Presentation on theme: "Polymerase chain reaction"— Presentation transcript:
1 Polymerase chain reaction Who would have thought a bacterium hanging out in a hot springin Yellowstone National Park would spark a revolutionary newlaboratory technique? The polymerase chain reaction, now widelyused in research laboratories and doctor's offices, relies on theability of DNA-copying enzymes to remain stable at hightemperatures. The polymerase from Thermus aquaticus (Taq),a bacterium from Yellowstone can produce millions of copies ofa single DNA segment in a matter of hours. In nature, most organismscopy their DNA in the same way. PCR mimics the natural process,only it does it in a test tube. When any cell divides, enzymes calledpolymerases make a copy of all the DNA in each chromosome.The first step in this process is to "unzip" the two DNA chains ofthe double helix. As the two strands separate, DNA polymerasemakes a copy using each strand as a template.
2 The role of the primerTo copy DNA, polymerase requires two other components:1. a supply of the four nucleotide bases2. a primer.DNA polymerases, whether from humans, bacteria, orviruses, cannot copy a chain of DNA without a shortsequence of nucleotides to "prime" the process, or get itstarted. So the cell has another enzyme called a primasethat actually makes the first few nucleotides of the copy.This stretch of DNA is called a primer. Once the primeris made, the polymerase can take over making the rest ofthe new chain.
3 Step I: DNA meltingThe three parts of the polymerase chain reaction arecarried out in the same vial, but at different temperatures.The first part of the process separates the two DNAchains in the double helix. This is done simply byheating the vial to 95 oC for 30 seconds.double-stranded DNA single-stranded DNA
4 Step II: Primer annealing The primers cannot bind to the DNA strands at such ahigh temperature, so the vial is cooled to 55 oC. Atthis temperature, the primers bind or "anneal" to theappropriate location in the DNA strands.This takes about 20 seconds.single-stranded DNA + primer annealed DNAThe final step of the reaction is to make a completecopy of the templates. Since the Taq polymerase worksbest at ca. 75 oC, the temperature of the vial is raised.
5 Step III: Primer extension The Taq polymerase begins adding nucleotides to theprimer and eventually makes a complementary copyof the section of the template that lies between theprimers. This completes one PCR cycle.primer-annealed DNA primer-extended DNAwith Taq polymerase anddATP, dTTP, dGTP, dCTP
6 Thermal cycling At the end of a cycle, each piece of DNA in the vial has been duplicated. Since the cycle can be repeated30 or more times and each newly synthesized DNApiece can act as a new template, one can obtain 230 orca. 1 million copies of a single piece of DNA.amplified regionNote that the region of the DNA between the twoprimers will be amplified. The flanking sequencesnot. The entire process takes about three hours.
7 PCR amplification The figure on the left shows the series of steps in a single cycle. The exponential growth ofthe double helical segment between the twoprimers is illustrated above.
8 Taq polymeraseTaq Polymerase In Complex With Tp7, An Inhibitory Fab