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Nucleic Acids Research Pages 860-861

PCR- and ligation-mediated synthesis of marker cassettes with long flanking homology regions for gene disruption in Saccharomyces cerevisiae
Acknowledgement
References
Related Papers Recently Published In Nucleic Acids Research


PCR- and ligation-mediated synthesis of marker cassettes with long flanking homology regions for gene disruption in Saccharomyces cerevisiae

PCR- and ligation-mediated synthesis of marker cassettes with long flanking homology regions for gene disruption in Saccharomyces cerevisiae Jun-ichi Nikawa* and Machiko Kawabata

Department of Biochemical Engineering and Science, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology, Iizuka, Fukuoka 820, Japan

Received September 30, 1997; Revised and Accepted December 10, 1997

ABSTRACT

We developed a novel method for synthesizing marker-disrupted alleles of yeast genes. The first step is PCR amplification of two sequences located upstream and downstream of the reading frame to be disrupted. Due to the addition of non-specific single A overhangs by Taq DNA polymerase, each PCR product can be ligated with a marker DNA which has T residues at its 3' ends. After amplification of individual ligation products through the second PCR, both products are mixed and annealed, and the single strand is converted to a double strand by an extension reaction. The final step is PCR amplification of the fragment composed of a selectable marker and two flanking sequences with the outermost primers. This method is rapid and needs only short oligonucleotides as primers.

Since the entire genome is known, the construction and disruption of specific sequences followed by analyses of the phenotypes is one of the most powerful genetic tools for the yeast, Saccharomyces cerevisiae. The classical strategy for gene disruption requires the isolation of a gene and digestion with restriction enzymes (1). Because of the use of restriction enzymes to digest the DNA fragments, it is sometimes very hard to construct appropriate fragments disrupted by a marker DNA. To overcome this problem, several advanced methods for constructing such deletions have been reported involving PCR. The synthesis of PCR-mediated DNA fragments consisting of a marker cassette flanked by short homology regions to the target locus is now a widely used method (2,3). However, the transformation efficiency with these PCR-products having small homology regions at both ends is inferior to that with DNA having long homology regions. Furthermore, the sequence polymorphism in different strain backgrounds will hinder homologous recombination when small homology regions are used. Elongation of the homology regions flanking the marker gene would solve this problem and several advanced methods have been reported. However, it is still necessary to isolate the DNA fragment of interest, or to use complicated procedures (4-6). Here we attempted to develop a new method for constructing a marker cassette with long flanking homology regions involving a simple procedure. Our method does not involve the purification of the DNA fragment, such as isolation on an agarose gel, phenol-extraction, and ethanol-precipitation of the DNA fragment. Taq polymerase exhibits a non-template-dependent activity, adding a single A residue to the 3' ends of PCR products (7). This allows us to ligate the PCR product with a marker DNA fragment having a T residue at each of its 3' ends. Our method is based on the PCR amplification of DNA fragments with Taq polymerase and the ligation of PCR products with marker DNAs.


Figure 1.Strategy for constructing gene disruption cassettes by means of PCR and ligation. Step 1: first PCR reaction. Two separate regions of a target gene are PCR amplified with oligonucleotide primers specific for the target sequence and genomic DNA as a template. Step 2: ligation. Two PCR products are ligated with a DNA fragment of a marker gene through two separate reactions. Step 3: second PCR reaction. The ligated fragments are PCR amplified separately. Step 4: annealing and extension. Two PCR-amplified fragments are mixed, denatured, annealed, and then extended with DNA polymerase. Step 5: third PCR reaction. The final product is PCR amplified with the outermost primers.

We first prepared marker DNA fragments having a single 3' T residue at both 3' ends. A pUC19-derived vector, pUC-T, having two adjacent Eam1105I restriction endonuclease sites was generated by inserting an annealed pair of synthetic oligonucleotides, 5'-AATTCGACAGACTGTCGACAAGCTTGACAGTCTGTC-3' and 5'-AGCTGACAGACTGTCAAGCTTGTCGACAGTCTGTCG-3', into pUC19 between the EcoRI and HindIII sites. pT-URA3 was then constructed by inserting the 1.2 kb HindIII fragment of URA3 into pUC-T at the HindIII site. pT-ADE8 and pT-HIS3 were constructed by inserting the 1.1 kb DraI fragment of ADE8 and the 1.3 kb Eam1105I/XhoI fragment of HIS3, which had been treated with Klenow fragment, into the HincII site of pUC-T, respectively. DNA fragments of marker genes having T residues at their 3' ends were prepared by digesting the respective plasmids with Eam1105I.


Figure 2. Disruption of yeast genes. (A) Fragments synthesized by PCR. Electrophoresis of PCR products (3 µl each) was performed on a 0.7% agarose gel. Lanes 1 and 2, fragments synthesized in the first PCR with genomic DNA as a template (~10 ng), and two sets of primers near the start and stop codons of EPT1, respectively. Lanes 3 and 4, fragments synthesized in the second PCR with ligation products as templates and two sets of primers, with the combination of a primer near the start codon plus one of the primers for the marker gene, URA3, and a primer near the stop codon plus the other primer for URA3, respectively. Lane 5, final product of the third PCR with the outermost primers. Lane 6, DNA size markers. The first PCR started with initial denaturation for 3 min at 94°C, then 25 cycles of: 94°C for 1 min, 50°C for 2 min, 72°C for 1 min. The second PCR consisted of 20 cycles of: 94°C for 30 s, 55°C for 30 s, 72°C for 2 min. Annealing and extension consisted of denaturation for 10 min at 94°C followed by decreasing the temperature to 37°C gradually (60 min), incubation at 37°C for 15 min, and extension at 72°C for 3 min. The third PCR consisted of 15 cycles of: 94°C for 30 s, 55°C for 30 s, 72°C for 3 min. (B) Verification of gene disruption. PCR was performed on yeast strains using ~20 base outside primers for the cassettes and genomic DNA as a template. More conveniently, gene disruption was verified by outermost primers used for constructing the cassettes and all transformants determined contained disrupted alleles (data not shown). The PCR products were analysed by 0.7% agarose gel electrophoresis. Lane 1, wild-type EPT1; lane 2, ept1::URA3; lane 3, wild-type INO2; lane 4, ino2::HIS3; lane 5, wild-type INO4; lane 6, ino4::ADE8; lane 7, DNA size markers.

The synthesis of marker cassettes with long flanking homology regions comprises three steps of PCR involving one ligase reaction (Fig. 1). In the first PCR step, two fragments of DNA were amplified with two sets of primers. The PCR products were both produced through two separate reactions. The 17mer in length of primers is sufficient and total yeast genomic DNA can be used as a template for the first PCR. In the second step, PCR products were ligated with the marker gene, followed by the second PCR. The first PCR products and ligation mixtures can be used for the ligation reaction and the second PCR reaction without purification, respectively. In the next step, two PCR products were mixed and annealed, and then the extension reaction was carried out with Taq polymerase. Finally, the marker cassettes with long flanking regions were amplified by the third PCR and used for replacement of the target gene in the genomic DNA. To obtain better results, excess primers should be removed from the second PCR products by centrifugation in a micro-concentrator (SUPRECTM-02; Takara Shuzo Co.). The PCR products used for constructing the URA3-disrupted EPT1 gene fragment are shown in Figure 2A. At each step of the PCR reaction, the sizes of the generated products observed on agarose gel electrophoresis were in good agreement with the predicted sizes of the fragments. Using the final PCR product we could disrupt the chromosomal EPT1 in strain D452-2 (a leu2 ura3 his3) by the lithium acetate method (8). We have also successfully deleted genes INO2 and INO4 in strain SP1 ([alpha] leu2 ura3 his3 trp1 ade8) by using this procedure with HIS3 and ADE8 as marker genes, respectively. We verified by PCR with outside primers for the replaced fragments that the transformants contained the constructs integrated into the expected loci (Fig. 2B).

The preparation of a marker module flanked by long homology regions of several hundred base pairs, through the simple method described here, would be advantageous for fast and high-throughput gene disruption. Furthermore, the ligation- and PCR-mediated synthesis of DNA fragments described here will be applicable to general methods for constructing DNA fragments.

ACKNOWLEDGEMENT

This work was supported in part by a Grant-in-Aid for Scientific Research from The Ministry of Education, Science and Culture of Japan.

REFERENCES

1 Rothstein,R. (1983) Methods Enzymol., 101, 202-211. MEDLINE Abstract

2 Baudin,A., Ozier-Kalogeropoulos,O., Denouel,A., Lacroute,F. and Cullin,C. (1993) Nucleic Acids Res., 21, 3329-3330. MEDLINE Abstract

3 Manivasakam,P., Weber,S.C., McElver,J. and Schiestl,R.H. (1995) Nucleic Acids Res., 23, 2799-2800. MEDLINE Abstract

4 Amberg,D.C., Botstein,D. and Beasley,E.M. (1995) Yeast, 11, 1275-1280. MEDLINE Abstract

5 Wach,A., Brachat,A., Pohlmann,R. and Philippsen,P. (1994) Yeast, 10, 1793-1808. MEDLINE Abstract

6 Wach,A. (1996) Yeast, 12, 259-265. MEDLINE Abstract

7 Clark,J.M. (1988) Nucleic Acids Res., 16, 9677-9686. MEDLINE Abstract

8 Ito,H., Fukuda,Y., Murata,K. and Kimura,A. (1983) J. Bacteriol., 153, 163-168.

RELATED PAPERS RECENTLY PUBLISHED IN NUCLEIC ACIDS RESEARCH

Oldenburg,K.R., Vo,K.T., Michaelis,S. and Paddon,C. (1997) Recombination-mediated PCR-directed plasmid construction in vivo in yeast. Nucleic Acids Res. 25, 451-452.

Lafontaine,D. and Tollervey,D. (1996) One-step PCR mediated strategy for the construction of conditionally expressed and epitope tagged yeast proteins. Nucleic Acids Res. 24, 3469-3472.

*To whom correspondence should be addressed. Tel: +81 948 29 7822; Fax: +81 948 29 7801; Email: nikawa@bse.kyutech.ac.jp

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