Site-Directed Mutagenesis - Kunkel Method

The Kunkel method for site-directed mutagenesis is a classic method for introducing mutations (either single base pairs or larger insertions, deletions, or substitutions) into a DNA sequence. There are three main steps to performing Kunkel mutagenesis.

In Vivo Process
1) Place the plasmid that contains your target sequence to be mutated into an ung- dut- strain of E. coli bacteria. dut- (lacking dUTPase) bacteria accumulate dUTP. ung- (lacking uracil deglycosidase) bacteria cannot remove dUTP that gets incorporated into new DNA strands. The end result is that your plasmid is converted to DNA that lacks T's and contains
U's instead:

Figure 1. dsDNA plasmid is transformed into ung- bacteria which converts the T's
U's. In this example, the GC base pair will be targeted for mutagenesis.
U-containing plasmid is converted to single-stranded DNA.

In Vitro Process
2) The U-containing target DNA is incubated with a mutagenic oligonucleotide that base pairs with the target except at the location of the desired mutation. Remember this could be an insertion, deletion, or base substitution of one or more nucleotides. Then this mixture is incubated with Klenow, dNTP's and later Ligase and ATP to produce double-stranded plasmid with one strand containing
U's and the new one containing only T's. Note how the desired nucleotide is not pairing with the old template strand.

Figure 2. Target plasmid is used as a template to produce a second strand
of DNA that contains the desired mutation and only

In Vivo Process
3) Finally, the hybrid old/new double-stranded DNA is transformed into bacteria that destroy the old
U-containing DNA and produce a T-containing strand using the new and mutagenized DNA strand as a template. Thus, all the plasmids will contain the newly mutated sequence.

Figure 3. Hybrid plasmid is transformed into wild-type bacteria that convert the original U-containing strand of DNA into a strand of T-containing DNA that is complement of the mutated sequence (in green). The original GC base pair has been converted to a new TA base pair.

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