An efficient PCR mutagenesis strategy without gel purificiation step that is
amenable to automation
An efficient PCR mutagenesis strategy without gel purificiation step that is amenable to automation
Bertrand
Séraphin*
and
Stefanie
Kandels-Lewis
EMBL, Meyerhofstrasse 1, D-69117
Heidelberg
,
Ge
rmany
*
To whom correspondence should be addressed
Received March 11, 1996;
Revised and Accepted June 21, 1996
ABSTRACT
We describe here an improved megaprimer PCR mutagenesis strategy. The cumbersome
gel purification step that is usually used can be omitted by appropriately
cleaving the first and second DNA templates with restriction enzymes and
enzymatically removing remaining primers from the first PCR reaction. We show
that this improved procedure is reproducible and highly efficient. Furthermore
this method is suitable for automation because all the steps are now carried
out in reaction tubes.
Among the many methods for site directed mutagenesis, the PCR-based megaprimer strategy (reviewed in
1
) is currently widely used because of its flexibility and the yield of mutants
approaches 100%. This method is relatively cheap, as only a single mutagenic
primer and two flanking primers are used per mutant. A limitation of the
megaprimer strategy is that the products of the first PCR reaction need to be
fractionated by gel electrophoresis and purified to remove left-over primers, before being used in the second round of PCR. This prevents
the amplification of the wild-type template during the second PCR reaction that would dramatically
decrease the mutant yield. This step is time consuming, expensive and error-prone, especially when multiple mutants are made in parallel. This step is
also a potential barrier to automation. A second problem associated with the
megaprimer strategy is the non-templated addition of nucleotide, most frequently but not always an A, at
the 3' end of the first PCR product. This can result in the presence of
additional unwanted mutations. Various strategies have been described to
eliminate these unwanted mutations (
2
-
4
) but they are time consuming and expensive, only applicable under particular conditions or sometime reduce but do not eliminate these mutations.
Here we describe an improved megaprimer mutagenesis strategy that circumvents
the gel electrophoresis step (Fig.
1
). This method is highly efficient with a mutant yield approaching 100%. All the
steps are now carried out in reaction tubes, making the method well suited for
automation. Our strategy relies on the observation that the gel purification
step used in the megaprimer strategy is only required to remove the primer used
during the first PCR reaction. The two modifications of the classical protocol
described below eliminate the amplification of wild-type DNA template during the second PCR reaction rendering the gel
purification step unnecessary. First, the templates used for PCR were cleaved
with restriction enzymes to eliminate full length template. In this way, wild-type DNA should not be amplified by the two external primers (Fig.
1
). Although the only requirement for choice of the restriction enzymes to be
used is that they should cut the fragment to be amplified once or multiple
times on only one side of the mutagenic primer, they are most conveniently
chosen as the enzyme that will subsequently be used for the cloning of the
mutated PCR fragment (Fig.
1
). A second modification was the treatment of the product from the first PCR
reaction with Klenow enzyme. The proofreading activity of the enzyme is used to
reduce the concentration of the primers used during the first PCR reaction as
well as reducing the level of non-templated nucleotides added by the
Taq
polymerase at the 3' end of the PCR product (Fig.
1
, step V). Using these modifications, the product of the first PCR reaction
could be used directly as a megaprimer in the second reaction (Fig.
2
). Both the cleavage of the template DNA and the reduction of the primer
concentration after the treatment with the Klenow enzyme are required to
prevent amplification of the wild-type sequence. Indeed, trace of full length DNA remaining after the
digestion could still be amplified by standard amounts of primers 1 and 3 while
the reduced levels of primers 1 and 3 obtained after the Klenow treatment could
still produce low amount of product on a non-cleaved template. Experimentally, only mutant clones (36/36) were recovered in six independent mutagenesis experiments (Table
1
). A few clones contained an additional unwanted mutation (T -> C) one nucleotide 5' to the mutagenic primer, suggesting that the proofreading activity
of the Klenow enzyme did not remove all non-templated nucleotides added by the
Taq
polymerase. Further study will resolve whether other enzymes (e.g. T4 DNA
polymerase) are better suited for this step.
REFERENCES
1 Tao, B.Y. and Lee, K.C.P. (1994) In Griffin, H.G. and Griffin, A.M. (eds), PCR Technology: Current Innovations. CRC Press, Boca Raton, Florida. pp. 69-83.
2 Kuipers, O.P., Boot, H.J. and de Vos, V.W. (1991) Nucleic Acids Res. 19, 4558.
3 Sharrocks, A.D. and Shaw, P.E. (1992) Nucleic Acids Res. 20, 1147.
4 Armengaud, J. and Jouanneau, Y. (1993) Nucleic Acids Res. 21, 4424-4425.MEDLINE Abstract
5 Good, L. and Nazar, R.N. (1992) Nucleic Acids Res. 20, 4934.MEDLINE Abstract
