Friday, May 23, 2008

This Week's Citation Classic

This week's citation classic is Clyde A. Hutchison, III, Sandra Phillips, Marshall H. Edge Shirley Gillam, Patricia Jahnke, and Michael Smith. Mutagenesis at a Specific Position in a DNA Sequence. The Journal of Biological Chemistry, 253: 6551-6560.

Nobel Laureate Michael Smith doesn't get enough recognition, and I attribute it to his relatively conventional name. His 1993 co-laureate Kary Mullis seems to get much more attention, perhaps because of his more unusual name*, but the technique that Smith developed, in my opinion, ranks with the PCR in terms of utility. This technique is site-directed mutagenesis (SDM).

Back in the old days, if you wanted to change the genotype of your study organism, you needed to bombard it with chemicals or radiation, then screen the mutants, hoping you might chance upon the right mutation. Obviously this technique is kind of inefficient. Joshua Lederberg once commented, "“The ignis futuus of Genetics has been the specific mutagen, the reagent that would penetrate to a given gene, recognize it, and modify it in a specific way. ”

SDM is this ignis futuus. Where radiation works like a sledgehammer, SDM works like a laser. One can target specific nucleotides for mutation into whatever change is desired.

SDM relies on constructing DNA oligionucleotides that are identical for the sequence of interest, but contain the mutation of interest.

Using the bacteriophage {phi}X174, the study's lead author Clyde Hutchinson "teamed up with Smith, and the pair realized that an obvious route to a mutagenic method was to use a mutant oligonucleotide primer for E. coli DNA polymerase I on a circular single strand template, which would produce a product that could be converted to a closed circular duplex by enzymatic ligation."

Today SDM is coupled with the PCR, and is used widely to study gene regulatory elements, DNA-protein interaction, and protein structure/function are all typical targets for mutagenesis studies, and in my own lab to study life history variation among phage lambda.

A Journal of Biological Chemistry article describing the discovery is available here.

Any connection between a recent Sandwalk post, the fact that Smith is Canadian and that this article is biochemical in bent is purely coincidental.

*I suppose there are other hypotheses, but let's be kind.


  1. Clyde Hutchinson "teamed up with [Michael] Smith"

    And now Clyde works down the hall from me at the J. Craig Venter Institute's San Diego campus along with another Nobel Smith -- Ham Smith!

  2. Clearly a classic paper. I would suggest that this technology can be (and should be) supplanted by "in vivo recombination" using Saccharomyces cerevisiae and the awesome power of homologous recombination to generate SDM.

    Here's an early description of the approach,
    Muhlrad D, Hunter R, Parker R. A rapid method for localized mutagenesis of yeast genes. Yeast. 1992 Feb;8(2):79-82.

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  4. Very cool. However, with computers, the line is that the rest is "a small matter of software". That is, though the solution is provably possible, it's still difficult, perhaps impractical. The trouble is, with software, we have some idea how the computer is going to behave. Biology isn't my primary field, so maybe there are already some techniques that are known. But it is doubtful it's to the point where to imagine it is to do it.