Mutagenesis Flashcards
Mutagenesis
change the sequence of a DNA fragment -> generate mutations that differ from the typical “wild-type”organism at one or more bases
Random Mutagenesis drawbacks
non-specific
low frequency of mutants for one gene
possible multiple mutations in different genes
uncertainty about phenotype-genotype relationship
Site-directed mutagenesi advantages
Protein structure and function
Modfying enzymes with improved functions
Regulatory regions of promoters/enhancers
Classical Methods for site-directed mutatgenesis
based on the original method of Zoller & Smith
improvements in selection methods
PCR-base Methods
Quikchange and other commerical kits
- overlap extension and Gibson assembly
Classical method single _____ and single ___________________, _____ for DNA synthesis and _______________ to detect mutants
primer
stranded DNA derived from M13-based vectors(as template)
Klenow
32P
Primers are specific for _________ and complimentary to ______________, usually ≥ __ nucleotides and longer if _____________, mismatch bases should be near the _________
template DNA target 20 more than one substitution is required middle of the primer
DNA polymerases should have high _______, be ________, and inactivated __________,
processivity (short reaction time)
accurate (Proof-reading ability)
5’ exonuclease activity (does not degrade primer)
Polymerases used in M13-based methods
Klenow fragments (large fragment of E. coli DNA polymerase I) T4 or T7 DNA Polymerase (better fidelity than Klenow)
Disadvantages to M13 site-directed mutagenesis
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
Advantages to current methods of sdm
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
Steps of sdm (Example in the quick change method of this step)
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
Temperature cycling in Quikchange SDM
Using the ______ of ________,
extend and incorporate the
___________ resulting
in ____________
nonstrand-displacing action
Pfu DNA polymerase
mutagenic primers
nicked circular strands
Quikchange mutagenesis kit Primers
two __________ which are ______ to each other
____ bases in length with ______ bases on each side of the mutation
GC content of _____
mutagenic primers, complimentary
25-45, 10-15
40%-60%
in quikchange selection, the enzyme used is ______ and the parental templates are selected against because they are ______
Dpn1
methylated
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 _____________
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
Gibson assembly is used for synthesis and cloning without __________
restriction sites
Random mutagenesis
Allows ______
Generates _________
Characterize ___________ or select for _________
analysis on critical residues when little is known about the protein
multiple mutations on a defined region of cloned DNA
mutations introduced, mutant phenotype
methods of random mutagenesis
- 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+)
Gene shuffling
prepare a gene library of recombinant chimeric genes from a set of related gene sequences
Steps in gene shuffling
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
Directed evolution
Multiple rounds of mutagenesis and selection for improved function and selection for improved function and activity of a protein
