Primer Annealing

The temperature and length of time required for primer annealing depends upon the base composition, length, and concentration of the amplification primers. In general, the annealing temperature is 5°C below the true T _m of the amplification primers. Because Taq DNA polymerase is active over a broad range of temperatures, primer extension will occur at low temperatures, including those used during the annealing step. The range of enzyme activity varies by two orders of magnitude between 20 and 85°C.

Annealing temperatures in the range of 55 to 72°C generally yield the best results. The higher annealing temperatures enhance discrimination against incorrectly annealed primers and reduce mis-extension of incorrect nucleotides at the 3' end of primers. Stringent annealing temperatures, especially during the first several cycles, will help increase specificity. The reaction temperature is briefly cooled to 40 to 60°C and then raised to 70 to 75°C. Primers anneal to the complementary sequence during the cooling phase and extend the primers with Taq polymerase during the heating phase.

Primer Extension

Extension time depends upon the length and concentration of the target sequence and temperature. Primer extensions are usually performed at 72°C. Estimates for the rate of nucleotide incorporation at 72°C vary from 35 to 100 nucleotides per second , depending upon the buffer, pH, salt concentration, and the nature of the DNA template. An extension time of one minute at 72°C is considered sufficient for products up to 2 kb in length.

Denaturation Time and Temperature

The most likely cause for failure of a PCR is incomplete denaturation of the target template and/or the PCR product.¹¹ Typical denaturation conditions are 95°C for 30 seconds or 97°C for 15 seconds; higher temperatures may be appropriate especially for targets containing a high GC percentage. Incomplete denaturation allows the DNA strand to snapback, reducing the product yield. Denaturation steps that are conducted at too high a temperature and for too long lead to unnecessary loss of enzyme activity.

The half-life of Taq DNA polymerase activity varies with temperature:

• Less than 2 hours at 92.5°C
• 40 minutes at 95°C
• 5 minutes at 97.5°C

Finally, the double-stranded DNA is denatured by briefly heating the samples to 90 – 95°C.

Cycle Number

The optimum number of cycles depends mainly on the starting concentration of the target DNA when other parameters are optimized. Too many cycles can increase the amount and complexity of nonspecific background products (plateau effect) and too few cycles can produce low product yield.

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