Procedure of PCR - Polymerase Chain Reaction,DNA Polymerase,DNA Denaturation,Annealing,Hybrid Formation,DNA Polymerase Synthesizes,Thermal Cyclers

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	Procedure of PCR - Polymerase Chain Reaction - 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. 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°e.
Annealing. The mixture is now cooled to a temperature (generally, between 40-60°C) that permits annealing of the primer to the complementary sequences in the DNA. As a rule, these sequences are located at the 3' -ends of the two strands of the segment to be amplified. 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 favoured over reannealing of the template strands.
Primer Extension. The temperature is now so adjusted that the DNA polymerase synthesizes the complementary strands by utilizing the 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. Taq polymerase usually amplifies DNA fragments of up to 2 Kb; special reaction conditions are necessary for the amplification of longer segments. 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.
Therefore, the products of this extension step, and indeed of every cycle based on the original template DNA, is of indefinite length; this PCR product is usually called the long product. At the second cycle of PCR, primers will anneal to the 'long product' much before its 3/-end, where sequences complementary to them are located. Extension step in this cycle will produce a product that will be much shorter than the 'long product'; this is the 'correct' PCR product and represents the target sequence. The original template sequence will also be copied during the second and all subsequent cycles to generate the 'long product'. Therefore, the long product continues to increase linearly, while the 'correct PCR' product will multiply exponentially.
	The next cycle of amplification is initiated by denaturation (Step 1), which separates the newly synthesized DNA strands from the old DNA strands. This step is usually of one minute as against 2 min in the first cycle. Annealing allows the primers to base-pair with both the new and old strands, the total number of strands being twice their original number. 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. 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 cycles 2n copies of the segment are expected; the real values are quite close to but lower than this expectation.

Usually, 30-45 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 cloning, DNA sequencing, etc. But Taq polymerase does not have proof reading function. Therefore, PCR products have much higher mutations (base substitutions) than DNA obtained by in vivo replication. This aspect has to be gaurded against while using PCR products.