Polymerase chain reaction
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[edit] Purpose
The polymerase chain reaction or PCR is a method used to isolate a specific sequence of DNA present on a strand or amplify an existing strand. It is a way of locating and purifying selected small areas in vast amounts of whole genomic DNA. Usually specific sites, either matching or surrounding the DNA of interest, are amplified and hybridized with whole DNA.
There are numerous reasons for purifying and exponentially reproducing a specific DNA fragment, usually falling into one of these there categories:
1) Comparing a known condition causing DNA sequence with a specific subject's DNA in order to confirm the presence or absence of the sequence. gemscool
2) Using a known stretch of DNA to look for related sequences, or to look for the presence or absence of that sequence in other populations
3) Using PCR to amplify DNA that was derived from a specific cell type's mRNA (actively expressed genes) to view/compare expression.
[edit] PCR Process
PCR consists of 3 steps: denaturing, annealing and extension. In this process the DNA is unpaired and the solution is heated in order to separate the two strands of DNA. Special primers which selectively bond to the target sequence, which is the segment of DNA to be copied are added. This allows the DNA polymerase to begin synthesizing the new DNA strands. Once the new strands of DNA have been synthesized the solution is heated again and the process repeated for a determined number of cycles. PCR is generally carried out using a machine called a thermocycler, which heats and cools the DNA throughout the programmed cycles. Common temperatures for PCR include a high temperature (94 degrees) for separating the DNA, 74 degrees for active amplification, and 4 degrees to hold the DNA after the process is complete. Cycle temperatures and length will vary a little bit based on the nature of the gene and test being tested. gemscool
[edit] PCR Components
- Primers (stretches of DNA surrounding the area of interest for PCR, that allow the polymerase to bind and start copying the DNA)
- nucleotides for building new DNA
- Polymerase Enzyme to make DNA copies
- Template DNA
- Buffer
- MgCl2
- There are also a few components you can add to stabilize the reaction and favor amplification of the chosen product over primer dimers or other homoduplexes.
- It is also very common to dilute DNA before combining it with the other components and moving the mixture to the thermocycler.
A special thermostable DNA polymerase called Taq polymerase is used because ordinary polymerase would be deactivated in the high temperatures required for this process.The number of new DNA strands synthesized increases exponentially every time the process is repeated. Billions of copies of the target sequence can be produced with this method.
The PCR process wasn't developed until 1985 because until then there were no known DNA polymerases that could withstand the high temperatures required for the process. A thermostable DNA polymerase was eventually discovered in the thermophilic archaea, Thermus aquaticus found in the hotsprings in Yellowstone National Park, USA.
[edit] Types of PCR
RT PCR: Uses reverse transcriptase to get DNA versions of mRNAs from a given cell type
Anchored PCR: Uses a known sequence as an anchor to amplify an unknown sequence next to it.
Hot Start PCR: Mixing the PCR reagents at high temperatures in order to avoid non specific binding that happens prior to the first hot denaturing step.
Real Time PCR: Uses fluorescence to detect the concentrations of the various products during the reaction.
Whole Genome PCR: Using linkers attached to fragments of genomic DNA to replicate all parts of the genome.
[edit] Limitations
Taq polymerase will not exclusively amplify the target DNA. Great caution must be exercised to make sure PCR reaction tubes are not contaminated with other DNA. Also, pre PCR mixtures must not be exposed to UV or any DNAse enzymes. Very frequently, laboratories will have separated pre and post PCR areas, with a separate set of equipment for each. PCR mixtures are generally mixed in a bio hood.
