All methods for DNA amplification have rested on the concept of DNA strand complementarity discovered by James Watson and Francis Crick. The polymerase chain reaction is an enzymatic process analogous to the replication process used by cells to copy their own DNA. It targets a specific region of DNA, using two oligonucleotide primers that are designed to flank the template DNA segment to be amplified. Amplification of the template DNA is achieved by repeating a 3-step process through 25-30 cycles in a thermal cycler.
Each cycle has three steps:
- The two DNA strands are denatured by heat.
- The sample is then cooled to allow the primers to anneal to the DNA segments.
- The temperature is raised to allow the DNA polymerase to add nucleotides to extend the primers to produce a copy of each DNA template strand.
This PCR product is sometimes referred to as an amplicon. Each cycle results in the doubling of amplicons. The result is an exponential accumulation of the specific target fragment, approximately 2 n, where n is the number of cycles of amplification performed. However, the process loses efficiency at higher cycle numbers. After 30 cycles, approximately a billion copies of the target DNA template are generated.
More than one region can be copied simultaneously by adding more than one primer set to the reaction;01 this is known as multiplexing. Primer design and the optimization of thermal cycling parameters are more complex with multiplex reactions than for a single-locus reaction.
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