The standard PCR reaction takes place in a closed tube that is subject to a series of temperature changes or cycles. The initial phase of a cycle involves heating the reaction mixture, including template, primers, polymerase, and free deoxynucleotides, to 92-96°C to denature the double-stranded template and, in subsequent cycles, the double-stranded reaction products. When the reaction mixture is cooled during the second phase of the cycle, oligonucleotide primers anneal to opposite strands of the template flanking the target sequence. The optimal annealing temperature ranges from 45°C to 72°C and is related to the melting temperature of the primers, which is determined by their length and G + C content, and the concentration of monovalent cations. In the final stage, at 72°C, the polymerase extends the primers from the 3' ends toward each other along the template strands. The entire cycle is ordinarily repeated 25-40 times, and because the products are also used as template in subsequent reactions, there is an exponential increase in the number of copies, resulting in the generation of a discrete portion of DNA, which is defined by the 5' ends of the primers.
Table 1
Equations Describing Electrophoretic Driving Force
Eq. (1)—Electrophoretic driving force F = XQ
VQ l
where F is the force driving the ion forward, X is the electric field strength or voltage drop, and V is the applied voltage, and l is the length of the gel. Eq. (2)—Stokes Law
where F is the counterforce, n is a constant (3.14159), r is the radius of the analyte, n is the viscosity of the matrix, n and, v is the velocity of the analyte movement (lit)
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