Mutagenesis Methods

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					Mutagenesis Methods

   Lily Peterson
   April 5th, 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

Use of M13 allows for single
strand recombinant DNA         Screen for
recovery                       mutant
                 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          With PCR based
    are useful in making        methods it is hard to
    specific mutations in       replicate the mutated
    the DNA                     DNA…in order for
                                replication to occur
   Which is useful when        super competent cells
    studying different          must be used and are
    aspects of protein          expensive!
                               Screening can be
                                tedious, usually
                                requires sequencing to
                                confirm if mutation
             Cassette Mutagenesis

   Used to introduce multiple mutations into the
    DNA sequence
   Uses blunt ended DNA for insertion site of
   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
             Uses and Limitations

   Typically used for          Requires the SapI
    protein structure but        restriction enzyme cut
    possibly used for gene       sites, and other cut
    function                     sites flanking the target
   Less expensive than          region for removal of
    site directed                DNA
    mutagenesis to create       Works best when target
    several mutations,           region is contained in a
    because there is no          small DNA fragment
    need for primers
             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      Not useful in large plasmids
    of gene function, though           because many recognition
    can be used to create gene         sites could be contained in
    fusions                            the a single plasmid
   Usually easy to see a             Suicide vectors are used,
    change in phenotype due to         though some may have
    gene knockout                      limited replication, so further
   Because the transposon             screening is needed
    inserts at a specific
    sequence, helps in
    determining where insertion

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