Polymerase chain reaction is a revolutionary method developed by Kary Mullis in the 1980s. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. Because DNA polymerase can add a nucleotide only onto a preexisting 3′-OH group, it needs a primer to which it can add the first nucleotide. This requirement makes it possible to delineate a specific region of template sequence that the researcher wants to amplify. At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies called amplicons.
Components of PCR:
- DNA template– the sample DNA that contains the target sequence. At the beginning of the reaction, high temperature is applied to the original double-stranded DNA molecule to separate the strands from each other.
- DNA polymerase– A type of enzyme that synthesizes new strands of DNA complementary to the target sequence. The first and most commonly used of these enzymes is TaqDNA polymerase (from Thermus aquaticus, a thermophilic eubacterium found in hot springs), whereas PfuDNA polymerase (from Pyrococcus furiosus) is used widely because of its higher fidelity when copying DNA.
Although these enzymes are subtly different, they both have two capabilities that make them suitable for PCR:
1) They can generate new strands of DNA using a DNA template and primers.
2) They are heat resistant.
The polymerase begins synthesizing new DNA from the end of the primer. There are two requirements for a suitable DNA polymerase enzyme for PCR. First, it should have 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. Naturally coming from a hot environment, it does not easily denature in the hot temperatures required in PCR. Second. it should have a good efficiency,that is, it should be 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.
- Nucleotides (dNTPs or deoxynucleotide triphosphates)– single units of the bases A, T, G, and C, which are essentially “building blocks” for new DNA strands. 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.
- Primers: These are short oligonucleotides of DNA, usually around 8-60 base pairs in length that are complementary to the target sequence. 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.
- A reaction buffer: is used to provide a stable pH. It may also contain magnesium chloride, if not, and 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.
Denaturation: At 94 C (201.2 F), the double-stranded DNA melts and opens into two pieces of single-stranded DNA.
Annealing: At medium temperatures, around 54 C (129.2 F), the primers pair up (anneal) with the single-stranded “template” (The template is the sequence of DNA to be copied.) On the small length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template.
Extension: At 72 C (161.6 F), the polymerase works best, and DNA building blocks complementary to the template are coupled to the primer, making a double stranded DNA molecule.
With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially.
Limitations of PCR:
The PCR reaction starts to generate copies of the target sequence exponentially. Only during the exponential phase of the PCR reaction is it possible to extrapolate back to determine the starting quantity of the target sequence contained in the sample. Because of inhibitors of the polymerase reaction found in the sample, reagent limitation, accumulation of pyrophosphate molecules, and self-annealing of the accumulating product, the PCR reaction eventually ceases to amplify target sequence at an exponential rate and a “plateau effect” occurs, making the end point quantification of PCR products unreliable. This is the attribute of PCR that makes Real-Time Quantitative RT-PCR so necessary.