Biotechnology: PCR

Question 1

Primers

Answer 1

Short, single-stranded DNA molecules used to mark the 3’ ends of a sequence of DNA, allowing the DNA Polymerase to begin synthesising a strand in a 5’ to 3’ direction

Question 2

Purpose of PCR

Answer 2

Used to rapidly amplify small amounts of particular sequences of DNA, making it easier to visualise and study

Question 3

Taq DNA Polymerase

Answer 3

Enzyme that catalyses the formation of new DNA molecules from free nucleotides, used for it’s ability to resist denaturing at high temperatures

Question 4

Components Required for PCR

Answer 4

• Template DNA to be copied
• DNA Polymerase enzyme
• Buffer solution
• Supply of four nucleotides
• Two sets of single stranded DNA primers

Question 5

Steps of PCR

Answer 5

1. Denaturation
2. Annealing
3. Extension/Elongation

Question 6

1. Denaturation

Answer 6

The double stranded DNA is heated to 95*C, breaking the hydrogen bonds between the bases and causing them to denature (separate)

Question 7

2. Annealing

Answer 7

Temperature is reduced to 50-60*C, allowing for primers to anneal to complementary sequences at opposite ends of each strand. The reduced temperature is necessary for base pairing and hydrogen bonds.

Question 8

3. Extension

Answer 8

Temperature is raised to 72*C, the optimum temperature for DNA polymerase to function. Starting from primers, new DNA strands are synthesised in a 5’ to 3’ direction using DNA Polymerase and available nucleotides.