Enzyme selection

Question 1

What are the 4 factors to choosing a polymerase?

Answer 1

Thermal stability
Extension rate
Fidelity
Processivity

Question 2

Describe extension rate

Answer 2

Influenced by extension temperature, DNA template sequence, and buffer composition

Question 3

Describe fidelity

Answer 3

How accurate it is

Proofreading

Question 4

Describe processivity

Answer 4

The probability that the polymerase will detach during extension

Question 5

Describe standard thermostable DNA polymerases

Answer 5

Suitable for routing PCR- detection, and estimation
Standard Taq produces fragments with a single ‘A’ overhand at the 3’ end enabling insertion to T/A cloning vectors
Good processivity
Fast extension rate
Lack proofreading

Question 6

Describe hot-start polymerase

Answer 6

Used to suppress nonspecific product during setup to increase yield as standard Taq can be active on ice and extend non-specific annealed primers
The polymerase is inactivated and becomes active either chemically or through antibody activation
Useful when there is a low concentration of DNA or several pairs of primers
Good processivity
Fast extension
lacks proofreading
Extremely stable

Question 7

Describe Hi-Fi polymerase

Answer 7

Has a 3’ to 5’ exonuclease activity and removes erroneously attached bases
Will remove the ‘A’ overhang
Low processivity rate
Expensive

Question 8

Describe long-range polymerase

Answer 8

Blend of thermostable and proofreading polymerase
Add optimised buffer with a high salt content
Gives a high yield