Chemical Components of PCR
Let’s now take a look at what the name 'Polymerase Chain Reaction' describes in order to better understand the details of the process. 'Polymerase' is chosen because PCR makes use of a DNA polymerase enzyme for constructing new DNA strands, just like in a living cell. 'Chain Reaction' is also used because this technique involves repeating different heating and cooling cycles over and over again, as many as 35 or more times. Each complete cycle doubles the amount of DNA present, so in just 20 cycles there are over one million (220) copies of that specific segment of DNA . For PCR there are five chemical components needed, including a DNA template, DNA polymerase enzyme, primers, nucleotides and reaction buffer. These are described here in detail.
1. The DNA template is that particular DNA sequence which you want copied.
2. There are two requirements for a suitable DNA polymerase enzyme for PCR. First, one is needed that has a good activity rate around 75°C. Second, it should be able to withstand temperatures of 95-100°C so that more enzyme does not have to be added at the beginning of each new cycle. The first to be discovered with these characteristics was Taq polymerase, isolated from Thermus aquaticus, a thermophilic eubacterium found in hot springs (Chien et al. 1976). Naturally coming from a hot environment, it does not easily denature in the hot temperatures required in PCR; plus it has a good efficiency, able to add 60 base pairs/sec at 70°C. Like all other DNA polymerases, Taq cannot begin DNA replication without the addition of a starting primer.
3. Primers are short oligonucleotides of DNA, usually around 8-60 base pairs in length. They can be random sequences if the project’s goal is for general genomic studies. However, if the purpose is to amplify a certain section of DNA in the genome, such as a known gene, then primers of specific sequences must be used.
4. The four different deoxyribonucleotide triphosphates (dNTPs), adenine (A), guanine (G), cytosine (C), and thymine (T) are needed to provide the building blocks for DNA replication. DNA polymerase will add each complementary base to the new growing DNA strand according to the original strand’s sequence following normal A-T and C-G pairings.
5. Finally, a reaction buffer is used to provide a stable pH. It may also contain magnesium chloride, if not, then MgCl2 must be added separately. Mg2+ plays a vital role in the PCR reaction, acting as a co-factor for Taq polymerase and thereby influencing enzyme activity.