The principles of the polymerase chain reaction (PCR) as an in vitro…

The idea

PCR copies one specific region of DNA in a thermal cycler. The mixture contains the DNA sample, a large excess of two primers, free DNA nucleotides and Taq DNA polymerase, an enzyme from a thermophilic bacterium that is not denatured at high temperature. Each cycle has three stages. The mixture is heated to about 95 degrees Celsius, which breaks the hydrogen bonds between the base pairs and separates the double helix into two single strands. It is cooled to about 55 degrees so that the primers, short single strands with bases complementary to the sequences at either end of the target region, anneal to each strand. It is then warmed to about 72 degrees, the optimum for Taq polymerase, which joins free nucleotides to the end of each primer by complementary base pairing, building the new strands.

What earns the marks

• Heating to about 95 degrees Celsius to break the hydrogen bonds and separate the two DNA strands.
• Cooling so that primers with complementary base sequences bind to the ends of the target strands.
• DNA polymerase joining free DNA nucleotides to form complementary strands, at about 72 degrees Celsius.
• The number of DNA molecules doubling each cycle, so n cycles produce two to the power n copies.
• The use of the product, such as providing enough DNA from a small sample to sequence, screen or fingerprint.

Common mistakes and how to put them right

• Saying DNA helicase separates the strands, when in PCR heat alone breaks the hydrogen bonds.

• Using ordinary human DNA polymerase, which would be denatured at 95 degrees Celsius.

• Saying the primers are RNA, or that they are complementary to the whole gene rather than to its ends.

• Calculating copies as n multiplied by two rather than two to the power n, or forgetting the original molecule.

• Writing that PCR amplifies protein or mRNA.

Exam tips

• Give a temperature with a reason attached; a bare list of numbers rarely scores.
• For calculations use two to the power n, and check whether the question counts the starting molecules in the total.
• Mention that the primers set the boundaries of the region copied - it explains why PCR is specific.