The PCR steps are all carried out, one after the other, in bouts of cycling. Cycle 1 is as follows:

• During denaturation (about 1 min at 95°C), the DNA strands separate to form single strands.

• During annealing (about 1 min at temperatures ranging between 45°C and 60°C), one primer binds to one DNA strand and another binds to the complementary strand. The annealing sites of the primers are chosen so that they will prime DNA synthesis in the region of interest during extension.

• During extension (about 1 min at 72°C), the DNA synthesis proceeds through the target region and for variable distances into the flanking region[Flanking regions: The DNA sequences extending on either side of a specific gene or locus.], giving rise to 'long fragments' of variable lengths


When the second cycle starts, there are effectively two types of template: (1) the original DNA strands; and (2) the newly synthesised DNA strands, consisting of the target region and variable lengths of the flanking region at the 3' end. When the latter template is used in this cycle, only the target region is replicated.

In the third cycle, the newly synthesised target region DNA (i.e. without flanking regions) acts as template. The original DNA molecule is still present, and will be until the end of the reaction. However, after a few cycles, the newly synthesised DNA fragment quickly establishes itself as the predominant template. Cycles are typically repeated 25 to 45 times. Standardisation of the thermocycler's running conditions is essential for the reproducibility of results.

上一页  [1] [2] [3] [4] [5] [6] [7] 下一页