How PCR Works

What is PCR?

The polymerase chain reaction (PCR) is a molecular genetic technique for making multiple copies of a gene, and is also part of the gene sequencing process. Gene copies are made using a sample of DNA and the technology is good enough to make multiple copies from one single copy of the gene found in the sample. PCR amplification of a gene to make millions of copies, allows for detection and identification of gene sequences using visual techniques based on size and charge (+ or -) of the piece of DNA.
Under controlled conditions, small segments of DNA are generated by enzymes known as DNA polymerases, that add complimentary deoxynucleotides (dNTPs) to a piece of DNA known as the "template". Even smaller pieces of DNA, called "primers" are used as a starting point for the polymerase. Primers are small man-made pieces of DNA (oligomers), usually between 15 and 30 nucleotides long. They are made by knowing or guessing short DNA sequences at the very ends of the gene being amplified. During PCR, the DNA being sequenced is heated and the double strands separate. Upon cooling, the primers bind to the template (called annealing) and create a place for the polymerase to begin.
PCR was made possible by the discovery of thermophiles and thermophilic polymerase enzymes (enzymes that maintain structural integrity and functionality after heating at high temperatures).

The Technique Explained

A mixture is created, with optimized concentrations of the DNA template, polymerase enzyme, primers and dNTPs. The ability to heat the mixture without denaturing the enzyme allows for denaturing of the double helix of DNA sample at temperatures in the range of 94 degrees Celsius. Following denaturation, the sample is cooled to a more moderate range, around 54 degrees, which facilitates the annealing (binding) of the primers to the single-stranded DNA templates. In the third step of the cycle, the sample is reheated to 72 degrees, the ideal temperature for Taq DNA Polymerase, for elongation. During elongation, DNA polymerase uses the original single strand of DNA as a template to add complimentary dNTPs to the 3’ ends of each primer and generate a section of double-stranded DNA in the region of the gene of interest. Primers that have annealed to DNA sequences that are not an exact match do not remain annealed at 72 degrees, thus limiting elongation to the gene of interest.