Restriction sites

Restriction sites

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I would like to know: how many restriction sites does a restriction enzyme use on a DNA molecule? In other words: If a sequence on a plasmid contains the following bases:


and I want to have only the bases in bold as a new, separate molecule, will I need two restriction enzymes (One to cut the molecule between the T and G and another one to cut it between the T and C)? Or one restriction enzyme (that will cut them both)?

It wasn't clear enough in Wikipedia and in my biology book.

Most restriction enzymes have a 6bp restriction site (some have 8bp site also). So two restriction sites generally have to span 12bp. However some restriction sites can overlap. For example BamHI and SmaI:

BamHI |______| GGATCCCGGG |______| SmaI

In this case the total length has reduced to 10bp. However such combinations are not that common and at max you will achieve a 2-3bp overlap. A restriction endonuclease only makes a single cut. To make double cuts you need two restriction sites.

Even if the sites are non-overlapping there has to be a minimum length of separation otherwise the binding of one enzyme will hinder the binding of the other enzyme in a double digest. A 6 nucleotide gap between the restriction sites should be fine. If there is no gap then double digest may not be very efficient.

You can also have two restriction sites of the same enzyme so that you need not use two separate enzymes.

BTW the sequence in your question does not have any restriction site.

Hope this answers your question.

Just to bring another light to the question: a restriction enzyme cuts a double-stranded DNA two times, but it only cuts it once on each strand.

So yes, you would need two different restriction enzymes to excise the sequence in bold (assuming you want that sequence in double-stranded-form): one enzyme that would cut between the T and the G on the strand that you have represented, and one that would cut between the T and the C on this same strand.

Plant Molecular Systematics

Restriction Site Analysis (RFLPs)

A restriction site is a sequence of approximately 6–8 base pairs of DNA that binds to a given restriction enzyme. These restriction enzymes, of which there are many, have been isolated from bacteria. Their natural function is to inactivate invading viruses by cleaving the viral DNA. Restriction enzymes known as type II recognize restriction sites and cleave the DNA at particular locations within or near the restriction site. An example is the restriction enzyme EcoRI (named after E. coli, from which it was first isolated), which recognizes the DNA sequence seen in Figure 14.8 and cleaves the DNA at the sites indicated by the arrows in this figure.

FIGURE 14.8 . A DNA restriction site, cleaved (at arrows) by the restriction site enzyme EcoRI.

Restriction fragment length polymorphism, or RFLP, refers to differences between taxa in restriction sites, and therefore the lengths of fragments of DNA following cleavage with restriction enzymes. For example, Figure 14.9 shows, for two hypothetical species, amplified DNA lengths of 10,000 base pairs that are subjected to (“digested with”) the restriction enzyme EcoRI. Note, after a reaction with the EcoRI enzyme, that the DNA of species A is cleaved into three fragments, corresponding to two EcoRI restriction sites, whereas that of species B is cleaved into four fragments, corresponding to three EcoRI restriction sites. The relative locations of these restriction sites on the DNA can be mapped one possibility is seen at the bottom of Figure 14.9 . (Note that there are other possibilities for this map precise mapping requires additional work.) Additional restriction enzymes can be used. Figure 14.10 illustrates how each of the DNA fragments from the EcoRI digests can be digested with the BAM HI restriction enzyme, yielding different fragments for the two species. These data can be added to the original in preparing a map (one possible map is shown in lower part of Figure 14.10 ).

FIGURE 14.9 . Example of restriction site analysis of species A and B, using restriction site enzyme EcoRI. Note differences in fragment lengths. Possible restriction site maps of species A and B are shown in the lower portion of the figure.

FIGURE 14.10 . Example of restriction site analysis of species A and B, using restriction site enzyme EcoRI, followed by restriction site enzyme BAM HI. Possible restriction site maps of species A and B are shown in the lower portion of the figure.

Restriction site fragment data can be coded as characters and character states in a phylogenetic analysis. For example, given that the restriction site maps of Figure 14.10 are correct, the presence or absence of these sites can be coded as characters, as seen in Figure 14.11 . Restriction site analysis contains far less data than complete DNA sequencing, accounting only for the presence or absence of sites 6–8 base pairs long. It has the advantage, however, of surveying considerably larger segments of DNA. However, with improved and less expensive sequencing techniques, it is less valuable and less often used than in the past.

FIGURE 14.11 . Character coding of restriction site map data of Figure 14.10 , derived by presence or absence of EcoRI or BAM sites at specific locations along DNA.

DNA: Restriction Mapping and Nucleotide Sequencing

Read this article to learn about the restriction mapping of DNA which involves the size analysis of restriction fragments and also learn about nucleotide sequencing of DNA for which two techniques have been developed:

(1) based on enzymatic method called Sanger sequencing and (2) based on chemical method called Maxam and Gilbert sequencing.

Restriction Mapping of DNA Fragments:

This involves the size analysis of restriction fragments produced by several restriction enzymes individually and in combination. For example, in Fig. 3.2 restriction sites of two enzymes A and B are being mapped. Cleavage with A gives fragments 2 and 7 kilo bases from a 9 kb molecule, hence we can have position of single A site from one end.

Similarly, B gives fragments 3 kb and 6 kb away, so it has a single site 3 kb from one end but it is still not clear whether this site is near A’s site or is at opposite end of DNA. This can be resolved by double digestion. If the resultant fragments are 2 kb, 3 kb and 4 kb away, then A and B cut at opposite ends of the molecule if they are 1 kb, 2 kb and 6 kb away the sites are near to each other. It is worth stating here that the mapping of real molecules is rarely as simple as this.

Nucleotide Sequencing of DNA:

The precise usage of codons, information regarding mutations and polymorphisms and identifica­tion of gene regulatory control sequences can only be elucidated by analyzing DNA sequences. Two techniques have been developed for this, one based on an enzymic method frequently termed Sanger sequencing and chemical method called Maxam and Gilbert sequencing.

Sanger’s Sequencing or Dideoxynucelotide Chain Terminators:

In this the reaction mixture is divided into four groups, representing the four dNTPs A, C, G and T. In addition to all the dNTPs being present in the A tube an analogue of dATP is added (2′, 3′ ddATP) is added that is similar to A but has no 3′ hydroxyl group and so will terminate the growing chain. Situation for other tubes ddC, ddG and ddT are identical except they contain ddCTP, ddGTP and ddTTp respectively.

Since the incorporation of ddNTP rather than dNTP is a random event, the reaction will produce new molecule varying widely in length, but all terminating at the same type of base. Thus four sets of DNA sequences are generated, each terminating at a different type of base, but all have a common 5′- end. The four labelled and chain-terminated samples are then denatured by heating and loaded next to each other for electrophoresis.

Electrophoresis is performed at 70°C in pres­ence of urea, to prevent renaturation of DNA. Very thin and long gels are used for maximum resolution over a wide range of fragment lengths. After electrophoresis, the position of radioactive DNA bands on the gel is determined by autoradiography.

Since every band in the track from dideoxyadenosine triphosphate must contain molecules that terminate at adenine, and those in ddCTP terminate at cytosine, etc., it is possible to read the sequence of the newly synthesized strand from autoradiograph, provided that the gel can resolve differences in length equal to single nucleotide. Under ideal conditions, sequences up to about 400 bases length can be read from one gel.

Direct PCR Sequencing:

It is possible to undertake nucleotide sequencing from double-stranded molecules such as plasmid cloning vectors and PCR products but the double-stranded DNA must be denatured prior to annealing with primer. In case of plasmids, an alkaline denaturation step is sufficient. However, for PCR products this is more problematic and a focus of much research. Unlike plasmids, PCR products are short and re-annealed rapidly, so preventing the re-annealing process or biasing the amplification towards one strand by using a primer ratio of 100:1 can overcome this problem to a certain extent.

Denaturants such as form amide or dimethylsulphooxide (DMSO) are usually em­ployed to prevent renaturation of PCR strands after their separation it is possible to physically separate and retain one PCR strands by incorporating a molecule such as biotin into one of the primer, which can be recover after PCR by affinity chromatography with streptavidin, leaving the complimentary PCR strand. Thus it provided high quality single stranded DNA for sequencing.

One of the most useful methods of sequencing PCR products is termed PCR cycle sequencing. This is not strictly a PCR, since it involves linear amplification with a single primer in about 20 PCR cycles. Radiolabeled or fluores­cent—labelled dideoxynucleotides are then introduced into the final stages of reaction to generate chain termination extension products. Automated direct PCR sequencing is increasingly being refined, allowing greater lengths of DNA to be analysed in one sequencing run.

Automated Fluorescent DNA Sequencing:

This involves dideoxynucleotides labelled with different flurochromes and are used to carry out chain termination as in standard re­actions. The advantage of this modification is that, since different labellel is incorporated in each ddNTP all the products are run on same denaturing electrophoresis gel.

Each product with their base specific dye is excited by a laser and dye then emits light at its characteristic wavelength. A diffraction grating separates the emissions, which are detected by charge couple device (CCD) and the sequence is interpreted by computer. In addition to real time sequencing, the length of the sequence that may be analysed is in excess of 500 bp.

Maxam and Gilbert Sequencing:

This is a chemical method of sequencing developed by Maxam and Gilbert and the method is often used for sequencing of small fragments of DNA such as oligonucleotides. A radioactive label is added to either the 3′ or the 5′ end of a double stranded DNA preparation. The strands are then separated by electrophoresis under denaturating conditions and analysed separately.

DNA labelled at one end is divided into four aliquots each is treated with chemicals that act on specific bases by methylation or removal of bases. Conditions are chosen such that each molecule is modified at only one position along its length and every base in the DNA strand has equal chances of being modi­fied.

After modification reactions separate samples are cleaved by piperidine, which breaks phosphodiester bonds exclusively at the 5′-side of nucleotides whose base has been modified. The result is similar to that produced by Sanger method, since each sample now contains radiolabelled molecules of various length, all with one end common (the labelled end), and with the other end cut at the same type of base. Analysis of the reaction products by electrophoresis is as already de­scribed for Sanger method.

Bgl2 - A'GATCT

Primary Function: Promoter Insertion

The Bgl2 site always flanks our promoters or promoter multiple cloning sites. We recommend using the closest unique restriction sites (e.g. AsiSI and NotI) before attempting to use the Bgl2 sites. This will increase the chances of your ligation yielding clones in the correct orientation. If you would like to insert your own promoter then you can use our promoter specific multiple cloning site plasmids (PromMCS).

Restriction site

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Situated on a pretty site, the camp consists of two lines of wooden buildings running along the shore for about a mile.

Restriction Digestion of DNA and DNA Samples | Molecular Biology

In this article we will discuss about the restriction digestion of DNA and DNA samples.

Restriction Digestion of DNA:

Restriction endonucleases cut DNA mol­ecules into smaller pieces. They cut DNA at the specific recognition sequence, so that it is no longer susceptible to cleavage by the endonucleases. These enzymes are present in vivo in bacteria and are involved in the recog­nition and destruction of foreign DNA like that of the invading phage. They recognise specific sequences called palindromic sequence in the DNA molecule (whenever that se­quence occurs in DNA) and cleave symmetri­cally both strands.

There are three major types of restriction enzymes which are I, II and III. Type I & III contain the restriction and modification ac­tivities in the same multi-subunit enzyme com­plex. They require ATP for cleavage and cleave the DNA at substantial distance from the recognition sequence. In contrast, type II endonucleases are not physically associated with the corresponding modification methylases. It does not require ATP for cleav­age and generally cleave within or very near
the recognition sequence. Because of this, type II endonucleases are preferred in cleaving spe­cific sites to generate defined fragments. There are more than 600 different commercially available Type II enzymes isolated by molecu­lar biologists. They cut only double stranded DNA and each of the specific sequence is a palindrome. These properties make these en­zymes extremely useful in RFLP and DNA finger printing.

Digestion of DNA Samples:

A typical restriction enzyme digestion reac­tion includes the incubation of a restriction enzyme with DNA in presence of an appro­priate buffer and ionic concentrate at a spe­cific temperature and duration.

1. To a clean centrifuge tube kept on ice, pipette out:

5 μl of appropriate 10x buffer.

5-10 μg of DNA sample. (Maniatis recommends 1 μg/20 μl)

Water to final volume of 50 μl.

2. Add 1-2 (typically 3 to 20 units) of desired enzyme and mix gently by tap­ping. The volume of the restriction en­zyme added should not exceed 10% of the total volume. If more enzyme is needed, the digestion should be done in a larger volume.

3. Incubate the reaction mixture at the rec­ommended temperature (for most of the enzymes it is 37°C) over night.

4. Run an aliquot of undigested DNA (without enzyme) and digested DNA on a mini agarose gel to ensure complete digestion.

5. If DNA is fully cleaved, the sample can be purified with phenol: chloroform and precipitate the cleaved DNA.

6. Add l/l0th volume of 3(M) Sodium ac­etate to the reaction mixture and twice the volume of chilled ethanol and keep at —20°C for two hours.

Creating primers to add restriction sites to vector backbone - (Apr/11/2013 )

I need to add restriction sites to where a single BamHI site sits on my backbone to be able to ligate an insert into that spot, no MCS on this plasmid. I have not done this and am wondering if anyone has experience modifying the recipient vector with additional sites? Any rules/strategies for primer design to amplify my 8kb vector by PCR and add two unique sites? Thanks.

It's as easy as any other pcr. Don't forget to add 4-6 bp of junk DNA 5' of the restriction site, to allow the enzyme to cut. Make sure you purify your pcr product before cutting with the enzymes.

The only complication is that the reverse primer anneals to a loxP site and its one of two loxP sites in the vector, so the primer is non-specific.

That is usually fatal. PCR will favor the shorter fragment. If you can extend the primer to the first base that mismatches in the two loxP sites, that is often sufficient. A single 3' base primer mismatch usually inhibits amplification.

I would do that, and have designed such primers however the reverse primer would have to be about 33bp for to get to the first mismatch. Think this is ok? I can match up the forward primer in Tm appropriately.

This should not be a problem. After the first cycle, longer primers perfectly match in any pcr which adds a 5' extension. You're just doing it a cycle earlier.

Thanks for the advice! I will try that. Seems easier to create new sites on my vector backbone that deleting a restriction site in the middle of my insert that I need for ligation.

Yes, and there's lower background from uncut circular plasmid transforming.

Restriction-site PCR: a direct method of unknown sequence retrieval adjacent to a known locus by using universal primers

Fast acquisition of unknown nucleotide sequences around a known sequence has important implication in molecular biology, especially in genome mapping. We have developed a method, termed restriction site polymerase chain reaction (RS-PCR), that utilizes specially designed primers that recognize, anneal, and sustain PCR. These primers, termed restriction site oligonucleotides (oligonucleotide primers specific for a given restriction enzyme recognition sequence or RSOs), could be generated corresponding to any restriction enzyme irrespective of the length of the recognition site and used as PCR primers corresponding to the unknown region of a DNA segment. In this method a first round of PCR is carried out in different tubes with a set of RSOs and a primer specific to the known region. A second round of PCR is then performed on the products of the first PCR with the same RSOs and another specific primer internal to the first one. Subsequently, the products of the last round of PCR are transcribed with an appropriate RNA polymerase and sequenced with a reverse transcriptase with an end-labeled specific primer internal to the second specific PCR primer. To demonstrate the applicability of RS-PCR in retrieving unknown sequences around a known sequence, we have used a set of four RSOs and three specific primers representing the known sequence and have successfully obtained hitherto unknown factor IX sequences (12 of 12 times) from three species starting from genomic DNA. The sequences obtained indicate the presence of a conserved stretch of 20 nucleotides in the 3' noncoding region of the factor IX gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Frequency of restriction enzyme sites in a genome - (Mar/18/2011 )

Hi all
I want to check one enzyme that on the average how many sites are there for this particular enzyme in human or mouse genome. is there any tool available for that?

Well you can usually calculate it yourself, take the length of the genome, and divide by how often an enzyme cuts on average. For example EcoRI cuts at a 6bp site, the frequency of cutting is 4^6, so it cuts every 4096bp on average, as it's sequnce will occur at random every 4096bp.

This does mean that in a genome of several billion such as humans, it will cut millions of times.

philman on Fri Mar 18 14:31:55 2011 said:

Well you can usually calculate it yourself, take the length of the genome, and divide by how often an enzyme cuts on average. For example EcoRI cuts at a 6bp site, the frequency of cutting is 4^6, so it cuts every 4096bp on average, as it's sequnce will occur at random every 4096bp.

This does mean that in a genome of several billion such as humans, it will cut millions of times.

philman on Fri Mar 18 14:31:55 2011 said:

Well you can usually calculate it yourself, take the length of the genome, and divide by how often an enzyme cuts on average. For example EcoRI cuts at a 6bp site, the frequency of cutting is 4^6, so it cuts every 4096bp on average, as it's sequnce will occur at random every 4096bp.

This does mean that in a genome of several billion such as humans, it will cut millions of times.

I dont think thats a valid method bcaz there are enzymes that cut with more frequency, even if they have same recognition sequence length. for example EcoRI cuts 3 times more frequently than MsPI

As a general rule that method is roughly correct, though it does vary. You can use it to estimate the general number of cut sites. If you want to know exactly, then you will have to get the genome and run it through a program like enzymeX or NEBcutter.

You can get a much more accurate estimate if you take into account the probability of GC and AT pairs independently. If the GC content of the organism is (say) 70%, and the recognition site of the enzyme is GAATTC (EcoRI site), then the probability of its presence will be (.35)(.15)(.15)(.15)(.15)(.35) = 6.2e-5, since the probability of G is half of the probability of GC, and the probability of A is half of the probability of AT.

Instead of the naively calculated 4096 bp between sites on average, the 70% GC content version will have an expected distance of 1/6.2e-5 = 16,129 bp.

This still won't be exact, nor does it account for digraphs or special genome sequences.

Restriction sites - Biology

A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (usually 20+) restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique. In other words, they only occur once within that particular plasmid. MCSs are commonly used during procedures involving.
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Watch the video: How Do Restriction Enzymes Interact With DNA? (September 2022).


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