Technician with rows of thermocyclers used in research into the human genome at Celera Genomics, Maryland, USA. Thermocyclers clone DNA using a polymerase chain reaction (PCR). Celera aimed to sequence every gene in human DNA (deoxyribonucleic acid) by 2001. Human DNA is made up of some 30,000 genes among over 3 billion base pairs. Celera sequences DNA fragments and uses supercomputers to put the pieces together. This is a cruder but faster method than that used by the publicly–funded Human Genome Project. The complete human genome map will allow improved drug design and a greater understanding of genetic diseases.
PCR is only feasible because of the discovery and isolation of thermostable DNA polymerase. The first thermostable DNA polymerase was isolated from the bacterium Thermus aquaticus, which normally grows in the water of hot springs. This enzyme, called Taq DNA polymerase, is optimally active at 72°C and can retain its activity after successive heating to denature the template DNA. With each successive cycle of DNA synthesis, the number of copies of the target sequence is doubled. Typically 20–30 cycles of DNA synthesis will be performed, resulting in 106–109 copies of the target sequence.
If we go into details of PCR mechanism, the procedure is as follows:
Step 1: At the start of PCR, the DNA from which a segment is to be amplified, an excess of the two primer molecules, the four deoxyriboside triphosphates and the DNA polymerase are mixed together in the reaction mixture. The following operations are now performed sequentially.
Step 2: The mixture is now cooled to a temperature (generally 40–60 degree C) that permits annealing of the primer to the complementary sequences in the DNA; these sequences are located at the 3' ends of the two strands of the desired segment. This step is called annealing.
Step 3: The temperature is now so adjusted that the DNA polymerase synthesizes the complementary strands by utilizing 3' – OH of the primers; this reaction is same as that occurs in vivo during replication of the leading strand of a DNA duplex. The primers are extended towards each other so that the DNA segment lying between the two primers is copied; this is ensured by employing primers complementary to the 3' ends of the segment to be amplified. In case of Taq polymerase, the optimum temperature for synthesis is between 70-75 degree C; the temperature of reaction mixture is, therefore, adjusted to this temperature. This situation has the following additional advantages. Between 70–75 degree C, the base pairing between about 20 bases long primers and the DNA is much more specific than at 37 degree C, the optimal temperature for E.coli DNA polymerase. This minimizes the chances of annealing of primers to imperfectly matched sequences and thereby, amplification of unwanted DNA. The specificity of annealing is further increased by selecting appropriate conditions like ionic strength, primer length etc. The completion of step 3 completes the first cycle of amplification; each cycle may take few (ordinarily 1–3) minutes.
Step 4: The next cycle of amplification is initiated by denaturation (step 1), which separates the newly synthesized DNA strands from the old DNA strands.
Step 5: Annealing allows the primers to base pair with both the new and old strands, the total number of strands being twice their original number.
Step 6: Synthesis of new strands takes place, which doubles the number of copies of the desired DNA segment present at the end of step 1. This completes the second cycle.