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Short PCR product yields improved by lower denaturation temperatures.

Polymerase chain reactions (PCR) typically involve template denaturation between 930 and 95°C for every cycle of amplification. Under these conditions Taq DNA polymerase is inactivated gradually, with a half life of 130 and 40 min at 92.50 and 95°C respectively (1) and its activity may be limiting in the later cycles. Since the amplified product acts as template in subsequent cycles, it may be unnecessary to use such high denaturation temperatures during the later cycles, when the original DNA template of longer length forms an insignificant fraction of the total template. The empirical melting point equations for DNA-DNA duplexes in solution predicts that hybrid length affects Tm by a factor of 650/L, where L is the length in base pairs (2). Hence, Tm of duplexes less han 350 base pairs in length would be expected to be 2°C or more lower than that for genomic DNA of similar GC content, though absolute Tm values have not been formally established for the conditions used in PCR. We discovered that lowering the denaturation temperature after the initial few cycles gives a much improved yield of product, and enables amplification to be performed for up to 70 cycles.