Polymerase chain reaction (PCR) technique was originally invented by Kary Mullis (1985). It results in the selective amplification of a specific region of DNA molecule (up to billion copies). It can generate micro-gram (pg) quantities of DNA copies. The PCR process has been completely automated and compact thermal cycles are available in market.
Principle : The basic principle underlying this technique is that when a double strand DNA molecule is heated to a high temperature, the two DNA strands separate, giving rise to a single stranded DNA molecules (templates). If these single stranded molecules are copied by a DNA polymerase, it would lead to the duplication of original DNA molecule and if these events are repeated many times, multiple copies of the original DNA sequence can be regenerated.
The PCR is carried out in vitro. It utilises the following:
• a DNA preparation containing the desired segment to be amplified (target sequence),
• two nucleotide primers (about 20 bases long) specific, i.e., complementary, to the two 3′-borders (the sequences present at or beyond the 3′-ends of the two strands) of the desired segment,
• the four deoxynucleoside triphosphates, viz. TTP (thymidine triphosphate, dCTP (deoxycytidine triphosphate), dATP (deoxyadenosine triphosphate) and dGTP (deoxyguanosine triphosphate), and
• a heat stable DNA polymerase, e.g., Taq (isolated from the bacterium Thermus acquaticus), Pfu (from Pyrococcus furiosus) and Vent (from Thermococcus litoralis) polymerases. Pfu and Vent polymerases are more efficient than the Taq polymerase.
Procedure of PCR:
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 that has appropriate quantities of Mg2+. The following operations are now performed sequentially (Fig.) Denaturation: The reaction mixture is first heated to a temperature between 90-98°C (commonly 94°C) that ensures DNA denaturation. This is the denaturation step. The duration of this step in the first cycle of PCR is usually 2 min at 94°C.
Annealing:
The mixture is now cooled to a temperature (generally 40-60°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. The duration of annealing step is usually 1 min during the first as well as the subsequent cycles of PCR. Since the primer concentration is kept very high relative to that of the template DNA, primer-template hybrid formation is greatly favored over reannealing of the template strands.
Primer Extension: The temperature is now so adjusted that the DNA polymerase synthesizes the complementary strands by utilizing 3′-OH of the primers; this reaction is the 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. The duration of primer extension is usually 2 min. at 72°C. It has been shown that in case of longer target sequences, best results are obtained when the period of extension is kept at the rate of 1 min per kb of the target sequence and the extension is carried out at 68°C in the place of usual 72°C. Taq polymerase usually amplifies DNA segments of upto 2 kb; special reaction conditions are necessary’ for amplification of longer DNA segments.
In case of Taq polymerase, the optimum temperature for synthesis is between 70° and 75°C ; the temperature of reaction mixture is, therefore, adjusted to this temperature.
The completion of the extension step completes the first cycle of amplification; each cycle may take few’ (ordinarily 4-5) minutes. It should be noted that extension of the primer continues till the strands are separated during the denaturation step of the next PCR cycle.
The next cycle of amplification is initiated by denaturation (Step 1), which separates the newly synthesised DNA strands from the old DNA strands. Synthesis of new strands takes place, which doubles the number of copies of the desired DNA segment present at the end of first amplification cycle. This completes the second cycle.
Thus at each cycle, both new and old strands anneal to the primers and serve as templates for DNA synthesis. As a result, at the end of each cycle, the number of copies of the desired segment becomes twice the number present at the end of the previous cycle. Thus at the end of n cy cles 2” copies of the segment are expected; the real values are quite close to the expectation. Usually 20-30 cycles are carried out in most PCR experiments.
In case of automated PCR machines, called thermal cyclers, the researcher has to only specify the number and duration of cycles, etc. after placing the complete reaction mixture for incubation, and the machine performs the entire programme of operations precisely. After PCR cycles, the amplified DNA segment is purified by gel electrophoresis and can be used for the desired purpose.
PCR is of immense value in generating abundant amount of DNA for analysis in the DNA fingerprinting technique used in forensic science to link a suspect’s DNA to the DNA recovered at crime scene, to solve murder and rape cases etc.
Applications of Polymerase Chain Reaction Technology :
• PCR enables rapid amplification of template DNA for screening of uncharacterised mutation
• PCR permits rapid genotyping for polymorphic markers.
• A wide variety of PCR based methods can be used to assay for known mutation.
• Degenerate oligonucleotide primers and primers specific for ligated linker sequences permit co-amplification of sequence families.
• Anchored PCR uses a target specific primer and a universal primer for amplifying sequences adjacent to a known sequence.
• PCR is used in molecular diagnostics.
