8 Thermal Cycling

To amplify just a particular segment of DNA the following steps are followed which requires a process of cycling between different temperatures - thermal cycling:

• The hydrogen bonds of the double strand are broken apart by heating the sample to over 94°C. This means that the bases are now exposed and available to pair with their complementary bases that are present in the solution as nucleotides.
• The short primer DNA chains bind or anneal to their target sequence on the DNA strand as determined by the order of complementary bases in them. This is achieved by cooling to a specific temperature. A short primer DNA molecule of only 18–20 bases in length will stick to only one site in the entire human genome. It is often said this is ‘finding the needle in the haystack’.
• As the Taq polymerase starts to add nucleotides to the end of each primer it does so using base-pairing rules so that each new strand is an exact complementary strand. This is achieved by heating the reaction to 72°C.
• When the completed double strands are heated again to separate them into single-stranded chains, each strand has the target site for the other primer in it. Hence, with each round of synthesis the number of copies of the region increases.

The number of copies of DNA grows exponentially with each cycle – after one cycle there are two copies, after two cycles there are four copies and so on. After just 30 cycles there will be 2³⁰ or over a billion copies. A ‘haystack’ has been created from the ‘needle’.