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[Yeast vol. 13 iss. 8] TAKITA, YOKO_ TAKAHARA, MANABU_ NOGAMI, SATORU_ ANRAKU, YASUHIRO - Applications of the Long and Accurate Polymerase Chain Reaction Method in Yeast Molecular Biology_ Direct Sequencing of the Ampl (1997) [ - lib
[Yeast vol. 13 iss. 8] TAKITA, YOKO_ TAKAHARA, MANABU_ NOGAMI, SATORU_ ANRAKU, YASUHIRO - Applications of the Long and Accurate Polymerase Chain Reaction Method in Yeast Molecular Biology_ Direct Sequencing of the Ampl (1997) [ - lib
A DNA fragment longer than 10 kb can be amplified by the long and accurate polymerase chain reaction (LA-PCR)
method. We demonstrate here applications of this technique in molecular biological studies of Saccharomyces
cerevisiae. We have shown that DNA fragments amplified by LA-PCR can be directly used as a template in the
chain-termination sequencing protocol, making it possible to quickly identify the DNA insert of yeast genomic
library clones. We have also shown that the amplified yeast DNA can easily be introduced into yeast by
co-transformation with linearized vector DNA. Overlapping DNA between the amplified yeast fragment and the
vector must be more than 20 bp long in order to obtain 90% or more correct recombinant plasmids. These results
suggest that simple amplification of yeast clones by LA-PCR can replace the previous procedures of yeast clone
recovery, consisting of transformation of Escherichia coli, propagation of plasmids in E. coli and preparation of
plasmid DNA. ? 1997 by John Wiley & Sons, Ltd.
— Saccharomyces cerevisiae; long and accurate (LA)-PCR; homologous recombination; co-
transformation
? 1997 by John Wiley & Sons, Ltd . 13: 763–768 (1997)
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Table 1. Primers used in this study.
Primera Sequence
Sequencing primers
N1 GCG ACC ACA CCC GTC CTG T
N2 CGC GAT CAT GGC GAC CAC ACC CG
N3 CGA TCA TGG CGA CCA CAC CC
N4 CTA CGC GAT CAT GGC GAC CAC ACC
N5 CGA CTA CGC GAT CAT GGC GA
C1 CCA CGA TGC GTC CGG CGT AG
C2 GGT GAT GCC GGC CAC GAT GCG TCC
C3 CAG CAA CCG CAC CTG TGG CGC
C4 GCC AGC AAC CGC ACC TGT GGC
C5 CGA TAT AGG CGC CAG CAA CCG
a
Suitable primers for sequencing analysis are shown in Figure 1.
b
The overlapping regions are underlined.
screened from a YEp-13-based genomic library Materials and Methods. We found that primers
(Yoshihisa and Anraku, 1989) based on its comp- efficient for amplification of the DNA insert are
lementation of a temperature-sensitive calmodulin the Tc-N and Tc-R oligonucleotides described in
mutation (Ohya and Botstein, 1994). Yeast DNA Table 1, which are complementary to segments
was isolated from the strain (YTY228) harboring upstream and downstream of the BamHI site in the
pST26 by using a smash-and-grab method tetracycline gene on YEp13 (Figure 1). After 0·7%
(Strathern and Higgins, 1991) as described in agarose gel electrophoresis, the LA-PCR product
? 1997 by John Wiley & Sons, Ltd . 13: 763–768 (1997)
766 . .
Figure 1. Schematic representation of the YEp13 library clone marked with primers
for LA-PCR amplification (Tc-N and Tc-R) and primers for sequencing analysis (N1,
N2, N3, N4, N5, C1, C2, C3, C4 and C5). Arrows indicate the 5* ] 3* orientation of
the primers. Bold arrows represent suitable primers for sequencing analysis. Only
DNA regions covered by Tc-N and Tc-R are to scale.
was purified with DNA prep, used in the sequenc- technique using a LA-PCR product as a template,
ing reaction, and applied to the automated DNA one can quickly obtain information about the
sequencing apparatus. yeast genomic clone. It takes only several hours to
Among ten primers that we prepared for the prepare the DNA template for sequencing, starting
DNA sequencing analysis (Table 1), the most from the grown yeast culture. This simple pro-
accurate base detection (>98%) was possible with cedure is substituted for the conventional 3-day
N1 for the analysis of the left end of the insert. procedure that includes transformation of the
Satisfactory base calling was provided equally with yeast plasmid DNA into E. coli, propagation of
C1, C2 and C4 for the right end in the automated E. coli cells harboring the plasmid, and purifi-
DNA sequencing system. The better sequencing cation of the plasmid. Now that the complete
primers contain a slightly higher GC content than sequence of the yeast genome is known, infor-
the other primers. Sequencing about 400 bases at mation from both ends of the DNA insert is
both ends of the pST26 insert revealed that the enough to provide the entire sequence of the insert.
DNA insert maps from 1207 bases downstream of In addition to determining the sequence of library
the end of YBR107 to 2545 bases downstream of inserts, this technique should be generally appli-
the end of YBR112 on chromosome II (Feldmann cable to analysis of any DNA at long range that
et al., 1994), which contains the calmodulin gene. the conventional PCR technique fails to cover.
Besides identification of the calmodulin clone, this When DNA clones are recovered from yeast,
protocol was utilized successfully in the easy iden- they must then be reintroduced into yeast. Taking
tification of several other genomic library clones this situation into account, we next examined
(unpublished results). whether the amplified DNA fragment could be
Isolation of S. cerevisiae genes by complement- introduced easily into yeast. LA-PCR is also
ation is one of the most powerful tools in yeast applicable here, because its high fidelity is sufficient
molecular biology. After finding genomic DNA for functional complementation experiments. The
library clones, the next step is determination of the average error rate was determined to be 1·05 per
complementing genes. With the direct sequence 105 bp for 16 cycles of LA-PCR (Barnes, 1994).
? 1997 by John Wiley & Sons, Ltd . 13: 763–768 (1997)
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Table 2. Frequency of complementing transformants Table 3. Frequency of complementing transformants
after co-transformation of the amplified RHO1 with after co-transformation of the amplified RHO1 with
linearized pRS314. linearized pRS314.
? 1997 by John Wiley & Sons, Ltd . 13: 763–768 (1997)
768 . .
effectively into yeast by co-transformation. These Cheng, S., Fockler, C., Barnes, W. M. and Higuchi, R.
experimental procedures will be useful not only for (1994). Effective amplification of long targets from
yeast genes which are difficult to propagate in cloned inserts and human genomic DNA. Proc. Natl.
E. coli, but also for yeast genes which are difficult Acad. Sci. USA 91, 5695–5699.
Feldmann, H., Aigle, M., Aljinovic, G., et al. (1994).
to manipulate with classical molecular biological
Complete DNA sequence of yeast chromosome II.
techniques such as restriction enzyme digestion EMBO J. 13, 5795–5809.
and ligation. Moreover, with this protocol, one Ma, H., Kunes, S., Schatz, P. J. and Botstein, D. (1987).
can reduce the time required for the DNA recovery Plasmid construction by homologous recombination
and move on to the functional analysis of the gene. in yeast. Gene 58, 201–216.
We assume, however, that this protocol is not Ohya, Y. (1996). LA-PCR-based quick method for the
suitable in all circumstances. When two different identification of genes responsible for the comp-
pBR322-derived plasmids are present in the same lementation of Saccharomyces cerevisiae mutations.
yeast strain, both insert DNAs would be amplified, BioTechniques 20, 778–779.
resulting in a mixed population. Also, if the yeast Ohya, Y. and Botstein, D. (1994). Diverse essential
functions revealed by complementing yeast calmodu-
strain contains plasmids or a DNA region homolo-
lin mutants. Science 263, 963–966.
gous to the end of the linearized vector, the linear Ohya, Y., Miyamoto, S., Ohsumi, Y. and Anraku, Y.
vector can circularize frequently without incorpor- (1986). Calcium-sensitive cls4 mutant of Saccharomy-
ating the amplified DNA fragment. Nevertheless, ces cerevisiae with a defect in bud formation. J.
we believe that correct understanding of the Bacteriol. 165, 28–33.
homologous recombination process will allow Qadota, H., Python, C., Inoue, S. B., et al. (1996).
application of this protocol to other yeast molecu- Identification of yeast RHO1 GTPase as a regulatory
lar biological techniques, including random and subunit of the 1,3-â-glucan synthase. Science 272,
site-directed mutagenesis, functional analysis of 279–281.
genes, and protein engineering. Ratzkin, B. and Carbon, J. (1977). Functional expres-
sion of cloned yeast DNA in Escherichia coli. Proc.
Natl. Acad. Sci. USA 74, 487–491.
ACKNOWLEDGEMENTS Saiki, R. K., Gelfand, D. H., Stoffel, S., et al. (1988).
Primer-directed enzymatic amplification of DNA
We would like to thank K. Richards for critical with a thermostable DNA polymerase. Science 239,
reading of the manuscript. This study was sup- 487–491.
ported in part by a grant-in-aid for Scientific Schiestl, R. H. and Gietz, R. D. (1989). High efficiency
Research from the Ministry of Education, Science, transformation of intact yeast cells using single
Sports and Culture of Japan (Y. O.). stranded nucleic acids as a carrier. Curr. Genetics 16,
339–346.
Strathern, J. N. and Higgins, D. R. (1991). Recovery of
plasmids from yeast into Escherichia coli: shuttle
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? 1997 by John Wiley & Sons, Ltd . 13: 763–768 (1997)