Mutagenesis Methods Lily Peterson April 5 th, 2010.
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Mutagenesis Methods Lily Peterson April 5 th, 2010
Overview: Types of Mutagenesis PCR Based Methods Site-directed mutagenesis Mismatched mutagenesis 5’ add on mutagenesis Cassette Mutagenesis Insertional Mutagenesis Trasposon mutagenesis In vivo Mutagenesis Direct Mutagenesis
Mismatched Mutagenesis Similar to Site-Directed Mutagenesis But only focuses on a single amino acid Important when trying to determine a particular missense mutation in known gene of a disease. Or when just trying to evaluate the contributation of the single amino acid to the function of the protein.
Gene or cDNA is cloned into M13 vector Screen for mutant Use of M13 allows for single strand recombinant DNA recovery
5’ add on Mutagenesis Involves adding on a new sequence or chemical group to the 5’end of a PCR product This involves a particular way of designing the primers: 3’ end of the primer matches the sequence of PCR product. 5’ end contains the novel sequence Suitable restriction site Addition of a functional sequence (promoter sequence) Modified nucleotide that contains labeled group, biotinlyated, or fluorophore.
Uses and Limitations PCR based methods are useful in making specific mutations in the DNA Which is useful when studying different aspects of protein function With PCR based methods it is hard to replicate the mutated DNA…in order for replication to occur super competent cells must be used and are expensive! Screening can be tedious, usually requires sequencing to confirm if mutation occurred.
Cassette Mutagenesis Used to introduce multiple mutations into the DNA sequence Uses blunt ended DNA for insertion site of mutation Where mutation is inserted a 3 base pair direct terminal repeat is created The mutagenic codon cassette has two head to head SapI sites allowing for removal of all DNA except for mutation.
Targeted codon removed using restriction enzyme that creates blunt cut SapI digestion creates 3’ overhang allowing for ligation. Ligation, which creates final mutation
Uses and Limitations Typically used for protein structure but possibly used for gene function Less expensive than site directed mutagenesis to create several mutations, because there is no need for primers Requires the SapI restriction enzyme cut sites, and other cut sites flanking the target region for removal of DNA Works best when target region is contained in a small DNA fragment
Transposon Mutagenesis Transposon: a piece of short DNA that replicates by inserting into other pieces of DNA (plasmids, chromosomes, etc…) Useful for studying gene function because when the transposon moves into different location in the DNA it may cause a disruption in a gene or a set of genes. Transposons also have many useful properties for mutagenesis: Cause clean mutations Can be random or specific mutations Typically encode for antibiotic resistance or some other advantageous gene. Can use a transposon that inserts at a high frequency When used in bacteria it causes selectable phenotypes Recognize specific sequence that is ~2-12 base pairs long
Uses and Limitations Primary use is for the study of gene function, though can be used to create gene fusions Usually easy to see a change in phenotype due to gene knockout Because the transposon inserts at a specific sequence, helps in determining where insertion occurred Not useful in large plasmids because many recognition sites could be contained in the a single plasmid Suicide vectors are used, though some may have limited replication, so further screening is needed