Polymerase chain reaction (PCR) is the principal technique used in molecular biology laboratories to make millions of copies of DNA. To look for a mutation it's first necessary to amplify the DNA so that we can easily measure it. It's hard to find a needle in a haystack, but if you can amplify the needle and make it very large, it becomes much easier to detect. PCR can reliably amplify a targeted sequence for analysis in an efficient manner. This makes the study of nucleic acid possible.
The basic principle of PCR uses a DNA polymerase, nucleotides (A, T, C, G), primers (short sequences of complementary nucleic acid), and necessary ions such as magnesium in a reaction that is heated and cooled rapidly in a thermal cycler to create millions of copies of DNA. The steps in PCR are:
- Heat causes the separation of the two DNA strands, called denaturation. This opens up the template for the primers to bind to a complementary sequence. The complementary nature of the primer sequence allows us to design a PCR reaction to selectively amplify any sequence that is known.
- The temperature is then reduced to allow the DNA polymerase to bind (anneal) and DNA synthesis to begin.
- Once bound, the DNA polymerase can attach where the primer meets the template and replicate a second strand of DNA that is complementary to the first strand (extension).
- The temperature continues to cycle allowing denaturation, annealing, extension phases to occur during each cycle. With each cycle, the number of DNA template strands made is doubled. This allows for exponential amplification of the original sequence.
There are many variations of the original PCR method. RNA can be amplified using RNA polymerase and nucleotides that include Uracil instead of Thymine. RNA sequences can be converted into complementary DNA (cDNA) using a reverse transcriptase enzyme instead of the DNA polymerase in a process fittingly called reverse-transcriptase PCR. The primers in the reaction can be fluorescently labeled to allow a laser to detect the rate of DNA synthesis during the reaction. This is called real-time PCR and is effectively used to quantitate the amount of nucleic acid present in the starting sample.
PCR is capable of single nucleotide discrimination, meaning PCR can distinguish between the nucleotides at each site interrogated. This makes it a very useful tool to determine the presence or absence of a single nucleotide polymorphism (SNP). PCR is widely used to determine the genotype of cancer cells to help guide treatment.
Image courtesy of Wikimedia
