Mutagenesis

Question 1

Mutagenesis

Answer 1

change the sequence of a DNA fragment -> generate mutations that differ from the typical “wild-type”organism at one or more bases

Question 2

Random Mutagenesis drawbacks

Answer 2

non-specific
low frequency of mutants for one gene
possible multiple mutations in different genes
uncertainty about phenotype-genotype relationship

Question 3

Site-directed mutagenesi advantages

Answer 3

Protein structure and function
Modfying enzymes with improved functions
Regulatory regions of promoters/enhancers

Question 4

Classical Methods for site-directed mutatgenesis

Answer 4

based on the original method of Zoller & Smith

improvements in selection methods

Question 5

PCR-base Methods

Answer 5

Quikchange and other commerical kits

- overlap extension and Gibson assembly

Question 6

Classical method single _____ and single ___________________, _____ for DNA synthesis and _______________ to detect mutants

Answer 6

primer
stranded DNA derived from M13-based vectors(as template)
Klenow
32P

Question 7

Primers are specific for _________ and complimentary to ______________, usually ≥ __ nucleotides and longer if _____________, mismatch bases should be near the _________

Answer 7

template
DNA target
20
more than one substitution is required
middle of the primer

Question 8

DNA polymerases should have high _______, be ________, and inactivated __________,

Answer 8

processivity (short reaction time)
accurate (Proof-reading ability)
5’ exonuclease activity (does not degrade primer)

Question 9

Polymerases used in M13-based methods

Answer 9

Klenow fragments (large fragment of E. coli DNA polymerase I)
T4 or T7 DNA Polymerase (better fidelity than Klenow)

Question 10

Disadvantages to M13 site-directed mutagenesis

Answer 10

Need single stranded DNA as a template
Low mutant to wild-type ratio
Klenow is low fidelity, T7 and klenow non-thermostable
Use of radioisotopes

Question 11

Advantages to current methods of sdm

Answer 11

Different forms of DNA can be used (ds, ss, linear or circular)
High rate of mutant recovery
High temperatures(if using a pcr method) reduce secondary structures in dna templates
Speed and ease of use

Question 12

Steps of sdm (Example in the quick change method of this step)

Answer 12

identify specific changes needed (Plasmid preparation, with target site for mutation)
Synthesis of mutant DNA (temperature cycling: denatures plasmid, anneals oligonucleotide primers w/ desired mutation)
Elimination or reduction of non-mutated DNA (Digestion, of methylated non-parental DNA template with Dpn1)
Transfer to host and detection of mutants by phenotype and sequencing

Question 13

Temperature cycling in Quikchange SDM

Using the ______ of ________,
extend and incorporate the
___________ resulting
in ____________

Answer 13

nonstrand-displacing action
Pfu DNA polymerase
mutagenic primers
nicked circular strands

Question 14

Quikchange mutagenesis kit Primers
two __________ which are ______ to each other
____ bases in length with ______ bases on each side of the mutation
GC content of _____

Answer 14

mutagenic primers, complimentary
25-45, 10-15
40%-60%

Question 15

in quikchange selection, the enzyme used is ______ and the parental templates are selected against because they are ______

Answer 15

Dpn1

methylated

Question 16

Overlap extension method
There are 2 __________ each containing __________ which should each contain an ____________ of at least __ bases
There are 3 ________. The first 2 create ___________
The third one creates _____________

Answer 16

sets of primer pairs
external and mutagenic primers
overlapping sequence
15

PCR reactions
overlapping DNA fragments containing the desired mutation
amplification of a single large DNA fragment containing the desired mutation

Question 17

Gibson assembly is used for synthesis and cloning without __________

Answer 17

restriction sites

Question 18

Random mutagenesis
Allows ______
Generates _________
Characterize ___________ or select for _________

Answer 18

analysis on critical residues when little is known about the protein
multiple mutations on a defined region of cloned DNA
mutations introduced, mutant phenotype

Question 19

methods of random mutagenesis

Answer 19

• chemical mutagenesis (DNA exposure to chemical mutagens)
• Error prone pcr and misincorporation (using error-prone polymerase, limiting concentrations of dNTP in the presence of dITP, presence of MN2+)

Question 20

Gene shuffling

Answer 20

prepare a gene library of recombinant chimeric genes from a set of related gene sequences

Question 21

Steps in gene shuffling

Answer 21

Select a set of related gene sequences
Treatment with DNase to generate random fragments
Run denature-annealing extension cycles as in PCR
Mutant selection for a function or phenotype

Question 22

Directed evolution

Answer 22

Multiple rounds of mutagenesis and selection for improved function and selection for improved function and activity of a protein