S H O R T C O M M U N I C AT I O N

Improvedmethod for thePCR-basedgene disruption inSaccharomyces cerevisiaeHiroshi Koyama, Eriko Sumiya, Takahiro Ito & Kazuhisa Sekimizu

Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo, Tokyo, Japan

Correspondence: Kazuhisa Sekimizu,

Laboratory of Microbiology, Graduate School

of Pharmaceutical Sciences, University of

Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo

113-0033, Japan. Tel.: 181 3 5841 4820;

fax: 181 3 5684 2973;

e-mail: sekimizu@mol.f.u-tokyo.ac.jp

Received 10 August 2007; revised 10 October

2007; accepted 10 October 2007.

First published online 20 November 2007.

DOI:10.1111/j.1567-1364.2007.00334.x

Editor: Monique Bolotin-Fukuhara

Keywords

Saccharomyces cerevisiae ; PCR-based gene

disruption; targeting vector; gene targeting.

Abstract

The PCR-based gene disruption strategy originally devised by Baudin et al. is

widely used for gene targeting in Saccharomyces cerevisiae. An advantage of this

strategy is its simplicity in making targeting constructs. The efficiencies of the

targeted disruption are highly variable from locus to locus, however, and often very

low. In this report, a method for improving the gene deletion efficiency is

described.

Disruption of a gene of interest in yeast and mice requires a

gene targeting vector composed of the 50- and 30-flanking

sequences of the target gene separated by a selectable marker,

such as genes conferring drug resistance or nutrient auto-

trophy (Fig. 1a) (Rothstein, 1991). The 50- and 30- flanking

sequences in the targeting vector are termed the 50-arm and

30-arm, respectively. In general, the longer the arm regions,

the more efficient the homologous recombination. Baudin

et al. (1993) previously reported a simple procedure for

targeting vector construction. In this method, targeting

vectors are constructed in a single PCR without any DNA

cloning steps, which are the most time-consuming steps in

the gene disruption experiments. Because of its simplicity,

this method was used in the genome-wide Saccharomyces

Genome Deletion Project (Winzeler et al., 1999). An exam-

ple for this simple strategy is shown in Fig. 1a; the PCR used

primers composed of both arm region and part of a

selectable marker gene (Fig. 1a-i). The resulting vector,

however, generally contains very short arms (c. 50 bp),

because of the difficulties in synthesizing a much longer

DNA chain. Thus, the efficiency of homologous recombina-

tion with the vectors is often quite low. For example, when

an attempt was made to disrupt the SUB1 gene encoding

a transcriptional coactivator (Henry et al., 1996; Knaus

et al., 1996) using this conventional method, no targeted

disruptant could be identified among 33 histidine autotroph

(HIS1) transformants (0/33). To improve the homologous

recombination frequency, a new method was developed to

obtain a targeting vector harboring longer arms. First, very

few correctly targeted yeast cells were assumed to be present

among many HIS1transformants (433 in this case) ob-

tained by the conventional method, most of which have

randomly integrated HIS3 gene disruption cassette. Then,

by setting up a PCR using the genomic DNA from the

correctly targeted transformants as a template, the DNA

region encoding the selectable marker gene was amplified

with longer flanking sequences, which can serve as the

targeting vector (Fig. 1a-ii). For this purpose, genomic

DNA was extracted from �200 HIS1transformants, and

PCR using the genomic DNA mixtures from the transfor-

mants as a template was performed. The resulting PCR

products were almost exclusively derived from the SUB1

wild-type allele, not from the sub1-disrupted allele (Fig. 1b,

lane 1). A trial to selectively amplify the DNA fragments

from the sub1-disrupted allele was attempted. Toward this

aim, the genomic DNA mixture was digested with a

FEMS Yeast Res 8 (2008) 193–194 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

restriction enzyme that cuts the wild-type allele, but not the

correctly targeted allele (Fig. 1a). When the EcoRV-digested

DNA was used as template DNA in PCR, genomic fragments

derived from the sub1-disrupted allele were amplified (Fig.

1b, lane 2). Using the DNA fragment as a targeting vector, the

correct disruptant at a frequency of nine of 15 HIS1trans-

formants (9/15) was obtained, which was at least 20-fold

higher than the frequency obtained with the targeting vector

prepared using the conventional method. This efficient gene

targeting method is not SUB1 locus-specific because higher

efficiency with another gene was also observed, YOR289W

(1/32 using the conventional method; 6/15 using the im-

proved method). Therefore, this method might be a useful

alternative to the conventional method when the frequency

of the correct gene disruption is very low.

The Saccharomyces Genome Deletion Project has been

completed, and construction and analyses of mutant strains

with multiple gene deletions will become more important. It

is believed that this improved method will aid in construct-

ing multiple genes deleted strains in S. cerevisiae. Moreover,

it can be assumed that the strategy described here could be

applied to other nonconventional yeasts including patho-

genic fungi Candida, Cryptococcus and Aspergillus species. In

these fungi, PCR-based gene disruption is utilized, but the

frequency of the correct gene disruption is often very low

compared with S. cerevisiae. Thus, this strategy could also

benefit in manipulating genes of these nonconventional

yeasts and fungi.

References

Baudin A, Ozier-Kalogeropoulos O, Denouel A, Lacroute F &

Cullin C (1993) A simple and efficient method for direct gene

deletion in Saccharomyces cerevisiae. Nucleic Acids Res 21:

3329–3330.

Henry NL, Bushnell DA & Kornberg RD (1996) A yeast

transcriptional stimulatory protein similar to human PC4.

J Biol Chem 271: 21842–21847.

Knaus R, Pollock R & Guarente L (1996) Yeast SUB1 is a

suppressor of TFIIB mutations and has homology to the

human co-activator PC4. EMBO J 15: 1933–1940.

Rothstein R (1991) Targeting, disruption, replacement, and allele

rescue: integrative DNA transformation in yeast. Methods

Enzymol 194: 281–301.

Winzeler EA, Shoemaker DD, Astromoff A et al. (1999)

Functional characterization of the S. cerevisiae genome by gene

deletion and parallel analysis. Science 285: 901–906.

SUB1

HIS3

50 bp50 bp

SUB1

HIS3446 bp 300 bp

1.8 kbp

2.5 kbp

EcoRVFwd primer 2 Rev primer 2

Fwd primer 2 Rev primer 2

(i) Targeting vector 1

(ii) Targeting vector 2

Fwd primer 1 Rev primer 1

(5′ Arm) (3′ Arm)

sub1::HIS3 allele

WT allele

1.8 kbp

1 3

2.5 kbp

1.8 kbp

(5′ Arm) HIS3 (3′ Arm)

2

(a)

(b)

Fig. 1. Schematic illustrations of targeting vector construction strategies using the conventional and improved methods. (a) The locations of primers for

PCR and the sizes of the DNA fragments are shown. The PCR fragment from the wild-type allele can be cut with the EcoRV restriction enzyme whereas

the fragment from the targeted allele cannot. The primer sequences are as follows: Fwd primer 1, 5 0-AATCTTTCCGTACACATCAATTTTTCGACATATA

TACAAACACAAGCGCTCCTAGCATGTACGTGAG-30; Rev primer 1, 50-TTCACTTATGTCGTCTTCAGCCTTGTTCATTTCAGCTTCCAAGCTTTGAGCGGG-

GACACCAAATATG-30; Fwd primer 2, 50-ACGTATCTCGAGCTCGTCAATG-30; Rev primer 2, 50-ACCCGGTACCATTCTCACTGTG-30. (b) Agarose

gelelectrophoresis of the PCR products amplified with Fwd primer 2 and Rev primer 2. The templates used were as follows: lane 1, nondigested genomic

DNA mixture from HIS1transformants; lane 2, EcoRV-digested genomic DNA mixture from HIS1transformants; lane 3, genomic DNA prepared from

wild-type yeast strain.

FEMS Yeast Res 8 (2008) 193–194c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

194 H. Koyama et al.