosomes. Before typical cell division, these chromosomes are duplicated in the process of DNA replication, providing a complete set of chromosomes for each daughter cell. Eukaryotic organisms (animals, plants, fungi and protists) store most of their DNA inside the cell nucleus as nuclear DNA, and some in the mitochondria as mitochondrial DNA or in chloroplasts as chloroplast DNA. In contrast, prokaryotes (bacteria and archaea) store their DNA only in the cytoplasm, in circular chromosomes. Within eukaryotic chromosomes, chromatin proteins, such as histones, compact and organize DNA. These compacting structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed.

DNA was first isolated by Friedrich Miescher in 1869. Its molecular structure was first identified by Francis Crick and James Watson at the Cavendish Laboratory within the University of Cambridge in 1953, whose model-building efforts were guided by X-ray diffraction data acquired by Raymond Gosling, who was a post-graduate student of Rosalind Franklin.

DNA is a long polymer made from repeating units called nucleotides, each of which is usually symbolized by a single letter: either A, T, C, or G. The structure of DNA is dynamic along its length, being capable of coiling into tight loops and other shapes. In all species it is composed of two helical chains, bound to each other by hydrogen bonds. Both chains are coiled around the same axis, and have the same pitch of 34 angstroms (Å) (3.4 nanometres). The pair of chains has a radius of 10 angstroms (1.0 nanometre). Although each individual nucleotide is very small, a DNA polymer can be very large and contain hundreds of millions, such as in chromosome 1. Chromosome 1 is the largest human chromosome with approximately 220 million base pairs, and would be 85 mm long if straightened.

DNA does not usually exist as a single strand, but instead as a pair of strands that are held tightly together. These two long strands coil around each other, in the shape of a double helix. The nucleotide contains both a segment of the backbone of the molecule (which holds the chain together) and a nucleobase (which interacts with the other DNA strand in the helix). A nucleobase linked to a sugar is called a nucleoside, and a base linked to a sugar and to one or more phosphate groups is called a nucleotide. A biopolymer comprising multiple linked nucleotides (as in DNA) is called a polynucleotide.

The backbone of the DNA strand is made from alternating phosphate and sugar residues. The sugar in DNA is 2-deoxyribose, which is a pentose (five-carbon) sugar. The sugars are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. These are known as the 3′-end (three prime end), and 5′-end (five prime end) carbons, the prime symbol being used to distinguish these carbon atoms from those of the base to which the deoxyribose forms a glycosidic bond. When imagining DNA, each phosphoryl is normally considered to “belong” to the nucleotide whose 5′ carbon forms a bond therewith. Any DNA strand therefore normally has one end at which there is a phosphoryl attached to the 5′ carbon of a ribose (the 5′ phosphoryl) and another end at which there is a free hydroxyl attached to the 3′ carbon of a ribose (the 3′ hydroxyl). The orientation of the 3′ and 5′ carbons along the sugar-phosphate backbone confers directionality (sometimes called polarity) to each DNA strand. In a nucleic acid double helix, the direction of the nucleotides in one strand is opposite to their direction in the other strand: the strands are antiparallel. The asymmetric ends of DNA strands are said to have a directionality of five prime end (5′ ), and three prime end (3′), with the 5′ end having a terminal phosphate group and the 3′ end a terminal hydroxyl group. One major difference between DNA and RNA is the sugar, with the 2-deoxyribose in DNA being replaced by the alternative pentose sugar ribose in RNA.

Twin helical strands form the DNA backbone. Another double helix may be found tracing the spaces, or grooves, between the strands (Figure 11.4). These voids are adjacent to the base pairs and may provide a binding site. As the strands are not symmetrically located with respect to each other, the grooves are unequally sized. One groove, the major groove, is 22 angstroms (Å) wide and the other, the minor groove, is 12 Å wide. The width of the major groove means that the edges of the bases are more accessible in the major groove than in the minor groove. As a result, proteins such as transcription factors that can bind to specific sequences in double-stranded DNA usually make contact with the sides of the bases exposed in the major groove.

Figure 11.4: DNA major and minor grooves. PDB 1bna rendered with open source molecular visualization tool PyMol.)

In a DNA double helix, each type of nucleobase on one strand bonds with just one type of nucleobase on the other strand. This is called complementary base pairing. Here, purines form hydrogen bonds to pyrimidines, with adenine bonding only to thymine in two hydrogen bonds, and cytosine bonding only to guanine in three hydrogen bonds (Figure 11.5). This arrangement of two nucleotides binding together across the double helix is called a Watson-Crick base pair. As hydrogen bonds are not covalent, they can be broken and rejoined relatively easily. The two strands of DNA in a double helix can thus be pulled apart like a zipper, either by a mechanical force or high temperature. As a result of this base pair complementarity, all the information in the double-stranded sequence of a DNA helix is duplicated on each strand, which is vital in DNA replication. This reversible and specific interaction between complementary base pairs is critical for all the functions of DNA in organisms.

Figure 11.5: Top, a GC base pair with three hydrogen bonds. Bottom, an AT base pair with two hydrogen bonds. Non-covalent hydrogen bonds between the pairs are shown as dashed lines. The two types of base pairs form different numbers of hydrogen bonds, AT forming two hydrogen bonds, and GC forming three hydrogen bonds (see figures, right). DNA with high GC-content is more stable than DNA with low GC-content.

As noted above, most DNA molecules are actually two polymer strands, bound together in a helical fashion by noncovalent bonds this double-stranded (dsDNA) structure is maintained largely by the intrastrand base stacking interactions, which are strongest for G,C stacks. The two strands can come apart—a process known as melting—to form two single-stranded DNA (ssDNA) molecules. Melting occurs at high temperature, low salt and high pH (low pH also melts DNA, but since DNA is unstable due to acid depurination, low pH is rarely used).

The stability of the dsDNA form depends not only on the GC-content (% G,C basepairs) but also on sequence (since stacking is sequence specific) and also length (longer molecules are more stable). The stability can be measured in various ways a common way is the “melting temperature”, which is the temperature at which 50% of the ds molecules are converted to ss molecules melting temperature is dependent on ionic strength and the concentration of DNA. As a result, it is both the percentage of GC base pairs and the overall length of a DNA double helix that determines the strength of the association between the two strands of DNA. Long DNA helices with a high GC-content have stronger-interacting strands, while short helices with high AT content have weaker-interacting strands. In biology, parts of the DNA double helix that need to separate easily, such as the TATAAT Pribnow box in some promoters, tend to have a high AT content, making the strands easier to pull apart.

In the laboratory, the strength of this interaction can be measured by finding the temperature necessary to break the hydrogen bonds, their melting temperature (also called Tm value). When all the base pairs in a DNA double helix melt, the strands separate and exist in solution as two entirely independent molecules. These single-stranded DNA molecules have no single common shape, but some conformations are more stable than others.

A DNA sequence is called a “sense” sequence if it is the same as that of a messenger RNA copy that is translated into protein. The sequence on the opposite strand is called the “antisense” sequence. Both sense and antisense sequences can exist on different parts of the same strand of DNA (i.e. both strands can contain both sense and antisense sequences). In both prokaryotes and eukaryotes, antisense RNA sequences are produced, but the functions of these RNAs are not entirely clear. One proposal is that antisense RNAs are involved in regulating gene expression through RNA-RNA base pairing.

A few DNA sequences in prokaryotes and eukaryotes, and more in plasmids and viruses, blur the distinction between sense and antisense strands by having overlapping genes. In these cases, some DNA sequences do double duty, encoding one protein when read along one strand, and a second protein when read in the opposite direction along the other strand. In bacteria, this overlap may be involved in the regulation of gene transcription, while in viruses, overlapping genes increase the amount of information that can be encoded within the small viral genome.

DNA can be twisted like a rope in a process called DNA supercoiling. With DNA in its “relaxed” state, a strand usually circles the axis of the double helix once every 10.4 base pairs, but if the DNA is twisted the strands become more tightly or more loosely wound. If the DNA is twisted in the direction of the helix, this is positive supercoiling, and the bases are held more tightly together. If they are twisted in the opposite direction, this is negative supercoiling, and the bases come apart more easily. In nature, most DNA has slight negative supercoiling that is introduced by enzymes called topoisomerases. These enzymes are also needed to relieve the twisting stresses introduced into DNA strands during processes such as transcription and DNA replication.

The expression of genes is influenced by how the DNA is packaged in chromosomes, in a structure called chromatin. Base modifications can be involved in packaging, with regions that have low or no gene expression usually containing high levels of methylation of cytosine bases. DNA packaging and its influence on gene expression can also occur by covalent modifications of the histone protein core around which DNA is wrapped in the chromatin structure or else by remodeling carried out by chromatin remodeling complexes. There is, further, crosstalk between DNA methylation and histone modification, so they can coordinately affect chromatin and gene expression.

For one example, cytosine methylation produces 5-methylcytosine, which is important for X-inactivation of chromosomes. The average level of methylation varies between organisms—the worm Caenorhabditis elegans lacks cytosine methylation, while vertebrates have higher levels, with up to 1% of their DNA containing 5-methylcytosine. Despite the importance of 5-methylcytosine, it can deaminate to leave a thymine base, so methylated cytosines are particularly prone to mutations. Other base modifications include adenine methylation in bacteria, the presence of 5-hydroxymethylcytosine in the brain, and the glycosylation of uracil to produce the “J-base” in kinetoplastids.

11.1.1 DNA Damage

DNA can be damaged by many sorts of mutagens, which change the DNA sequence. Mutagens include oxidizing agents, alkylating agents and also high-energy electromagnetic radiation such as ultraviolet light and X-rays. The type of DNA damage produced depends on the type of mutagen. For example, UV light can damage DNA by producing thymine dimers, which are cross-links between pyrimidine bases. On the other hand, oxidants such as free radicals or hydrogen peroxide produce multiple forms of damage, including base modifications, particularly of guanosine, and double-strand breaks. A typical human cell contains about 150,000 bases that have suffered oxidative damage. Of these oxidative lesions, the most dangerous are double-strand breaks, as these are difficult to repair and can produce point mutations, insertions, deletions from the DNA sequence, and chromosomal translocations. These mutations can cause cancer. DNA damage that is naturally occurring, due to normal cellular processes that produce reactive oxygen species, the hydrolytic activities of cellular water, etc., also occurs frequently. Although most of this damage is repaired, in any cell some DNA damage may remain despite the action of repair processes. This DNA damage accumulates with age in mammalian postmitotic tissues. This accumulation appears to be an important underlying cause of aging.

Many mutagens fit into the space between two adjacent base pairs, this is called intercalation. Most intercalators are aromatic and planar molecules examples include ethidium bromide, acridines, daunomycin, and doxorubicin. For an intercalator to fit between base pairs, the bases must separate, distorting the DNA strands by unwinding of the double helix. This inhibits both transcription and DNA replication, causing toxicity and mutations. As a result, DNA intercalators may be carcinogens, and in the case of thalidomide, a teratogen. Others such as benzo[a]pyrene diol epoxide and aflatoxin form DNA adducts that induce errors in replication. Nevertheless, due to their ability to inhibit DNA transcription and replication, other similar toxins are also used in chemotherapy to inhibit rapidly growing cancer cells.

DNA usually occurs as linear chromosomes in eukaryotes, and circular chromosomes in prokaryotes. The set of chromosomes in a cell makes up its genome the human genome has approximately 3 billion base pairs of DNA arranged into 46 chromosomes. Transmission of genetic information in genes is achieved via complementary base pairing. For example, in transcription, the DNA sequence is copied into a complementary RNA sequence. Usually, this RNA copy is then used to make a matching protein sequence in a process called translation. In alternative fashion, a cell may simply copy its genetic information in a process called DNA replication.

11.1.2 Genes And Genomes

Genomic DNA is tightly and orderly packed in the process called DNA condensation, to fit the small available volumes of the cell. In eukaryotes, DNA is located in the cell nucleus, with small amounts in mitochondria and chloroplasts. In prokaryotes, the DNA is held within an irregularly shaped body in the cytoplasm called the nucleoid.

In many species, only a small fraction of the total sequence of the genome encodes protein. For example, only about 1.5% of the human genome consists of protein-coding exons, with over 50% of human DNA consisting of non-coding repetitive sequences. The reasons for the presence of so much noncoding DNA in eukaryotic genomes and the extraordinary differences in genome size, or C-value, among species, represent a long-standing puzzle known as the “C-value enigma”. However, some DNA sequences that do not code protein may still encode functional non-coding RNA molecules, which are involved in the regulation of gene expression.

Some noncoding DNA sequences play structural roles in chromosomes. Telomeres and centromeres typically contain few genes but are important for the function and stability of chromosomes. An abundant form of noncoding DNA in humans are pseudogenes, which are copies of genes that have been disabled by mutation. These sequences are usually just molecular fossils, although they can occasionally serve as raw genetic material for the creation of new genes through the process of gene duplication and divergence.

11.1.3 Transcription And Translation

A gene is a sequence of DNA that contains genetic information and can influence the phenotype of an organism. Within a gene, the sequence of bases along a DNA strand defines a messenger RNA sequence, which then defines one or more protein sequences. The relationship between the nucleotide sequences of genes and the amino-acid sequences of proteins is determined by the rules of translation, known collectively as the genetic code. The genetic code consists of three-letter ‘words’ called codons formed from a sequence of three nucleotides (e.g. ACT, CAG, TTT).

In transcription, the codons of a gene are copied into messenger RNA by RNA polymerase. This RNA copy is then decoded by a ribosome that reads the RNA sequence by base-pairing the messenger RNA to transfer RNA, which carries amino acids. Since there are 4 bases in 3-letter combinations, there are 64 possible codons. These encode the twenty standard amino acids, giving most amino acids more than one possible codon. There are also three ‘stop’ or ‘nonsense’ codons signifying the end of the coding region these are the TAA, TGA, and TAG codons.

11.1.4 DNA Replication

Cell division is essential for an organism to grow, but, when a cell divides, it must replicate the DNA in its genome so that the two daughter cells have the same genetic information as their parent. The double-stranded structure of DNA provides a simple mechanism for DNA replication. Here, the two strands are separated and then each strand’s complementary DNA sequence is recreated by an enzyme called DNA polymerase. This enzyme makes the complementary strand by finding the correct base through complementary base pairing and bonding it onto the original strand. As DNA polymerases can only extend a DNA strand in a 5′ to 3′ direction, different mechanisms are used to copy the antiparallel strands of the double helix. In this way, the base on the old strand dictates which base appears on the new strand, and the cell ends up with a perfect copy of its DNA.

In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the basis for biological inheritance.

DNA is made up of a double helix of two complementary strands. During replication, these strands are separated. Each strand of the original DNA molecule then serves as a template for the production of its counterpart, a process referred to as semi-conservative replication. As a result of semi-conservative replication, the new helix will be composed of an original DNA strand as well as a newly synthesized strand. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication.

In a cell, DNA replication begins at specific locations, or origins of replication, in the genome. Unwinding of DNA at the origin and synthesis of new strands, accommodated by an enzyme known as helicase, results in replication forks growing bi-directionally from the origin. A number of proteins are associated with the replication fork to help in the initiation and continuation of DNA synthesis. Most prominently, DNA polymerase synthesizes the new strands by adding nucleotides that complement each (template) strand. DNA replication occurs during the S-stage of interphase.

DNA replication (DNA amplification) can also be performed in vitro (artificially, outside a cell). DNA polymerases isolated from cells and artificial DNA primers can be used to start DNA synthesis at known sequences in a template DNA molecule. Polymerase chain reaction (PCR), ligase chain reaction (LCR), and transcription-mediated amplification (TMA) are examples.

The replisome is a complex molecular machine that carries out replication of DNA. The replisome first unwinds double stranded DNA into two single strands. For each of the resulting single strands, a new complementary sequence of DNA is synthesized. The net result is formation of two new double stranded DNA sequences that are exact copies of the original double stranded DNA sequence.

In terms of structure, the replisome is composed of two replicative polymerase complexes, one of which synthesizes the leading strand, while the other synthesizes the lagging strand. The replisome is composed of a number of proteins including helicase, RFC, PCNA, gyrase/topoisomerase, SSB/RPA, primase, DNA polymerase III, RNAse H, and ligase.

For prokaryotes, each dividing nucleoid (region containing genetic material which is not a nucleus) requires two replisomes for bidirectional replication. The two replisomes continue replication at both forks in the middle of the cell. Finally, as the termination site replicates, the two replisomes separate from the DNA. The replisome remains at a fixed, midcell location in the cell, attached to the membrane, and the template DNA threads through it. DNA is fed through the stationary pair of replisomes located at the cell membrane.

For eukaryotes, numerous replication bubbles form at origins of replication throughout the chromosome. As with prokaryotes, two replisomes are required, one at each replication fork located at the terminus of the replication bubble. Because of significant differences in chromosome size, and the associated complexities of highly condensed chromosomes, various aspects of the DNA replication process in eukaryotes, including the terminal phases, are less well-characterised than for prokaryotes.

The replisome is responsible for copying the entirety of genomic DNA in each proliferative cell. This process allows for the high-fidelity passage of hereditary/genetic information from parental cell to daughter cell and is thus essential to all organisms. Much of the cell cycle is built around ensuring that DNA replication occurs without errors.

In G1 phase of the cell cycle, many of the DNA replication regulatory processes are initiated. In eukaryotes, the vast majority of DNA synthesis occurs during S phase of the cell cycle, and the entire genome must be unwound and duplicated to form two daughter copies. During G2, any damaged DNA or replication errors are corrected. Finally, one copy of the genomes is segregated to each daughter cell at mitosis or M phase. These daughter copies each contain one strand from the parental duplex DNA and one nascent antiparallel strand.

11.1.5 Eukaryotic DNA Replication

Eukaryotic DNA replication is a conserved mechanism that restricts DNA replication to once per cell cycle. Eukaryotic DNA replication of chromosomal DNA is central for the duplication of a cell and is necessary for the maintenance of the eukaryotic genome.

DNA replication is the action of DNA polymerases synthesizing a DNA strand complementary to the original template strand. To synthesize DNA, the double-stranded DNA is unwound by DNA helicases ahead of polymerases, forming a replication fork containing two single-stranded templates. Replication processes permit the copying of a single DNA double helix into two DNA helices, which are divided into the daughter cells at mitosis. The major enzymatic functions carried out at the replication fork are well conserved from prokaryotes to eukaryotes, but the replication machinery in eukaryotic DNA replication is a much larger complex, coordinating many proteins at the site of replication, forming the replisome.

After the replicative helicase has unwound the parental DNA duplex, exposing two single-stranded DNA templates, replicative polymerases are needed to generate two copies of the parental genome. DNA polymerase function is highly specialized and accomplish replication on specific templates and in narrow localizations. At the eukaryotic replication fork, there are three distinct replicative polymerase complexes that contribute to DNA replication: Polymerase α, Polymerase δ, and Polymerase ε. These three polymerases are essential for viability of the cell.

Because DNA polymerases require a primer on which to begin DNA synthess, polymerase α (Pol α) acts as a replicative primase. Pol α is associated with an RNA primase and this complex accomplishes the priming task by synthesizing a primer that contains a short 10 nucleotide stretch of RNA followed by 10 to 20 DNA bases. Importantly, this priming action occurs at replication initiation at origins to begin leading-strand synthesis and also at the 5’ end of each Okazaki fragment on the lagging strand.

However, Pol α is not able to continue DNA replication and must be replaced with another polymerase to continue DNA synthesis. Polymerase switching requires clamp loaders and it has been proven that normal DNA replication requires the coordinated actions of all three DNA polymerases: Pol α for priming synthesis, Pol ε for leading-strand replication, and the Pol δ, which is constantly loaded, for generating Okazaki fragments during lagging-strand synthesis.

  • Polymerase α (Pol α): Forms a complex with a small catalytic subunit (PriS) and a large noncatalytic (PriL) subunit. First, synthesis of an RNA primer allows DNA synthesis by DNA polymerase alpha. Occurs once at the origin on the leading strand and at the start of each Okazaki fragment on the lagging strand. Pri subunits act as a primase, synthesizing an RNA primer. DNA Pol α elongates the newly formed primer with DNA nucleotides. After around 20 nucleotides, elongation is taken over by Pol ε on the leading strand and Pol δ on the lagging strand.
  • Polymerase δ (Pol δ): Highly processive and has proofreading, 3’->5’ exonuclease activity. In vivo, it is the main polymerase involved in both lagging strand and leading strand synthesis.
  • Polymerase ε (Pol ε): Highly processive and has proofreading, 3’->5’ exonuclease activity. Highly related to pol δ, in vivo it functions mainly in error checking of pol δ.

DNA replication, like all biological polymerization processes, proceeds in three enzymatically catalyzed and coordinated steps: initiation, elongation and termination.

11.1.6 Initiation

For a cell to divide, it must first replicate its DNA. DNA replication is an all-or-none process once replication begins, it proceeds to completion. Once replication is complete, it does not occur again in the same cell cycle. This is made possible by the division of initiation into two temporally distinct steps: formation of the pre-replication complex and the preinitiation complex.

11.1.7 Pre-replication complex

In late mitosis and early G1 phase, a large complex of initiator proteins assembles into the pre-replication complex at particular points in the DNA, known as “origins”. In E. coli the primary initiator protein is DnaA in yeast, this is the origin recognition complex. Sequences used by initiator proteins tend to be “AT-rich” (rich in adenine and thymine bases), because A-T base pairs have two hydrogen bonds (rather than the three formed in a C-G pair) and thus are easier to strand-separate. In eukaryotes, the origin recognition complex catalyzes the assembly of initiator proteins into the pre-replication complex. Cdc6 and Cdt1 then associate with the bound origin recognition complex at the origin in order to form a larger complex necessary to load the Mcm complex onto the DNA. The Mcm complex is the helicase that will unravel the DNA helix at the replication origins and replication forks in eukaryotes. The Mcm complex is recruited at late G1 phase and loaded by the ORC-Cdc6-Cdt1 complex onto the DNA via ATP-dependent protein remodeling. The loading of the Mcm complex onto the origin DNA marks the completion of pre-replication complex formation.

If environmental conditions are right in late G1 phase, the G1 and G1/S cyclin-Cdk complexes are activated, which stimulate expression of genes that encode components of the DNA synthetic machinery. G1/S-Cdk activation also promotes the expression and activation of S-Cdk complexes, which may play a role in activating replication origins depending on species and cell type. Control of these Cdks vary depending cell type and stage of development.

In a similar manner, Cdc7 is also required through S phase to activate replication origins. Cdc7 is not active throughout the cell cycle, and its activation is strictly timed to avoid premature initiation of DNA replication. In late G1, Cdc7 activity rises abruptly as a result of association with the regulatory subunit Dbf4, which binds Cdc7 directly and promotes its protein kinase activity. Cdc7 has been found to be a rate-limiting regulator of origin activity. Together, the G1/S-Cdks and/or S-Cdks and Cdc7 collaborate to directly activate the replication origins, leading to initiation of DNA synthesis.

11.1.8 Preinitiation complex

In early S phase, S-Cdk and Cdc7 activation lead to the assembly of the preinitiation complex, a massive protein complex formed at the origin. Formation of the preinitiation complex displaces Cdc6 and Cdt1 from the origin replication complex, inactivating and disassembling the pre-replication complex. Loading the preinitiation complex onto the origin activates the Mcm helicase, causing unwinding of the DNA helix. The preinitiation complex also loads α-primase and other DNA polymerases onto the DNA.

After α-primase synthesizes the first primers, the primer-template junctions interact with the clamp loader, which loads the sliding clamp onto the DNA to begin DNA synthesis. The components of the preinitiation complex remain associated with replication forks as they move out from the origin.

11.1.9 Elongation

DNA polymerase has 5′–3′ activity. All known DNA replication systems require a free 3′ hydroxyl group before synthesis can be initiated (note: the DNA template is read in 3′ to 5′ direction whereas a new strand is synthesized in the 5′ to 3′ direction—this is often confused). Four distinct mechanisms for DNA synthesis are recognized:

All cellular life forms and many DNA viruses, phages and plasmids use a primase to synthesize a short RNA primer with a free 3′ OH group which is subsequently elongated by a DNA polymerase. The 5′ end of the nicked strand is transferred to a tyrosine residue on the nuclease and the free 3′ OH group is then used by the DNA polymerase to synthesize the new strand. The first is the best known of these mechanisms and is used by the cellular organisms. In this mechanism, once the two strands are separated, primase adds RNA primers to the template strands. The leading strand receives one RNA primer while the lagging strand receives several. The leading strand is continuously extended from the primer by a DNA polymerase with high processivity, while the lagging strand is extended discontinuously from each primer forming Okazaki fragments. RNase removes the primer RNA fragments, and a low processivity DNA polymerase distinct from the replicative polymerase enters to fill the gaps. When this is complete, a single nick on the leading strand and several nicks on the lagging strand can be found. Ligase works to fill these nicks in, thus completing the newly replicated DNA molecule.

Multiple DNA polymerases take on different roles in the DNA replication process. In E. coli, DNA Pol III is the polymerase enzyme primarily responsible for DNA replication. It assembles into a replication complex at the replication fork that exhibits extremely high processivity, remaining intact for the entire replication cycle. In contrast, DNA Pol I is the enzyme responsible for replacing RNA primers with DNA. DNA Pol I has a 5′ to 3′ exonuclease activity in addition to its polymerase activity, and uses its exonuclease activity to degrade the RNA primers ahead of it as it extends the DNA strand behind it, in a process called nick translation. Pol I is much less processive than Pol III because its primary function in DNA replication is to create many short DNA regions rather than a few very long regions.

In eukaryotes, the low-processivity enzyme, Pol α, helps to initiate replication because it forms a complex with primase. In eukaryotes, leading strand synthesis is thought to be conducted by Pol ε however, this view has recently been challenged, suggesting a role for Pol δ. Primer removal is completed by Pol δ while repair of DNA during replication is completed by Pol ε.

As DNA synthesis continues, the original DNA strands continue to unwind on each side of the bubble, forming a replication fork with two prongs. In bacteria, which have a single origin of replication on their circular chromosome, this process creates a “theta structure” (resembling the Greek letter theta: θ). In contrast, eukaryotes have longer linear chromosomes and initiate replication at multiple origins within these.

11.1.10 Replication Fork

The replication fork is a structure that forms within the long helical DNA during DNA replication. It is created by helicases, which break the hydrogen bonds holding the two DNA strands together in the helix. The resulting structure has two branching “prongs”, each one made up of a single strand of DNA. These two strands serve as the template for the leading and lagging strands, which will be created as DNA polymerase matches complementary nucleotides to the templates the templates may be properly referred to as the leading strand template and the lagging strand template.

DNA is always synthesized by adding nucleotides to the 3′ end of a strand. Since the leading and lagging strand templates are oriented in opposite directions at the replication fork, a major issue is how to achieve synthesis of nascent (new) lagging strand DNA, whose direction of synthesis is opposite to the direction of the growing replication fork.

11.1.11 Replication of The Leading Strand

The leading strand is the strand of nascent DNA which is synthesized in the same direction as the growing replication fork. This sort of DNA replication is continuous.

11.1.12 Replication of The Lagging Strand

The lagging strand is the strand of nascent DNA whose direction of synthesis is opposite to the direction of the growing replication fork. Because of its orientation, replication of the lagging strand is more complicated as compared to that of the leading strand. As a consequence, the DNA polymerase on this strand is seen to “lag behind” the other strand.

The lagging strand is synthesized in short, separated segments. On the lagging strand template, a primase “reads” the template DNA and initiates synthesis of a short complementary RNA primer. A DNA polymerase extends the primed segments, forming Okazaki fragments. The RNA primers are then removed and replaced with DNA, and the fragments of DNA are joined together by DNA ligase.

In all cases the helicase is composed of six polypeptides that wrap around only one strand of the DNA being replicated. The two polymerases are bound to the helicase heximer. In eukaryotes the helicase wraps around the leading strand, and in prokaryotes it wraps around the lagging strand.

As helicase unwinds DNA at the replication fork, the DNA ahead is forced to rotate. This process results in a build-up of twists in the DNA ahead. This build-up forms a torsional resistance that would eventually halt the progress of the replication fork. Topoisomerases are enzymes that temporarily break the strands of DNA, relieving the tension caused by unwinding the two strands of the DNA helix topoisomerases (including DNA gyrase) achieve this by adding negative supercoils to the DNA helix.

Bare single-stranded DNA tends to fold back on itself forming secondary structures these structures can interfere with the movement of DNA polymerase. To prevent this, single-strand binding proteins bind to the DNA until a second strand is synthesized, preventing secondary structure formation.

Clamp proteins form a sliding clamp around DNA, helping the DNA polymerase maintain contact with its template, thereby assisting with processivity. The inner face of the clamp enables DNA to be threaded through it. Once the polymerase reaches the end of the template or detects double-stranded DNA, the sliding clamp undergoes a conformational change that releases the DNA polymerase. Clamp-loading proteins are used to initially load the clamp, recognizing the junction between template and RNA primers.

11.1.13 DNA Replication Proteins

At the replication fork, many replication enzymes assemble on the DNA into a complex molecular machine called the replisome. The following is a list of major DNA replication enzymes that participate in the replisome:

Table 9.1: A list of major DNA replication enzymes that participate in the replisome
Enzymes Function in DNA replication
DNA helicase Also known as helix destabilizing enzyme. Helicase separates the two strands of DNA at the Replication Fork behind the topoisomerase.
DNA polymerase The enzyme responsible for catalyzing the addition of nucleotide substrates to DNA in the 5′ to 3′ direction during DNA replication. Also performs proof-reading and error correction. There exist many different types of DNA Polymerase, each of which perform different functions in different types of cells.
DNA clamp A protein which prevents elongating DNA polymerases from dissociating from the DNA parent strand.
Single-strand DNA-binding protein Bind to ssDNA and prevent the DNA double helix from re-annealing after DNA helicase unwinds it, thus maintaining the strand separation, and facilitating the synthesis of the nascent strand.
Topoisomerase Relaxes the DNA from its super-coiled nature.
DNA gyrase Relieves strain of unwinding by DNA helicase this is a specific type of topoisomerase
DNA ligase Re-anneals the semi-conservative strands and joins Okazaki Fragments of the lagging strand.
Primase Provides a starting point of RNA (or DNA) for DNA polymerase to begin synthesis of the new DNA strand.
Telomerase Lengthens telomeric DNA by adding repetitive nucleotide sequences to the ends of eukaryotic chromosomes. This allows germ cells and stem cells to avoid the Hayflick limit on cell divisi

11.1.14 Termination

Eukaryotes initiate DNA replication at multiple points in the chromosome, so replication forks meet and terminate at many points in the chromosome. Because eukaryotes have linear chromosomes, DNA replication is unable to reach the very end of the chromosomes. Due to this problem, DNA is lost in each replication cycle from the end of the chromosome. Telomeres are regions of repetitive DNA close to the ends and help prevent loss of genes due to this shortening. Shortening of the telomeres is a normal process in somatic cells. This shortens the telomeres of the daughter DNA chromosome. As a result, cells can only divide a certain number of times before the DNA loss prevents further division. (This is known as the Hayflick limit.) Within the germ cell line, which passes DNA to the next generation, telomerase extends the repetitive sequences of the telomere region to prevent degradation. Telomerase can become mistakenly active in somatic cells, sometimes leading to cancer formation. Increased telomerase activity is one of the hallmarks of cancer.

Termination requires that the progress of the DNA replication fork must stop or be blocked. Termination at a specific locus, when it occurs, involves the interaction between two components: (1) a termination site sequence in the DNA, and (2) a protein which binds to this sequence to physically stop DNA replication. In various bacterial species, this is named the DNA replication terminus site-binding protein, or Ter protein.

Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome. E. coli regulates this process through the use of termination sequences that, when bound by the Tus protein, enable only one direction of replication fork to pass through. As a result, the replication forks are constrained to always meet within the termination region of the chromosome.

11.1.15 Regulation of DNA Replication

Within eukaryotes, DNA replication is controlled within the context of the cell cycle. As the cell grows and divides, it progresses through stages in the cell cycle DNA replication takes place during the S phase (synthesis phase). The progress of the eukaryotic cell through the cycle is controlled by cell cycle checkpoints. Progression through checkpoints is controlled through complex interactions between various proteins, including cyclins and cyclin-dependent kinases.

The G1/S checkpoint (or restriction checkpoint) regulates whether eukaryotic cells enter the process of DNA replication and subsequent division. Cells that do not proceed through this checkpoint remain in the G0 stage and do not replicate their DNA.

After passing through the G1/S checkpoint, DNA must be replicated only once in each cell cycle. When the Mcm complex moves away from the origin, the pre-replication complex is dismantled. Because a new Mcm complex cannot be loaded at an origin until the pre-replication subunits are reactivated, one origin of replication can not be used twice in the same cell cycle.

Activation of S-Cdks in early S phase promotes the destruction or inhibition of individual pre-replication complex components, preventing immediate reassembly. S and M-Cdks continue to block pre-replication complex assembly even after S phase is complete, ensuring that assembly cannot occur again until all Cdk activity is reduced in late mitosis.

Replication of chloroplast and mitochondrial genomes occurs independently of the cell cycle, through the process of D-loop replication.

11.1.16 Interactions of DNA with Proteins

All the functions of DNA depend on interactions with proteins. These protein interactions can be non-specific, or the protein can bind specifically to a single DNA sequence. Enzymes can also bind to DNA and of these, the polymerases that copy the DNA base sequence in transcription and DNA replication are particularly important.

11.1.17 DNA-binding proteins

Structural proteins that bind DNA are well-understood examples of non-specific DNA-protein interactions. Within chromosomes, DNA is held in complexes with structural proteins. These proteins organize the DNA into a compact structure called chromatin. In eukaryotes, this structure involves DNA binding to a complex of small basic proteins called histones, while in prokaryotes multiple types of proteins are involved. The histones form a disk-shaped complex called a nucleosome, which contains two complete turns of double-stranded DNA wrapped around its surface. These non-specific interactions are formed through basic residues in the histones, making ionic bonds to the acidic sugar-phosphate backbone of the DNA, and are thus largely independent of the base sequence. Chemical modifications of these basic amino acid residues include methylation, phosphorylation, and acetylation. These chemical changes alter the strength of the interaction between the DNA and the histones, making the DNA more or less accessible to transcription factors and changing the rate of transcription. Other non-specific DNA-binding proteins in chromatin include the high-mobility group proteins, which bind to bent or distorted DNA. These proteins are important in bending arrays of nucleosomes and arranging them into the larger structures that make up chromosomes.

A distinct group of DNA-binding proteins is the DNA-binding proteins that specifically bind single-stranded DNA. In humans, replication protein A is the best-understood member of this family and is used in processes where the double helix is separated, including DNA replication, recombination, and DNA repair. These binding proteins seem to stabilize single-stranded DNA and protect it from forming stem-loops or being degraded by nucleases.

In contrast, other proteins have evolved to bind to particular DNA sequences. The most intensively studied of these are the various transcription factors, which are proteins that regulate transcription. Each transcription factor binds to one particular set of DNA sequences and activates or inhibits the transcription of genes that have these sequences close to their promoters. The transcription factors do this in two ways. Firstly, they can bind the RNA polymerase responsible for transcription, either directly or through other mediator proteins this locates the polymerase at the promoter and allows it to begin transcription. Alternatively, transcription factors can bind enzymes that modify the histones at the promoter. This changes the accessibility of the DNA template to the polymerase.

As these DNA targets can occur throughout an organism’s genome, changes in the activity of one type of transcription factor can affect thousands of genes. Consequently, these proteins are often the targets of the signal transduction processes that control responses to environmental changes or cellular differentiation and development. The specificity of these transcription factors’ interactions with DNA come from the proteins making multiple contacts to the edges of the DNA bases, allowing them to “read” the DNA sequence. Most of these base-interactions are made in the major groove, where the bases are most accessible.

11.1.18 DNA-modifying Enzymes

11.1.19 Nucleases And Ligases

Nucleases are enzymes that cut DNA strands by catalyzing the hydrolysis of the phosphodiester bonds. Nucleases that hydrolyse nucleotides from the ends of DNA strands are called exonucleases, while endonucleases cut within strands. The most frequently used nucleases in molecular biology are the restriction endonucleases, which cut DNA at specific sequences. For instance, the EcoRI enzyme recognizes the 6-base sequence 5′-GAATTC-3′ and cuts each strand after the G creating 4 nucleotide sticky ends with a 5’ end overhang of AATT. In nature, these enzymes protect bacteria against phage infection by digesting the phage DNA when it enters the bacterial cell, acting as part of the restriction modification system. These sequence-specific nucleases are used in molecular cloning and DNA fingerprinting.

Enzymes called DNA ligases can rejoin cut or broken DNA strands. Ligases are particularly important in lagging strand DNA replication, as they join together the short segments of DNA produced at the replication fork into a complete copy of the DNA template. They are also used in DNA repair and genetic recombination.

11.1.20 Topoisomerases And Helicases

Topoisomerases are enzymes with both nuclease and ligase activity. These proteins change the amount of supercoiling in DNA. Some of these enzymes work by cutting the DNA helix and allowing one section to rotate, thereby reducing its level of supercoiling the enzyme then seals the DNA break. Other types of these enzymes are capable of cutting one DNA helix and then passing a second strand of DNA through this break, before rejoining the helix. Topoisomerases are required for many processes involving DNA, such as DNA replication and transcription.

Helicases are proteins that are a type of molecular motor. They use the chemical energy in nucleoside triphosphates, predominantly adenosine triphosphate (ATP), to break hydrogen bonds between bases and unwind the DNA double helix into single strands. These enzymes are essential for most processes where enzymes need to access the DNA bases.

11.1.21 Polymerases

Polymerases are enzymes that synthesize polynucleotide chains from nucleoside triphosphates. The sequence of their products is created based on existing polynucleotide chains—which are called templates. These enzymes function by repeatedly adding a nucleotide to the 3′ hydroxyl group at the end of the growing polynucleotide chain. As a consequence, all polymerases work in a 5′ to 3′ direction. In the active site of these enzymes, the incoming nucleoside triphosphate base-pairs to the template: this allows polymerases to accurately synthesize the complementary strand of their template. Polymerases are classified according to the type of template that they use.

In DNA replication, DNA-dependent DNA polymerases make copies of DNA polynucleotide chains. To preserve biological information, it is essential that the sequence of bases in each copy are precisely complementary to the sequence of bases in the template strand. Many DNA polymerases have a proofreading activity. Here, the polymerase recognizes the occasional mistakes in the synthesis reaction by the lack of base pairing between the mismatched nucleotides. If a mismatch is detected, a 3′ to 5′ exonuclease activity is activated and the incorrect base removed. In most organisms, DNA polymerases function in a large complex called the replisome that contains multiple accessory subunits, such as the DNA clamp or helicases.

RNA-dependent DNA polymerases are a specialized class of polymerases that copy the sequence of an RNA strand into DNA. They include reverse transcriptase, which is a viral enzyme involved in the infection of cells by retroviruses, and telomerase, which is required for the replication of telomeres. For example, HIV reverse transcriptase is an enzyme for AIDS virus replication. Telomerase is an unusual polymerase because it contains its own RNA template as part of its structure. It synthesizes telomeres at the ends of chromosomes. Telomeres prevent fusion of the ends of neighboring chromosomes and protect chromosome ends from damage.

Transcription is carried out by a DNA-dependent RNA polymerase that copies the sequence of a DNA strand into RNA. To begin transcribing a gene, the RNA polymerase binds to a sequence of DNA called a promoter and separates the DNA strands. It then copies the gene sequence into a messenger RNA transcript until it reaches a region of DNA called the terminator, where it halts and detaches from the DNA. As with human DNA-dependent DNA polymerases, RNA polymerase II, the enzyme that transcribes most of the genes in the human genome, operates as part of a large protein complex with multiple regulatory and accessory subunits.

11.1.22 DNA Recombination

A DNA helix usually does not interact with other segments of DNA, and in human cells, the different chromosomes even occupy separate areas in the nucleus called “chromosome territories”. This physical separation of different chromosomes is important for the ability of DNA to function as a stable repository for information, as one of the few times chromosomes interact is in chromosomal crossover which occurs during sexual reproduction, when genetic recombination occurs. Chromosomal crossover is when two DNA helices break, swap a section and then rejoin.

Recombination allows chromosomes to exchange genetic information and produces new combinations of genes, which increases the efficiency of natural selection and can be important in the rapid evolution of new proteins. Genetic recombination can also be involved in DNA repair, particularly in the cell’s response to double-strand breaks.

The most common form of chromosomal crossover is homologous recombination, where the two chromosomes involved share very similar sequences. Non-homologous recombination can be damaging to cells, as it can produce chromosomal translocations and genetic abnormalities. The recombination reaction is catalyzed by enzymes known as recombinases, such as RAD51. The first step in recombination is a double-stranded break caused by either an endonuclease or damage to the DNA. A series of steps catalyzed in part by the recombinase then leads to joining of the two helices by at least one Holliday junction, in which a segment of a single strand in each helix is annealed to the complementary strand in the other helix. The Holliday junction is a tetrahedral junction structure that can be moved along the pair of chromosomes, swapping one strand for another. The recombination reaction is then halted by cleavage of the junction and re-ligation of the released DNA. Only strands of like polarity exchange DNA during recombination. There are two types of cleavage: east-west cleavage and north-south cleavage. The north-south cleavage nicks both strands of DNA, while the east-west cleavage has one strand of DNA intact.

11.1.23 Evolutionary History of DNA

DNA contains the genetic information that allows all forms of life to function, grow and reproduce. However, it is unclear how long in the 4-billion-year history of life DNA has performed this function, as it has been proposed that the earliest forms of life may have used RNA as their genetic material. RNA may have acted as the central part of early cell metabolism as it can both transmit genetic information and carry out catalysis as part of ribozymes. This ancient RNA world where nucleic acid would have been used for both catalysis and genetics may have influenced the evolution of the current genetic code based on four nucleotide bases. This would occur, since the number of different bases in such an organism is a trade-off between a small number of bases increasing replication accuracy and a large number of bases increasing the catalytic efficiency of ribozymes. However, there is no direct evidence of ancient genetic systems, as recovery of DNA from most fossils is impossible because DNA survives in the environment for less than one million years, and slowly degrades into short fragments in solution.

Building blocks of DNA (adenine, guanine, and related organic molecules) may have been formed extraterrestrially in outer space. Complex DNA and RNA organic compounds of life, including uracil, cytosine, and thymine, have also been formed in the laboratory under conditions mimicking those found in outer space, using starting chemicals, such as pyrimidine, found in meteorites. Pyrimidine, like polycyclic aromatic hydrocarbons (PAHs), the most carbon-rich chemical found in the universe, may have been formed in red giants or in interstellar cosmic dust and gas clouds.

11.1.24 Genetic engineering

Methods have been developed to purify DNA from organisms, such as phenol-chloroform extraction, and to manipulate it in the laboratory, such as restriction digests and the polymerase chain reaction. Modern biology and biochemistry make intensive use of these techniques in recombinant DNA technology. Recombinant DNA is a man-made DNA sequence that has been assembled from other DNA sequences. They can be transformed into organisms in the form of plasmids or in the appropriate format, by using a viral vector. The genetically modified organisms produced can be used to produce products such as recombinant proteins, used in medical research, or be grown in agriculture.

11.1.25 DNA profiling

Forensic scientists can use DNA in blood, semen, skin, saliva or hair found at a crime scene to identify a matching DNA of an individual, such as a perpetrator. This process is formally termed DNA profiling, also called DNA fingerprinting. In DNA profiling, the lengths of variable sections of repetitive DNA, such as short tandem repeats and minisatellites, are compared between people. This method is usually an extremely reliable technique for identifying a matching DNA. However, identification can be complicated if the scene is contaminated with DNA from several people. DNA profiling was developed in 1984 by British geneticist Sir Alec Jeffreys, and first used in forensic science to convict Colin Pitchfork in the 1988 Enderby murders case.

DNA profiling is also used in DNA paternity testing to determine if someone is the biological parent or grandparent of a child with the probability of parentage is typically 99.99% when the alleged parent is biologically related to the child. Normally, paternity testing is performed after birth, but recently developed methods allow isolation and sequencing of fetal DNA from the blood of the mother.


Chromosomes and Genes

Each species has a characteristic number of chromosomes. Chromosomes are coiled structures made of DNA and proteins called histones (Figure below). Chromosomes are the form of the genetic material of a cell during cell division. See the "Chromosomes" section for additional information.

The human genome has 23 pairs of chromosomes located in the nucleus of somatic cells. Each chromosome is composed of genes and other DNA wound around histones (proteins) into a tightly coiled molecule.

The human species is characterized by 23 pairs of chromosomes, as shown in Figure below. You can watch a short animation about human chromosomes at this link:http://www.dnalc.org/view/15520-DNA-is-organized-into-46-chromosomes-including-sex-chromosomes-3D-animation.html.

Human Chromosomes. Humans have 23 pairs of chromosomes. Pairs 1-22 are autosomes. Females have two X chromosomes, and males have an X and a Y chromosome.

Autosomes

Of the 23 pairs of human chromosomes, 22 pairs are autosomes (numbers 1&ndash22 in Figureabove). Autosomes are chromosomes that contain genes for characteristics that are unrelated to sex. These chromosomes are the same in males and females. The great majority of human genes are located on autosomes. At the link below, you can click on any human chromosome to see which traits its genes control.http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/chooser.shtml

Sex Chromosomes

The remaining pair of human chromosomes consists of the sex chromosomes, X and Y. Females have two X chromosomes, and males have one X and one Y chromosome. In females, one of the X chromosomes in each cell is inactivated and known as a Barr body. This ensures that females, like males, have only one functioning copy of the X chromosome in each cell.

As you can see from Figure above and Figure above, the X chromosome is much larger than the Y chromosome. The X chromosome has about 2,000 genes, whereas the Y chromosome has fewer than 100, none of which are essential to survival. (For comparison, the smallest autosome, chromosome 22, has over 500 genes.) Virtually all of the X chromosome genes are unrelated to sex. Only the Y chromosome contains genes that determine sex. A single Y chromosome gene, called SRY (which stands for sex-determining region Y gene), triggers an embryo to develop into a male. Without a Y chromosome, an individual develops into a female, so you can think of female as the default sex of the human species. Can you think of a reason why the Y chromosome is so much smaller than the X chromosome? At the link that follows, you can watch an animation that explains why:www.hhmi.org/biointeractive/g. evolution.html.

Human Genes

Humans have an estimated 20,000 to 22,000 genes. This may sound like a lot, but it really isn&rsquot. Far simpler species have almost as many genes as humans. However, human cells use splicing and other processes to make multiple proteins from the instructions encoded in a single gene. Of the 3 billion base pairs in the human genome, only about 25 percent make up genes and their regulatory elements. The functions of many of the other base pairs are still unclear. To learn more about the coding and noncoding sequences of human DNA, watch the animation at this link: www.hhmi.org/biointeractive/d. sequences.html.

The majority of human genes have two or more possible alleles, which are alternative forms of a gene. Differences in alleles account for the considerable genetic variation among people. In fact, most human genetic variation is the result of differences in individual DNA bases within alleles.


Difference Between DNA and Chromosome

Both DNA and chromosomes lie behind our basic understanding of the human body. However, there are subtle differences between the two that determine their actions to a significant extent.

So, what do you understand by DNA anyway? DNA can be described as a long fiber that resembles a hair under a powerful microscope. The only difference is that they are much thinner and longer. The whole structure is made of two strands that are intertwined together. When cells get ready to divide, proteins attach themselves to the DNA and leads to the creation of a chromosome.

The DNA in a human body is organized into many stretches of genes. Proteins attach themselves to these stretches and coil them so that they form chromosomes. These stretches are very important in the formation of an organism. Do you know why?

This is because these are the stretches that determine which genes are going to be turned on and which are to be turned off. When a gene is turned on, it determines how the proteins are going to be formed in a cell. This in turn determines many aspects of a human being- starting from the color of his eye to the inheritance of a number of diseases and conditions.

A chromosome is simply the product of the DNA and the proteins that are attached to it. There are 23 pairs of the chromosomes in every human being. One set is inherited from the father, and one set is inherited from the mother. A DNA is a sort of a bio molecule. The entire DNA in cells can be found in individual pieces that are called chromosomes.

The main difference between DNA and chromosome is regarding the role of genes. DNA stands for deoxyribonucleic acid. The DNA is basically made up of cytosine, adenine, thymine and guanine. When you arrange these four bases to create a particular segment, it is called a gene. When these segments are coiled in a form that can be easily duplicated, they are known as chromosomes.

Confused? Try to remember it like this- a gene is composed of tiny chromosomes, each of which determines a particular characteristic in a human. These chromosomes are further divided into pieces of DNA. Chromosomes are basically pieces of DNA. If we looked at a chromosome as an intertwined necklace, the beads on it would be the different DNA. The pattern that is formed by this intertwining of the strands is called a double helix pattern.

All of these are basic building blocks of the body. DNA is the smallest part that, together with proteins, forms a chromosome. A chromosome is therefore, nothing but a chain of DNA that has been made compact enough to fit into a cell.

Summary:
1. Both chromosomes and DNA make up an important part of a person’s genes
2. A chromosome is a subpart of a person’s genes, while DNA is a part of the chromosome.
3. When proteins add to DNA, a chromosome is formed.


Difference Between Chromosome and Chromatid

Condensation

Chromosome: DNA is condensed 10, 000 times to form a chromosome. Thus, a chromosome is the most condensed form of DNA

Chromatid: DNA is condensed 50 times to form a chromatid. Thus, a chromatid is less condensed than a chromosome.

Content

Chromosome: A chromosome consists of a single, double-stranded DNA molecule.

Chromatid: A chromatid consists of two DNA strands joining together by their centromere.

Structure

Chromosome: A chromosome is a thin, ribbon-like structure.

Chromatid: A chromatid is a thin and long, fibrous structure.

Genetic Material

Chromosome: Homologous chromosomes are not identical. They might have different alleles of the same gene.

Chromatid: Homologous sister chromatids are identical.

Stage

Chromosome: Chromosomes appear in M phase.

Chromatid: Chromatids appear in the interphase.

Function:

Chromosome: Chromosomes are involved in the distribution of genetic material.

Chromatid: Chromatids are involved in metabolism and other activities of the cell.

Conclusion

A chromosome consists of a single DNA molecule whereas a chromatid consists of two identical DNA strands joined together by the centromere. Chromosomes generally participate in the distribution of genetic material at the nuclear division. Chromatids participate in metabolism and regulation of gene expression. Nevertheless, DNA is condensed 10,000 times in a chromosome while it is condensed 50 times itself in a chromatid. Thus, the key difference between a chromosome and a chromatid is in the level of condensation.

Reference:
1. Higgins, N. P., Chromosome Structure. ENCYCLOPEDIA OF LIFE SCIENCES. 2015 http://smcg.ccg.unam.mx/enp-unam/03-EstructuraDelGenoma/cromosomeStructure.pdf 08 Feb. 2017
2. “Chromosome”. National Human Genome Research institute. 08 Feb. 2017 https://www.genome.gov/26524120/chromosomes-fact-sheet/

Image Courtesy:
1. “Sky spectral karyotype” from National Human Genome Research Institute – Found on:National Human Genome Research (USA) – copied from wikipedia:en. (Public Domain) via Commons Wikimedia
2. � DNA Macrostructure” By OpenStax – (CC BY 4.0) via Commons Wikimedia

About the Author: Lakna

Lakna, a graduate in Molecular Biology & Biochemistry, is a Molecular Biologist and has a broad and keen interest in the discovery of nature related things


Watch the video: How CRISPR lets us edit our DNA. Jennifer Doudna (September 2022).


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