L18 - Mutagenesis

Question 1

what is mutagenesis

Answer 1

deliberate modifications to DNA to produce mutant genes and proteins to modify organisms

Question 2

how can mutations be introduced

Answer 2

spontaneously or induction with a mutagen - UV

or experimentally with PCR

Question 3

which areas of a gene might we want to mutate

Answer 3

regulatory sequences = effect on DNA

coding sequences = effect on protein

Question 4

what conditions of PCR could e changed to introduce mutations

Answer 4

use of low fidelity enztme - Taq pol

unbalanced dNTPs

changes in magnesium conc

Question 5

what is Magnesium’s tole in DNA synthesis/PCR

Answer 5

DNA polymerase cofactor:

stabilises complex by binding in DNA pol active site and stabilising negative charges of DNA

= too hig conc of Mg allows incorrect dNTPs to be incorparated due to high stability

Question 6

what do low fidelity DNA polymerases lack

Answer 6

3’ to 5’ exonuclease activity

= no proofreading

Question 7

what do we call ‘controlled’ mutagenis sexperiments

Answer 7

Site-directed mutagenesis

= have a desired change we wish to make = not as easy as random

Question 8

in Site-directed mutagenesis where should the mutations NOT be in the primer

Answer 8

primers with the mutation should NOT have the mutation at the 3’ end of the primer

= would affect annealing affinity at the growing end = may prevent polymerase growing at all

at least 15 nucleotides should be identical to template = need to be able to anneal to target sequence efficiently

Question 9

how can we use PCR to ‘delete’ a sequence - create targeted mutation

Answer 9

the primers designed do NOT include the sequence of the bit we want deleted

= forces the bit not included in primer to ‘loop out’

due to the primer not having this sequence the polymerase has no template to copy that sequence onto the new strand in the SECOND round of PCR = its skipped

Question 10

to add or change a sequence using PCR to introduce desired mutations what would we do ?

Answer 10

add:
include region we want added in the primer

= this will act as a template and the new strand will have the added mutation

change:
include a sequence that is different = eg a tag

this will be copied onto the new strand from the template primer in the SECOND step of PCR

Question 11

describe the differences between 1 and 2 step CR and when you might use them

Answer 11

describes the number of cycles of PCR needed for final fragment of interest with desired mutation is produced

1 step:
primers contain mutation IN them

= one round is enough to produce fragment with desired mutation

2 step:
2 rounds of PCR needed

1. 2 overlapping fragments are amplified with a overlapping region = each fragment contains HALF of the desired mutation
2. anneal the 2 fragments and perform PCR on the fully formed fragment with new primers

Question 12

what are the 2 main strategies/ideas for making new and better proteins

Answer 12

Rational protein design:
“we know what change we want”
= purposeful mutations

Directed evolution:
“let random mutations and natural selection find it”
= random/slow but powerful