DNA polymerase in PCR (polymerase chain reaction)

DNA polymerase in PCR (polymerase chain reaction)

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Can the DNA polymerase in PCR (polymerase chain reaction) recognize both DNA and RNA for use as a template?

I want to know is it possible if my primers bind to contaminant RNA and then any DNA polymerase like Taq DNA polymerase use this strand as the template for elongation?

No, this does not happen, as the DNA polymerases used for PCR are DNA dependent. This means that they only synthesize DNA when it is bound to DNA. Even if your primers bind to the RNA, the polymerase will not starting new strands here. To use RNA as a template for PCR you first need to reverse transcribe it.

Polymerase Chain Reaction Notes

The principle of PCR is based on the enzymatic replication of nucleic acids. PCR involves the use of primer mediated enzymes for the amplification of DNA. DNA Polymerase synthesises new strands of DNA complementary to the template DNA. The DNA polymerase can add a nucleotide to the pre-existing 3’-OH group only. Therefore, a primer is required. Thus, more nucleotides are added to the 3’ prime end of the DNA polymerase.

DNA polymerase fidelity and the polymerase chain reaction

High-fidelity DNA synthesis conditions are those that exploit the inherent ability of polymerases to discriminate against errors. This review has described several experimental approaches for controlling the fidelity of enzymatic DNA amplification. One of the most important parameters to consider is the choice of which polymerase to use in PCR. As demonstrated by the data in Tables 2 and 3, high-fidelity DNA amplification will be best achieved by using a polymerase with an active 3'-->5' proofreading exonuclease activity (Fig. 1E). For those enzymes that are proofreading-deficient, the in vitro reaction conditions can significantly influence the polymerase error rates. To maximize fidelity at the dNTP insertion step (Fig. 1A,B), any type of deoxynucleoside triphosphate pool imbalance should be avoided. Similarly, stabilization of errors by polymerase extension from mispaired or misaligned primer-termini (Fig. 1D) can be minimized by reactions using short synthesis times, low dNTP concentrations, and low enzyme concentrations. Additional improvements in fidelity can be made by further manipulating the reaction conditions. To perform high-fidelity PCR with Taq polymerase, reactions should contain a low MgCl2 concentration, not in large excess over the total concentration of dNTP substrates, and be buffered to approximately pH 6 (70 degrees C) using Bis-Tris Propane or PIPES (Table 2). These buffers have a pKa between pH 6 and pH 7 and a small temperature coefficient (delta pKa/degree C), allowing the pH to be maintained stably throughout the PCR cycle. For amplifications in which fidelity is the critical issue, one should avoid the concept that conditions generating more DNA product are the better conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Routine PCR is used to produce an amplified amount of DNA for downstream applications, including clone length verification. Our products offer a variety of options to perform routine PCR. Our ReadyMix™ PCR reaction mixes and REDTaq ® dye options provide an added convenience for your amplification reaction.

  • Taq DNA Polymerases
  • PCR Reaction Mixes
  • Roche AptaTaq Fast Start DNA Polymerase
  • Roche Expand™
  • Roche FastStart™ Taq

BioChain’s dNTP Mix Products

The dNTP products and mixes provided by BioChain are specially manufactured and tested for molecular biology applications. They can be used in PCR, RT-PCR, DNA labeling, and DNA sequencing processes. The dNTPs are purified with preparative HPLC and possess at least 99.5% purity.

Rigorous control standards and state-of-the-art technology ensures the best quality of product. The concentration is verified by optical density spectrophotometry. Preparation is careful to avoid DNase and RNase contamination. This is determined by incubation of the quality with radioactive substrates.

Each lot of dNTP is also tested for performance with Taq/Pfu/T7 DNA polymerase and Sequenase. Further, dNTP has been performance tested in BioChain’s on-site lab by long PCR to amplify 15 kb (E. coli DNA template) and 30 kb (Lambda DNA template).

Individually separated base nucleotide mixes are available, along with Hotstart Taq DNA polymerase reagents for all PCR needs.

BioChain offers the most competitive price on the market. Discount for large quantity/bulk purchase is available.

Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction

Polymerase chain reaction (PCR) has been a defining tool in modern biology. Towards realizing mirror-image PCR, we have designed and chemically synthesized a mutant version of the 352-residue thermostable Sulfolobus solfataricus P2 DNA polymerase IV with l-amino acids and tested its PCR activity biochemically. To the best of our knowledge, this enzyme is the largest chemically synthesized protein reported to date. We show that with optimization of PCR conditions, the fully synthetic polymerase is capable of amplifying template sequences of up to 1.5 kb. The establishment of this synthetic route for chemically synthesizing DNA polymerase IV is a stepping stone towards building a d-enzyme system for mirror-image PCR, which may open up an avenue for the creation of many mirror-image molecular tools such as mirror-image systematic evolution of ligands by exponential enrichment.

Keywords: Sulfolobus solfataricus P2 DNA polymerase IV native chemical ligation polymerase chain reaction protein chemical synthesis thermostable.

Conflict of interest statement

The authors have filed a provisional patent application related to this work.

Purpose of PCR / Why Do we do PCR

To have the reaction performed nicely, the native Deoxy Ribonucleic Acid strand that is going to be replicated/copied required not necessarily to be refined or in excess.

It is most probably be a super minute particle in a material micture. So PCR is recognized to have the innumerable & widespread uses to figure out genetic disorders, bacterial & viral disorders, evolution study, DNA cloning, and fingerprinting.

PCR is a core tool for biologists and researchers in certain types of laboratories including forensic, pathology genetics, and virology.

Polymerase Chain Reaction

Polymerase Chain reaction is a technique ubiquitously found in molecular biology labs worldwide and allows biologists to selectively amplify a single molecule of DNA. The utility of PCR is tremendous with uses in DNA forensics, functional analysis of genes, diagnosis of viral and bacterial diseases, genetic testing for hereditary disease and etc. The full utility of PCR is still being explored as new variations of PCR still continue to be published in scientific journals worldwide.

Polymerase Engineering

Given our current understanding of DNA polymerase and replication, the selection of polymerases available for PCR and amplification are tremendous. Among them, NEB remains one of the largest suppliers of polymerases for molecular biology labs. Check out the New England Biolabs’ catalogue of polymerases to see what advancements have made in polymerase engineering.

PCR Overlap Extension

PCR overlap extension is a method to assemble multiple linear fragments of DNA together and remains a powerful technique to assemble synthetic circuits. PCR extension utilises two sets of primers to fuse two DNA fragments together. The first reaction utilises primers to add homologous end to each DNA fragment, allowing them to anneal together to form the fully assembled construct. The second reaction serves to amplify the full DNA construct.

If you still have some questions about PCR overlap extension, check out the paper below:

Primer Design Tools

Need help designing primers? Here are some helpful resources to guide you in designing any primers for PCR.

Primer3Plus – This software automatically generates primer sequences for a given nucleotide sequence. Very helpful, when designing primers to amplify large genes

IDT oligoanalyzer – this program analyzers your primers and determines if your primers form any hairpin loops which may interfere with the PCR reaction

NEB Tm calculator – This website calculates the melting temperature of your primers and recommends temperature settings for your reaction. Scientists often try to design primers with close annealing temperatures to maximize the efficiency of their primers

Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) is a biochemical technology in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

Developed in 1983 by Kary Mullis,[1][2] PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.[3][4] These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes the diagnosis of hereditary diseases the identification of genetic fingerprints (used in forensic sciences and paternity testing) and the detection and diagnosis of infectious diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR.[5]

The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.

Figure: Schematic drawing of the PCR cycle. (1) Denaturing at 94–96 °C. (2) Annealing at

65 °C (3) Elongation at 72 °C. Four cycles are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA polymerase (light green circles), to give shorter DNA products (green lines), which themselves are used as templates as PCR progresses

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample through a defined series of temperature steps. In the first step, the two strands of the DNA double helix are physically separated at a high temperature in a process called DNA melting. In the second step, the temperature is lowered and the two DNA strands become templates for DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.

1. Bartlett, J. M. S. Stirling, D. (2003). "A Short History of the Polymerase Chain Reaction". PCR Protocols 226. pp. 3–6. doi:10.1385/1-59259-384-4:3. ISBN 1-59259-384-4. edit

3. Jump up to: a b Saiki, R. Scharf, S. Faloona, F. Mullis, K. Horn, G. Erlich, H. Arnheim, N. (1985). "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia". Science 230 (4732): 1350–1354. doi:10.1126/science.2999980. PMID 2999980. edit

4. Jump up to: a b Saiki, R. Gelfand, D. Stoffel, S. Scharf, S. Higuchi, R. Horn, G. Mullis, K. Erlich, H. (1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science 239 (4839): 487–491. doi:10.1126/science.2448875. PMID 2448875. edit

Module 1 : DNA Tools and Biotechnology

Let us switch here and talk about another revolutionary technique, which is polymerasechain reaction or PCR, something very similar to gene cloning but here you are using somechemicals to make multiple copies of a given gene.A scientist which is we have, who has made, it has a mile contribution to this field,Kary Mulis in 1984, he first time discover this process polymerase chain reaction andsome of these discoveries happen sometime accidentally, if you remember I were mentioningto you in you know old environment when we had these RK bacteria and some of the veryyou know, the organism living in the extreme conditions especially, you know the halophilesand some of the thermophiles.Those who are living in the very extreme hot condition, if you observe those particularorganism you might be able to derive certain properties from it and therefore, Kary Muliswas able to isolate some bacteria from a very hot spring of the sulphur hot springs fromwhich he was able to get some bacteria, thermus aquaticus who can withstand and still reproduceat 95 degrees, 100 degrees temperature, so now these enzymes gives that bacteria thatproperty, which can actually make it still live in those hot temperature.So, he used isolated that enzyme, taq polymerase and that was actually better instrumentalin developing this particular technique known as polymerase chain reaction.So, broadly in polymerase chain reaction, you have 3 processes one is the DNA strandsto separate, your double-stranded DNA, you will heat them, denature them, so that thedouble-stranded DNA become single standard, they get separated.Then you want to have some primers or a short stretch of nucleotides, which will bind onboth the opposite end of the pair from 5 degree 5 primes to 3 primes and then you want tohave a DNA synthesis using taq polymerase, all the nucleotides, which are required magnesiumchloride etc. everything you add in the reaction mix.So, the success of polymerase chain reaction came from the key discovery of knowing abouttaq polymerase which was isolated from thermus aquaticus, living in the hot hot springs.The stability of this DNA polymerase at very high temperature was very useful to derivethis process of polymerase chain reaction because this bacteria was able to live andreproduce even at 95 degrees temperature.And therefore, the enzyme taq polymerase which was isolated from it became very useful forthis process.So, broadly these are the 3 steps which happen in a polymerase chain reaction, first youhave the double-stranded DNA which you are denaturing at very high temperature 95 degreesor 100 degrees and now, this becomes 2 single standard DNA.Then you are adding a short stretch of nucleotides, which can hybridised to the complimentarybase pairing rule in the same manner.And now, from both the sides, you are giving you know that situation where now the secondstrand of the DNA can be synthesised, to do this part you are adding the taq polymerase.You are also providing the right temperature here which is annealing temperature and youare adding all the dNTP’s, or the nucleotides which are required for the synthesis.This thing is happening as the part of extension, when now a new a strand is being formed, sonow the single-stranded DNA became double-stranded and one copy of DNA became 2 copies of DNAhere.In this whole thing what is most important to understand is primers, what are the primersyou have an idea or understanding for what are the primers?Alright, these are if you are a large gene sequence, within the large gene sequence,you do not amplify the full gene, you might want to amplify a, you know a large regionof that.So, you are finding some region which can be used to amplify that the gene and you aresynthesising some nucleotides which are having some complimentary opposite base pairs.And you are using those short stretch of nucleotides which could be 18 to 28 nucleotide lengthas a starting point for the DNA synthesis to happen, now these you are you know, insome way, you are just adding ATGC and putting that based on the complimentary sequence ofDNA, so you want to ensure that you are picking up the sequence from a region which is nothaving too much GC contents or you are not having the same base pair in multiples presentlike you know, not A is continuous or G is continuous present.So and they should not be self-complimentary as well, one more important thing here itis known as the Tm or the melting temperature because if you go back, this process whichis annealing, you are giving a specific temperature for this primer to bind to the DNA strandsand this happens at a specific temperature which is known an annealing temperature, soyou can actually calculate what can be possible annealing temperature by looking at calculatingthe AT and GC contents.So, this is the formula for doing that you can have A + T times 2 + G and C contentstimes 4 that will give you the Tm value and that Tm value could be 65 degrees or 70 degrees,can be used as annealing temperature, so initially you used a very high temperature which wasfor denaturation, then you are reducing the temperature down now, you are bringing a primerto bind to that particular DNA strands using annealing temperature.And then you on to extends its further, again you are changing temperature around 72 degreefor the taq polymerase to work.So, let us look at one of the sequence and let us assume that you want to design theprimers for this particular gene sequence and you want to amplify this you know, startingfrom this arrow region to this arrow region, you want to amplify their gene, so for boththe DNA strands, we have mentioned here the, the full sequence here, so for the forwardprimer you have to have the complimentary opposite base pairs.So, if A is here or the G is here, what will be the complimentary base pair?C, right, if you have A then, C then, what will be the complimentary, so can you startwriting about what can be the forward primer sequence, please write that that will in the5 prime to 3 prime direction, this is a gene sequence we have on to amplify the gene fromthis part here, to do this we are saying that you can start making a primer, you can synthesisea primer that will be the forward primer will be complimentary to this particular segmentwhich you want to amplify.So, if you have just opposite sequence of that in 5 prime to 3 prime direction thatcan be your forward primer, now the reverse primer is much more is simpler and easy becausewe are deriving everything from 5 prime to 3 prime, you are synthesising in fact, chemicallysynthesising the primers, we now know how to synthesise chemically ATGC bases, so aprimer nucleotide sequence can be synthesised, let comes in the powder form.And then, you can actually you know add some water to make your primer mix, so now everythingyou want to have always in the 5 prime to 3 prime direction this is what you have usedhere for the forward primer.Now, if a opposite primer you want to design from this part here, it become much more simplerbecause you are just writing the sequence of the other DNA strand in this case for thereverse primer.And therefore, your sequence for the reverse primer will become a starting from C, it willbecome CAT GCC, A and you can continue with that.Are you with me?So, you want to amplify a given gene segment and I have shown you the arrows from thispart to this part you want to amplify that DNA, to do that you are adding a smalleststretch of nucleotides which you want to chemically synthesise along with those chemical synthesisedprimers, you will add the enzyme, you will add nucleotides, you will make the mixtureeverything provide the right temperature conditions inside the instrument.And perform polymerase chain reaction, so that your DNA can keep multiplying multiplecopies that is the intention here.To do this, the forward primer you have taken from 5 prime to 3 prime directions, you havejust use a complimentary sequence of it and you got this particular sequence derived forthe forward primer.Reverse primer the opposite strand of this which we have use for the DNA and becausewe have to derive in 5 prime to 3 prime in formation, we are just simply writing thesequence from the C 80 onwards.So, this is where you can synthesise and design these primers now, if I am giving you thisparticular primer sequence which is from the 5 prime to 3 prime, what will be the meltingtemperature, given that you have this formula which will not be shown in the exam, whatwill be the melting temperature Tm for this particular primer, a straight forward, justcount A, T’s, G’s and C’s.So, if this was a temperature to be used for PCR, so the second condition which is annealing,you are going to use 64 degrees for annealing because you have some theoretical ideas thatthis is the right temperature how my base pairs will have the best annealing or thebinding conditions, so you will use 64 degrees for the annealing condition to happen.Yes, “Professor – student conversation starts”.Alright, I think his question is right that you know let us say, you have derived sometheoretical value of 64 degrees, how exactly it actually will help into amplifying thatgene of interest, that is way very I think you know, practical question theoretically,you should see a ban amplified because of the Tm but usually, you know plus minus 2degrees can happen, so sometime that you are deriving 64 degrees that it may happen 66degree can be the right temperature for that gene to amplify, it is possible.So, you have to play with certain temperature conditions to find out what is the best temperaturefor your gene of interest to bind.“Professor – student conversation ends”.So, now you had started from the double helix DNA, the double-stranded DNA, after doingthe denaturation, you got the single stranded once, you added this primers which are theshort stretch of nucleotides which you have designed yourself.Because you wanted to study their gene of lamin A for example and now you are amplifyingtheir gene of interest now, you have added dNTP’s and nucleotides and providing theright temperature conditions, so that the nucleotides are getting synthesised and newstrands are being made, so now this became double-stranded DNA, this whole thing is onlyone cycle of PCR.Now, same way you are repeating the PCR cycle second time, third time and now you can doit n number of times.Ideally, people go for at least 30 to 35 or 40 cycles to make multiple copies of the geneof interest, so just imagine that after each cycle of performing the polymerase chain reaction,you are generating n number of fragments and therefore, for many of the forensic applications,think about any kind of you know, the crime scene, when there is some heroine is fallenor some sort of blood spot is detected over there, you do not have too much DNA to dolot of investigation.So, they use only the small part of those you know, the DNA extracted out of those biospecimens and then amplify those using these kind of conditions with the polymerase chainreaction, so that they have enough of the DNA, to then do further testing which canresult into very accurate deduction.So, we are performing here multiple cycles in any of the polymerase chain reaction.A 3 steps cycle brings about the chain reaction which produces the DNA chains in the exponentialmanner and after each successive cycle, you will have the target sequence which will doublethe numbers and these numbers will doubled 2 to the power n, so if you have done 30 cycleor 40 cycle, ideally, it looks only 10 cycle difference but if you think about 2 to thepower 30 or 2 to the power 40, there is a huge number of difference in how many copiesyou are producing for that gene of interest.So, once you do the PCR, there are many things to be optimised, of course as somebody rightlymentioned, you have to look at the annealing temperature, what is the best temperaturein which your primers are going to bind and you may have to play within a range of temperaturesfrom 60 to 65 or 70 to find out where your gene binds the best with the primers and thenyou have to see that at which cycle numbers, you can still see enough of the DNA beingproduced.So, then what you can do after doing the polymerase chain reaction, you can run yoursamples on the gel, so many time people do this gradient PCR, where they will use differentgradient of temperature and now, they will run the each PCR condition, let us say startinghere 60, 62, 64 and 66 and these are my lanes and what I am finding it you know, the veryfine band is appearing at 62 probably, you know a good band I can see at 64.So, then probably this 64 is the right temperature for me to take my experiments forward, sothis how people first tried to visualise where there you know the primers are going to getbest bind to the, the gene of interest and now, once you have done that then, you willdo 30 or 35 or 40 cycles to amplify and make enough number of copies of the gene of interest.Alright, so within a few hours of doing this PCR or polymerase chain reaction, you canactually amplify your DNA sufficient, so that you can make multiple copies of that specifictarget and then you can do lot of gene testing based on the amplify DNA, which is presentin the given sample.Now, just imagine that you know, so far we have been talking about all the things happeningat the gene level, now let us think about you wanted to study in aberration or the changehappening at the protein level especially, if you think about the context of progeria,there was a protein which was lamin A, which is defective, coming because of the defectsfrom the lamin A gene, so if you think about the, the DNA sequence, all this triplet codonsare going to make one amino acid.So, let us say we have glycine from triple G, phenyl alanine from TTC and like that wehave you know multiple amino acids derived from this triplet codons sequence, so the3 letter nucleotide, they are corresponding to a given amino acid sequence and these nucleotidesequences could be translated to give you amino acid sequence or looking at the polypeptidechain of that given protein.So, if you want to study let us say you know, change happening at the protein level fromthe same kind of cloning experiment and the same idea of what we have discuss for thedoing cloning, can you now think about, can you study those change at the protein levelthat something I think you have to now pay attention.So, let us imagine that you have a particular protein which you want to study and becausein that protein, there is some change happening at one amino acid level and that each aminoacid is derived from the triplet codon of the gene sequence, so if you are looking atthat gene sequence for example, TCT is the gene sequence for serine and you are justreplacing a C with the G therefore, the TCT becomes TGT, which becomes another amino acid,which is 16.Just by changing one base pair, you have changed a triplet codon, you have changed from oneamino acid to other amino acid, which will introduce so much change in the living system,rght, so now if you think about even when you are designing the primer, even when youare studying the thing at the gene level, if you want to introduce the changes at theprotein level, subsequently you can think about what changes you can make in the tripletcodons, which may result into the changes at the protein level, right.So, if you had this particular template is strand, if this is the strand, which is normaland now, you want to create a protein which is having slight difference only with oneparticular place, now this one nucleotide you have made a change and now, a proteinswhich is going to be derive from it or amino acid sequence going to be derived from it,will have mismatch, will have different as compared with the parental strand, right.So, this is how if you, you are designing the primers at the site of primer designingitself, you can make some small changes and those may result into the variations whichcan be seen after doing the cloning, then you can see those changes happening even atthe protein level, so to study the particular proteins, here still people have used bacterialas the system and just imagine it is very complex concept because think about we areyou know, eukaryotes and very complex human system.And now, for our human proteins to grow, we are still using bacterial system, which isprokaryotic system right, so but, but somehow with the genetic engineering, we have beenable to overcome these barriers and we are still able to even grow, the proteins of ourinterest in the bacteria, so proteins of eukaryotic interest into prokaryotic origin.So, the cells express the different versions of the proteins and result into the phenotypeswhich can actually inform about the normal versus the abundant function of the givenproteins.And we can use those information to express eukaryotic proteins in bacterial system although,it is very challenging and is still a question that how different eukaryotic and bacterialcells are and how you can use the bacterial system to even make the human protein or theeukaryotic protein that is really a challenging question, so if you want to use the bacterialsystem to express a given protein of interest, you have to use certain promoters which aregoing to overcome this problem in the expression vector.So, expression vector is a cloning vector which contains certain bacterial promoterwhich can provide the eukaryotic genes in the correct reading frame, so there are lotof difference in the prokaryotic and eukaryotic system and of course, you would not assumethat your eukaryotic proteins are going to made in bacteria very correctly and goingto be properly folded but somehow, you are still trying to use certain expression vectorssay, some of the cloning vectors where you have some of the promoters inserted upstreamof the restriction site which help you to at least put the things in the right frame.So that the right amino acids can be synthesised based on the that particular expression,so bacterial host cell would recognise a promoter and express a foreign genes or the eukaryoticgenes which are linked to that promoter.So, now let us take this particular situation which is really, really complex situationbut we want to study a complex mutant protein, think about this part which is the cloningpart which we have already pretty much familiar with now, right so, all the things which Ihave talked so far is now summarise in this particular image, let us pay attention tothis image and see which are all things which we can discuss from here.So, we have discussed, we had a plasmid vector where you wanted to insert a gene of yourinterest, you made this recombinant DNA and now you have done the transformation, youselected that and now you got the bacterial colony which are having your gene of interestright, this is what we discussed earlier.Now, this DNA which is a fragment, now this particular DNA you can also amplify usingpolymerase chain reaction, if you have very small copy of that DNA you can make multiplecopies of it using PCR.For doing PCR or polymerase chain reaction, you had use certain primers which are fromboth the sides right, you have anneal certain primers and those primers are the one whichare going to amplify the gene of interest, now in the primer sequence itself, if youcan introduce some variation, which can change your triplet codon, so that your protein whichare going to be synthesised from it will have some changes.You already know from which codon, what amino acid is going to be synthesised, so if youonly make even one change in the base pair of the primer sequence, even that will resultinto mutation or different change, so therefore at the primer designing level itself, peoplecan do lot of innovations, lot of ideas comes that you want to study a different form ordifferent gene, now you can make those changes at the primary designing level.Now, you do PCR, so your gene of interest will now contain certain added base pair orcertain less base pairs, right that you can do using polymerase chain reaction and onceyou have done that then, the rest of the step of cloning remain same, now you can have restof the step in the exactly same format, the way we have been discussing.Now, all of these things whatever we are talking for the DNA work everything you have to relyon your simple electrophoretic apparatus.You have to amplify your gene, you have to run on the gel and you have to see that were,what the size of this particular band is am I able to amplify by right gene, now letus say, if you have made a change in the gene because of the primer sequence which you haveadded, now is there some amplification you can see or some deletion you can see, a smallbase pair change, those you can against monitor on the agarose gels.So, these are the kind of some certain you know technique which are very interestingfor us to study, do you have a PCR polymerase chain reaction, alright, so shortly, we aregoing to show you a thermo cycler, the instrument which is very simple you know, in generalinnovation, it is like 3 simple thermo states and in those thermo states, you are just veryprecisely changing the temperature, so while PCR looks like you know a big technique.But you know shortly, we are going to show you the instrument, the polymerase chain reaction,it is very simple instrument thermo cycler, where we are just precisely regulating ourtemperature first initially, you are heating it at very high temperature, 100 degrees,then you are lowering the temperature based on your annealing temperature could be 55or 60 degrees and then, you are again doing extension at 72 degrees.So, by using these temperature changes, you are able to synthesise DNA using PCR, so itis again a very simple small instrument but with just works on a very much precision ofthe temperature and that has to be monitor.

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