Molecular Biology

What is Polymerase Chain Reaction

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Polymerase chain reaction (PCR) is a revolutionary molecular biology technique that is currently used in a wide array of medical and biological laboratories for a variety of applications. Introduced in 1983 by Kary Mullis, it is a technique that employs the basic machinery of cellular DNA replication to amplify a specific sequence of DNA. As DNA samples are generally very small, the amplification of a DNA sequence is necessary for further analysis and experimentation. PCR is used for many downstream applications such as hybridization probes for Southern or northern hybridization, DNA cloning, DNA sequencing, genetic fingerprinting, and many more.

The DNA sequence to be amplified can be a single gene, multiple genes, part of a gene or a non-coding sequence, usually up to 10 kilo base (kb) pairs. Some PCR methods allow the amplification of larger DNA fragments, up to 40 kb. PCR requires specific equipment, conditions and reagents that include:

1. DNA template that contains the DNA sequence to be amplified (may be genomic or plasmid in origin)
2. Oligonucleotides (or primers) that are small stretches of DNA that are complimentary to the 5' and 3' ends of the target DNA
3. Deoxynucleoside triphosphates (or dNTPs), the building blocks of DNA used to produce the new strands of DNA
4. A thermo-stable DNA polymerase, usually Taq polymerase, an enzyme isolated from the bacterium Thermos aquaticus that "fills in" the DNA strand in between the primers using the dNTPs
5. PCR buffer, providing an optimum environment for DNA polymerase to perform
6. Divalent cations, usually magnesium
7. Monovalent potassium ions
8. Thermal cycler

PCR usually occurs in 10 - 100uL reaction volumes in small, thin-walled tubes which are placed in a thermal cycler. The thermal cycler heats and cools the tubes according to the required temperature for each reaction step in a cycle. The thin walls of the tubes provide thermal conductivity for faster thermal equilibrium, providing a more efficient amplification. PCR consists of 20 to 40 repeated temperature changes called cycles. Each cycle has 3-5 reaction steps of differing durations and temperatures. The steps in a PCR method include:

Initial denaturation step: This step is only required when the DNA polymerase requires heat activation by "hot-start PCR". It consists of heating the reaction to 94 - 96C for 1 to 9 minutes. This step is not included in the cycling procedure.

Denaturation step: This is the first step in the cycle and consists of heating the sample to 94 - 96C for approximately 30 seconds. This causes melting of the template and primers, disrupting the hydrogen bonds between the base pairs, yielding single-stranded DNA.

Annealing step: This step lowers the temperature of the reaction to approximately 3 - 5C below the melting temperature of the primers being used. This temperature is usually 50 - 65C and allows the primers to anneal to the template DNA. Hydrogen bonds between DNA strands only form when the sequences of the primers and the template DNA are complimentary. DNA polymerase binds to the primer-template hybrid and begins DNA synthesis.

Elongation step: The temperature for this step is dependent upon the DNA polymerase being used. Commonly, an elongation temperature of 72 - 75C is used for Taq polymerase. During this step, the DNA polymerase synthesizes a new DNA strand by adding dNTPs that are complementary to the DNA template in a 5' to 3' direction. The elongation time is dependent upon the DNA polymerase and the length of DNA to be synthesized. Generally speaking, most methods call for 1 minute of elongation time for every 1000 base pairs.

Final elongation step: This is a single step that occurs after the last cycle has finished at the elongation temperature for 5 15 minutes. This ensures that any remaining single-stranded DNA is fully elongated.

To analyze the amplified product, agarose gel electrophoresis is usually employed to separate the DNA fragments according to size. The size of the fragments is determined by running the gel with a DNA ladder with known DNA fragment lengths. At times, the desired DNA fragment is not detected or the DNA product appears as a "smear" on the gel. Reasons for these results include the wrong annealing temperature, poorly designed primers, excess DNA template or degraded DNA template, among others. There are many ways to optimize PCR, but usually the first step is to optimize the annealing temperature of the primers. The PCR may be run again with a different annealing temperature, or a gradient thermal cycler may be used. Gradient thermal cyclers are specialized machines that allow a temperature gradient (usually a 20 - 30C difference) across the block containing the PCR tubes. Usually, this gradient is applied to the annealing temperature, while the denaturing and elongating temperatures are constant. This method allows for the determination of the ideal annealing temperature for the primers being used. Once the desired fragment of DNA is identified with agarose gel electrophoresis, many different methods may be employed to purify the amplified product for further analyses.

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