Lecture 3 GiM

Question 1

What are we expected to do if we don’t know the identity of the mutation?

Answer 1

• discovery methods (esp. “Sanger” DNA sequencing)

- “clonal” (next generation) sequencing

Question 2

What is polymerase chain reaction?

Answer 2

In vitro synthesis of large amounts of DNA by copying from small starting quantities

Question 3

What is the function of the small synthetic primers ‘oligonucleotides’?

Answer 3

Define the boundaries of DNA synthesis

Question 4

What is the enzyme used in DNA synthesis and what is the monomers?

Answer 4

• DNA polymerase

- deoxyribonucleotides

Question 5

What is the temperature used to denature DNA?

Answer 5

94 Celsius

Question 6

What is the temperature used in primer annealing?

Answer 6

55 celsius

Question 7

What is the temperature used in primer extension?

Answer 7

72 celsius

Question 8

What is the function of allele-specific PCR?

Answer 8

Determine the presence/absence of product
(The basic principle is that one of your primers covers the variant you’re interested in, so that, for example, if your primer matches the mutant allele then it will bind to mutant DNA but not WT DNA, so that you only get amplification if the mutant allele is present.)

Question 9

Which is more prefered? Allele specific or oligonucleotide ligation assay

Answer 9

OLA