How the Polymerase Chain Reaction (PCR) Works
What PCR Has to Do With DNA Sequencing and Amplifying Genes
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.
How Does Polymerase Chain Reaction Work?
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.
The PCR Technique
The polymerase chain reaction (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 steps involved in the PCR technique are as follows:
- 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 complementary 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.
This process of denaturing, annealing and elongation are repeated multiple (30-40) times, thereby increasing exponentially the number of copies of the desired gene in the mixture. Although this process would be quite tedious if performed manually, samples can be prepared and incubated in a programmable Thermocycler, now commonplace in most molecular laboratories, and a complete PCR reaction can be performed in 3-4 hours.
Each denaturing step stops the elongation process of the previous cycle, thus truncating the new strand of DNA and keeping it to approximately the size of the desired gene.
The duration of the elongation cycle can be made longer or shorter depending on the size of the gene of interest, but eventually, through repeated cycles of PCR, the majority of templates will be restricted to the size of the gene of interest alone, as they will have been generated from products of both of the primers.
There are several different factors for successful PCR that can be manipulated to enhance the results. The most widely used method to test for the presence of PCR product is agarose gel electrophoresis. Which is used to separate DNA fragments based on size and charge. The fragments are then visualized using dyes or radioisotopes.
Since the discovery of PCR, DNA polymerases other than the original Taq have been discovered. Some of these have better “proofreading” ability or are more stable at higher temperatures, thus improving the specificity of PCR and reducing errors from the insertion of the incorrect dNTP.
Some variations of PCR have been designed for specific applications and are now used regularly in molecular genetic laboratories. Some of these are Real-Time PCR and Reverse-Transcriptase PCR. The discovery of PCR has also lead to the development of DNA sequencing, DNA fingerprinting and other molecular techniques.