Site directed mutagenesis kits -recommend?

[Eckhard Boles]

You need no kit for doing site directed mutagenesis. We have just 
published a very simple, cheap and highly efficient method for PCR-based 
site-directed mutagenesis which works very well. However, in contrast 
to the manuscript we propose now to use a polymerase with 3'-5'- 
exonuclease activity but which does not degrade the mismatched primers 
(for example, Eurogentec sells such a polymerase which works very well). 
The method is published in Curr.Genet. (1995) 28, 197-198. I will add the 
manuscript at the end of this message.

Good luck for your experiments
Eckhard


A rapid and highly efficient method for PCR-based site-directed 
mutagenesis using only one new primer


Eckhard Boles and Thomas Miosga

Institut für Mikrobiologie, Technische Hochschule Darmstadt,
Schnittspahnstr. 10, D-64287 Darmstadt, Germany




Site-directed mutagenesis using two subsequent PCR reactions with one 
mutagenic and two flanking primers has become a method of choice for 
studying the roles of critical residues in DNA and protein sequences 
(1,2). In the first PCR a mutagenic DNA fragment is amplified which 
itself is used as a megaprimer in the second PCR. Recently, a method has 
been described (3) that takes advantage of exponential amplification in 
the second PCR. It requires two different vectors in which the target 
sequence must be cloned. However, a severe limitation of this method is 
that the sequence of the first flanking PCR-primer must not be present in 
the second PCR-template. This however, as judged by the authors, 
unfortunately is an infrequent circumstance. Here we describe an improved 
method which does not show this limitation because pairs of vectors with 
inverted multiple cloning sites such as pUC18/19 or pBluescript II 
KS+/SK+ (Stratagene) are used. Moreover, the new method is more economic 
because it requires only two primers: one new mutagenic primer and a 
second primer which can be one of the usual sequencing primers that flank 
the multiple cloning site (MCS) of many vectors.

The target DNA fragment must be cloned into both vectors (for example 
pUC19 and pUC18). Alternatively, if the fragment has compatible ends it 
can be cloned into the same vector in two different orientations. In the 
first PCR, vector 1 (pUC19 + insert) is the template for amplification 
with the mutagenic primer (MutP) and one of the sequencing primers (in 
this example M13 universal primer, UP) (Fig. 1-1). In the second PCR, the 
isolated amplified fragment of the first PCR is used as a megaprimer 
together with vector 2 (pUC18 + insert) as the template and, again, M13 
universal primer. During the first cycles megaprimer / vector 2 
heteroduplexes are extended by DNA polymerase resulting in only a few 
hybrid molecules (Fig. 1-2). In the subsequent cycles, however, only 
mutagenic sequences will be amplified exponentially with universal primer 
because they contain universal primer-binding sites at both ends (Fig. 
1-3). On the other hand, wild-type molecules can be only linearly 
amplified because they contain only one universal primer binding site. 
This results in a very high frequency of mutated sequences.

We have successfully applied this method to substitute in the yeast 
6-phosphofructo-2-kinase serine 644, thought to be involved in protein 
kinase A regulation (4, 5), for random amino acids. The mutagenic primer 
was completely degenerate in the three positions encoding the wild-type 
TCT serine codon (5'-GAAAGAAGATATNNNGTTATACCAACAGC-3'). A 1.4-kb internal 
SphI/SalI fragment of the PFK26 gene (4) was cloned into pUC18 and pUC19. 
Conditions for both PCR's were as follows: 10 mM Tris-HCl pH 8.3, 50 mM 
KCl, 1.5-2.5 mM MgCl2, 20 ng denatured template DNA, 0.2 mM dNTPs, 2 µM 
primers, 2.5 units Taq-DNA polymerase (Boehringer-Mannheim) in a volume 
of 50 µl. PCR 1: pUC19-PFK26 as template, M13 universal primer, mutagenic 
primer, 95°C 40 sec, 42°C 40 sec, 72°C 1.5 min, 25 cycles. PCR 2: 
pUC18-PFK26 as template (linearized with BamHI), 40 ng of the 0.9-kb 
amplified megaprimer fragment from PCR 1, 95°C 45 sec, 50°C 30 sec, 72°C 
45 sec, 5 cycles, then addition of M13 universal primer and 25 cycles 
with 95°C 40 sec, 52°C 40 sec, 72°C 1.75 min. A 1.4-kb fragment was 
isolated, cut with SphI and SalI, and cloned into pUC19 or was directly 
substituted for the wild-type sequence in the original vector. Sequencing 
revealed that out of 14 clones 13 clones did not contain the TCT codon at 
position 644 but all contained different codons demonstrating the very 
high efficiency of the new method.

In further attempts to test the efficiency of the described method we 
successfully replaced in the yeast PFK26 gene as well codons 665 and 666 
as codons 770 and 771 by two successive stop codons, and in the yeast 
putative reductase YPR1p (Miosga, unpublished) either lysine 241 or 
lysine 264 by glutamate. In both cases, the orientations of the two 
mutagenic primers were directed against each other. This allowed to use 
mutagenic primer 1 as the sequencing primer to verify the mutation 
introduced by mutagenic primer 2, and vice versa. The flanking 
PCR-primers were M13 universal primer and reverse primer, respectively, 
and Taq-DNA polymerase (Boehringer, Mannheim) was used in both PCR 
reactions. Sequencing of the cloned PCR products of, alltogether, five 
different transformants revealed in all cases only the desired 
substitutions. However, in a further attempt and using Vent-DNA 
polymerase (Stratagene) in the PCR reactions only one of sixteen cloned 
PCR products carried the expected exchanges. This might be explained by a 
3'-5' exonuclease activity of Vent-DNA polymerase.


ACKNOWLEDGEMENTS
We thank Prof. F.K. Zimmermann for his kind support and helpful 
suggestions. The work in our laboratory is supported by the 
Volkswagen-Stiftung and the Bundesminister für Forschung und Technologie.

REFERENCES
1. Kammann,M., Laufs,J., Schell,J. and Gronenborn,B. (1989) Nucleic Acids 
Res. 17, 5404.
2. Landt,O., Grunert,H.-P. and Hahn,U. (1990) Gene 96, 125-128.
3. Barrettino,D., Feigenbutz,M., Valcárcel,R. and Stunnenberg,H.G. (1994) 
Nucleic Acids Res. 22, 541-542.
4. Kretschmer,M. and Fraenkel,D.G. (1991) Biochemistry 30, 10663-10672.
5. Boles,E., Heinisch,J. and Zimmermann,F.K. (1993) Yeast 9, 761-770.