DNA amplification: does 'small' really mean 'efficient'?

The advent of the polymerase chain reaction (PCR) has, without a shadow of a doubt, hugely accelerated the progress of studies on the genetic structure of a diversity of organisms. PCR is an enzyme catalyzed amplification technique that allows any nucleic acid sequence to be generated in vitro and in abundance.1 It was first reported in early 1986 at the 51st Cold Spring Harbour laboratory Symposium on Quantitative Biology by Kary Mullis, and since has become an indispensable tool in basic molecular biology, genome sequencing, clinical research and evolutionary studies.2 The reason for the almost immediate acceptance of PCR as a DNA building tool lies in the beautiful simplicity of its underlying mechanism. Briefly, high temperature (normally in excess of 95 °C) is used to separate (denature) double stranded DNA into two single strands. Synthetic sequences of single stranded DNA (normally 20–30 nucleotides long), known as primers, are used to define or bracket the target region to be amplified. One primer is complementary to one DNA strand (at the start of the target region) with the second primer being complementary to the other DNA strand (at the end of the target region). The primers are hybridized (annealed) to the single stands by reducing the local temperature to between 50 and 65 °C. This is followed by an extension step at a slightly elevated temperature (approximately 72 °C) in which a complementary strand from each primer is extended by the catalytic action of a thermostable DNA polymerase enzyme (in the presence of free deoxynucleoside triphosphates) to form complementary strands of the template. This three-step process constitutes one PCR cycle, and if repeated n times will, in theory, lead to 2n21 copies of the target duplex. In other words, amplification is exponential, and consequently after only 20 cycles over one million copies of the original target DNA can be synthesized. In reality, amplification is never truly exponential and the copy yield is given by Y = (1 + x)n (1)