Enzyme selection Flashcards Preview

BIO2030 Biotechnology > Enzyme selection > Flashcards

Flashcards in Enzyme selection Deck (8)
Loading flashcards...
1

What are the 4 factors to choosing a polymerase?

Thermal stability
Extension rate
Fidelity
Processivity

2

Describe extension rate

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

3

Describe fidelity

How accurate it is
Proofreading

4

Describe processivity

The probability that the polymerase will detach during extension

5

Describe standard thermostable DNA polymerases

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

6

Describe hot-start polymerase

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

7

Describe Hi-Fi polymerase

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

8

Describe long-range polymerase

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