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MBY364 Theme 8 summary
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Full summary of theme 8 (site-directed mutagenesis) made using lecture notes, the textbook and class notes.
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SITE-DIRECTED MUTAGENESISINTRODUCTION
- Why construct mutants - to study gene structure, function and the production
of mutant proteins
- Proof that the correct mechanism has been elucidated requires the analysis of
mutants that have amino acid or nucleotide changes at key residues
- Mutants were previously generated by chemical or physical means
- Disadvantages to original methods:
- Any gene could be mutated so there is need for selection systems -
frequency with which mutation occurs in the target gene can be low
- The mutation might not occur in the target gene even if the mutant has
the desired phenotype
- Identity of the mutation is unknown - whether it is a single base
mutation, DNA insertion or deletion
- Modern molecular biology provides a solution in creating specific mutants
- Any single gene could be studied and any given base int hat gene could be
specifically mutated by using site-directed mutagenesis
- Allows for mutant proteins with novel properties t be created - protein
engineering
CASSETTE MUTAGENESIS
- A portion of the wild-type gene is exchanged for a synthetic DNA fragment
containing the mutation
- Technique is simple and near 100% efficient
- Simplified screening is used to identify mutant clones
- Disadvantage - unique restriction sites are needed flanking the target region
- Therefore it depends on the restriction enzyme used
- Replace the excised part of the DNA with a chemically synthesised DNA - will
already contain the required sticky ends for ligation
, PRIMER EXTENSION: SINGLE PRIMER METHOD
- Method consists of priming in vitro DNA synthesis with a synthetic
oligonucleotide containing the mutation (5’ end phosphorylated)
- DNA to be mutated should be single stranded - can be cloned into M13-based
vectors
- DNA cloned in a plasmid and obtained in duplex form can also be used
- The synthetic oligonucleotide is incorporated in the heteroduplex molecule in
the presence of dNTPs and DNA polymerase enzyme (Klenow polymerase)
- Ends of the newly synthesised DNA strand is sealed by DNA ligase
- Following transformation of E. coli with the heteroduplex molecules, it gives
rise to a homoduplex molecule that is either a wild-type or mutant
- Wild type will not contain the mutation
- Frequency with which mutated clones arise compared to wild type clones may
be low
- Selection of clones can be done using hybridization analysis using the
synthetic oligonucleotide as a probe
- Can sequence the mutated DNA to verify the presence of the introduced
mutation
- Clones can be screened by nucleic acid hybridization with 32P-labelled
oligonucleotide as a probe
- Under suitable conditions of stringency, a positive signal will be obtained only
with mutant clones
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