SATURATION MUTAGENESIS WITH SYNTHETIC OLIGONUCLEOTIDES
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The general approach to site-specific mutagenesis, which is targeted to a piece
of DNA inserted in an appropriate vector, employs an oligonucleotide with one base
altered from the complementary structure of the wild-type DNA. Using the oligonucleotide
as a primer, heteroduplex DNA is generated with DNA polymerase and DNA ligase.
Semi-conservative replication of the heteroduplex in a suitable host leads to a
population of DNAs up to half of which are site-specifically mutated.
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While the originally described and most widely used procedure involves transversions
(1), site-directed insertions and deletions are also possible. Using oligonucleotides
that give perfect base-pairing over approximately 12 nucleotides on either side of the
desired site, deletions of the order of 10 or more intervening bases have been achieved (2).
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Recently, a report of a new approach, termed saturation mutagenesis, was published (3).
In this technique, an entire region of the DNA, up to the entire length of the synthetic
oligomer, is mutagenized. This is achieved by synthesizing a DNA oligonucleotide from the
four monomers, each of which is deliberately contaminated with a small amount of the other
three. The desired result is a population of synthetic oligonucleotides, each member of
which contains statistically a single-base alteration at one position (4), and in which
each position over the entire length of the oligomer is so altered.
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The solution to a statistical equation (3) gives the required level of "contamination" of
each base with the other three so as to maximize, based on the size of the oligonucleotide,
the fraction of the total oligomers which will have one (4) mutated site.
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We at MCRC have added a refinement to this approach, taking into account the differential
reactivities of the four deoxynucleoside phosphoramidites, so as to maximize the number of
molecules with different single-base substitutions.
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MCRC is pleased to offer optimized oligonucleotides for saturation mutagenesis. We always
welcome the opportunity to discuss with you any aspect or application of synthetic DNA to
the unique needs of your research program.
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1. Hutchison, C.A., III, Phillips, S., Edgell, H.E., Gillam, S., Jahnke, P., and Smith, M. (1978) in J. Biol. Chem. 253 (18), 6551-6560.
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2. Starcich, B., personal communication.
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3. Derbyshire, K.M., Salvo, J.J., and Grindley, N.D.F. (1986) in Gene 46, 145-152.
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4. Typically it is desired to have only one mutated base per oligonucleotide; the calculation maximizing those oligomers with two or more altered sites per molecule can also be made.
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