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Pioneers of Site-Directed Mutagenesis

Pioneers of Site-Directed Mutagenesis - Biochemistry Forum

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Old 12-06-2008, 11:31 PM
Pipette Filler
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Question Pioneers of Site-Directed Mutagenesis



I'm giving a lecture called "The Biochemists' Toolbox." My lecture will consist of the aspects of protein engineering (expression vectors, expression systems, and probing protein structure/function via mutagenesis). I'm having trouble finding the pioneers of this field. I'm well aware of PCR (Kary Mullis), however, I don't know where to look for the rest. Any suggestions? Any classical experiments in this field anyone's aware of?
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Old 06-16-2009, 07:55 PM
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Default Re: Pioneers of Site-Directed Mutagenesis

Look for Kunkel mutagenesis

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:

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.

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.
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