Recombination-mediated PCR-directed plasmid construction
in vivo
in yeast
Recombination-mediated PCR-directed plasmid construction in vivo in yeast
Kevin R.
Oldenburg
+
,
Kham T.
Vo
,
Susan
Michaelis
1
and
Chris
Paddon*
Affymax Research Institute, 4001 Miranda Avenue,
Palo Alto
, CA 94304,
USA
and
1
Department of Cell Biology and Anatomy, The Johns Hopkins School of Medicine,
725 N. Wolfe Street,
Baltimore
, MD 21205,
USA
Received August 14, 1996;
Revised and Accepted November 19, 1996
ABSTRACT
We have extended the technique of PCR-directed recombination in
Saccharomyces cerevisiae
to develop a simple method for plasmid or gene construction in the absence of suitable
restriction sites. The DNA to be cloned is PCR-amplified with 30-40 bp of homology to a linearized yeast plasmid. Co-transformation into yeast results in homologous recombination
at a position directed by the PCR oligonucleotides.
The observation that linear DNA fragments can efficiently stimulate
recombination in
Saccharomyces cerevisiae
(
1
) has led to the rapid development of powerful methods for DNA manipulation in
yeast (
2
). Widely used recombination-based DNA manipulation methods include integrative DNA transformation, which is used to produce a chromosomal null allele of a gene of
interest (
3
), and allele rescue which involves the transplacement of a mutation from the
chromosome onto a plasmid-borne copy of that gene (
1
). A third use for recombination-based DNA manipulation in yeast is for straightforward cloning purposes.
In this case, a restriction fragment whose ends bear homology to plasmid
sequences can be directly `ligated' into a linearized vector by
in vivo
recombination, alleviating the need for an i
n vitro
ligation reaction (
4
). The three recombinational gene manipulation methods described above rely on
sufficient homology at the cut ends of a restriction fragment or plasmid to
engage the recombination machinery. Until recently, it was assumed that the
amount of homology necessary to mediate such events was significant (>100 bp,
but see below).
An important recent advance in the use of recombination-based methods for gene manipulation in yeast involves the use of PCR,
rather than restriction enzymes, to generate the DNA fragment to be
manipulated. Baudin (
5
) and Weber (
6
) demonstrated that the amount of homology necessary to promote efficient
recombination-mediated gene disruption in yeast is actually quite small (in the order of
30-50 bp), indeed small enough to be synthesized as part of a PCR primer.
Schiestl and co-workers (
7
) systematically examined the length of homology necessary to mediate such
events and found that as few as 15 bp was sufficient to mediate homologous
integration.
We have found that the recombination-mediated PCR targeting method can also be effectively used to (i) clone a
desired gene onto a plasmid, and (ii) to generate chimeric genes with precise
fusion junctions. We systematically examined the amount of homology needed to
efficiently carry out such PCR fragment-directed homology-mediated cloning experiments. The general procedure is outlined in
Figure
1
. Basically, a linearized target plasmid containing a selectable marker (e.g.
URA3
) is co-transformed with a PCR fragment. By homologous recombination a subset of
the cut plasmids are recircularized and simultaneously acquire a DNA segment of
interest; recombinants are selected as Ura
+
transformants.
REFERENCES
1 Orr-Weaver,T.L. et al. (1981) Proc. Natl. Acad. Sci. USA78, 6354-6358.
