Benchmarks

Benchmarks
Extraction of genomic DNA from yeasts for PCR-based applications
Marko Loke, Kersti Kristjuhan, and Arnold Kristjuhan Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia
BioTechniques 50:325-328 (May 2011) doi 10.2144/000113672 Keywords: DNA extraction; yeast; PCR Supplementary material for this article is available at www.BioTechniques.com/article/113672.

We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1 107 cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of 3500 bp.
The cell wall is the main obstacle for quick and easy lysis of yeast cells and therefore must be disrupted for efficient recovery of genomic DNA (gDNA). Conventional methods for gDNA preparation from yeast cells utilize either enzymatic degradation (1) or beating with glass beads (2), followed generally by lysis of cells with detergent and extraction of gDNA with phenolchloroform. When analyzing a large number of samples, these methods are time-consuming and relatively expensive. For quick genotyping, cells can also be lysed by repeated freeze-thaw cycles in a buffer containing Triton X-100 and SDS, followed by extraction of gDNA with chloroform (3). Although this method is considerably faster than conventional gDNA preparation methods, it requires transfer of the sample to a new test tube after chloroform extraction, which slows down the protocol and makes it inconvenient for simultaneous handling of large number of samples. Alternatively, gDNA can be prepared in a single tube by
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simple SDS treatment (4). However, as we demonstrate, the yield of gDNA by this protocol is relatively low and the results are poorly reproducible. In addition, a large number of cells is required for the protocol and the buffer for subsequent PCR reactions has to be supplemented with Triton X-100 (4). As lithium acetate (LiOAc) is commonly used in yeast transformation protocols to weaken cell walls (5,6), we decided to combine it with SDS to develop a quick, efficient, and robust method for gDNA extraction from yeasts. Eight single colonies of Saccharomyces cerevisiae were picked from a yeast peptone dextrose (YPD) plate, suspended in 100 L 200 mM LiOAc 1% SDS solution, and incubated at 70C for 15 min. After incubation, 300 L 96% ethanol was added, the samples were mixed by brief vortexing, and DNA was collected by centrifugation at 15,000 g for 3 min. Precipitated DNA was dissolved in 100 L TE, cell debris was spun down by brief centrifugation

(15,000 g for 1 min), and 1 L supernatant was used for PCR. Two different gDNA fragments, of 489 and 2383 bp, were amplified in separate reactions and the PCR products were analyzed on 0.9% TAE-agarose-ethidium bromide gel electrophoresis (Figure 1A; lanes 18). For comparison, gDNA from another eight S. cerevisiae colonies was prepared by the simple SDS treatment (4) (see Supplementary Material for detailed protocols of other DNA preparation protocols). As shown on Figure 1A, both the 489-bp and 2383-bp fragments were efficiently amplified from all samples prepared by LiOAc-SDS method (Method A in Figure 1A; lanes 18), while variable signals of the 489-bp fragment and no signal of the 2383-bp fragment was detected from samples prepared by the simple SDS treatment (Method B in Figure 1A; lanes 916). We also determined the yield and purity of the gDNA prepared by our LiOAc-SDS method and by the simple SDS method. S. cerevisiae cells were grown in liquid YPD media and aliquots of the culture were used for the preparation of gDNA. Cells (1 10 8) were collected for both methods, and the sample corresponding to gDNA from 1 10 7 cells was run on 0.9% TAE-agarose gel together with serial dilutions of pure yeast gDNA prepared by zymolyase-SDS treatment (7) (Method D) as concentration standards (Figure 1B; lanes 14). Approximately 100 nanograms of gDNA can be extracted from 1 10 7 cells by the LiOAc-SDS method, while the yield of gDNA by the simple SDS method is ~10 lower (Figure 1B; lanes 56). Both preparations contain high amounts of RNA that can be removed by subsequent RNase A treatment (Figure 1B; compare lanes 56 to lanes 78). Spectrophotometric quantification of gDNA after RNase A and proteinase K treatments of the samples confirmed the yield of 100 ng of gDNA per 1 10 7 cells by LiOAc-SDS method (data not shown). Together, we conclude that the lysis of cells by LiOAc-SDS treatment is a simple, efficient, and reliable method for extraction of yeast gDNA. Next, we optimized the protocol to find out the critical components for effective gDNA extraction by LiOAc-SDS lysis. We tested different concentrations of SDS (Figure 1C) and lithium acetate (Figure 1D) in the lysis solution. We also used different incubation times (Figure 1E) and temperatures (Figure 1F). In all experiments, 100-L aliquots of mid-log phase liquid culture were collected and
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Benchmarks

Figure 1. Optimization of LiOAc-SDS lysis in S. cerevisiae. (A) PCR amplification of S. cerevisiae gDNA prepared from single yeast colonies by LiOAc-SDS (Method A; lanes 18) or by SDS treatment (Method B; lanes 916). gDNA fragments (489 bp and 2383 bp) from VPS13 locus were amplified with FirePol Taq polymerase (Solis BioDyne, Tartu, Estonia) for 30 cycles (95C for 15 s, 58C for 30 s, and 72C for 150 s). The reaction buffer was supplemented with 1% Triton X-100 for samples 916 (as recommended in Reference 4). (B) Total lysate of 1 107 cells prepared by Methods A and B was analyzed on a 0.9% TAE-agarose gel. Lanes 14: dilution series of yeast gDNA concentration standards prepared by Method D; lane 5: gDNA prepared by Method A; lane 6: gDNA prepared by Method B; lanes 78: gDNA prepared by Methods A and B were purified further by RNase A and proteinase K treatments, followed by phenol:chloroform extraction and ethanol precipitation of gDNA. A longer exposure of the gDNA bands is shown underneath the main gel. (CF) Optimization of LiOAc-SDS lysis protocol conditions. Concentrations of SDS and LiOAc, lysis time and temperature were tested (panels C, D, E, and F, respectively). Variable parameters are indicated below the panels and constant parameters are shown on top. gDNA fragment from VPS13 locus (489 bp) was amplified by PCR. RT, room temperature. See Figure 2B for DNA size marker (lanes M). PCR products were analyzed on 0.9% TAE-agarose gel electrophoresis, stained with ethidium bromide, and photographed. Sequences of PCR primers are listed in the Supplementary Material.

Figure 2. PCR amplicon size of LiOAc-SDS extracted DNA and the lysis of various yeast species. (A) Comparison of genomic DNA amplification efficiencies of various amplicon sizes. gDNA was prepared by LiOAc-SDS (Method A), by glass bead beating (Method C) and by zymolyase-SDS (Method D) from cells grown overnight in 10 mL liquid YPD media. 718-bp to 5920-bp DNA fragments were amplified from S. cerevisiae VPS13 locus. (B) LiOAc-SDS lysis of S. cerevisiae (strain W303), P. pastoris (strain GS115), C. albicans (strain CAI4), S. pombe (strain 972), H. polymorpha (strain CBS4732), and K. lactis (strain IFO 1267) cells grown for three days on YPD-agarose plates. A single colony from each species was taken for DNA extraction by LiOAc-SDS method. The following loci were amplified: VPS13 (S. cerevisiae ; 979 bp), URA3 (P. pastoris; 1365 bp), MIP1 (C. albicans; 350 bp), cut11+ (S. pombe ; 990 bp), HpMAL1 (H. polymorpha; 655 bp), and TRP1 (K. lactis; 1010 bp). Lane M on both panels: GeneRuler 1-kb DNA Ladder (Fermentas, Vilnius, Lithuania). PCR products were analyzed by 0.9% TAE-agarose gel electrophoresis, stained with ethidium bromide, and photographed. Sequences of PCR primers are listed in the Supplementary Material.

suspended in 100 L of lysis solution. In these experiments, we varied the test conditions so that the tested component was the limiting factor in the reaction. For example, short incubation time was used for testing of different temperatures (Figure 1F); reactions were carried out at room temperature for testing of SDS concentration (Figure 1C) and incubation time (Figure 1E); and long incubation time at high temperature was used for determination of optimal LiOAc concentration (Figure 1D). To summarize, we recommend using 200 mM LiOAc and 1% SDS in the lysis solution and carrying out the lysis at 70C for 5 min (See
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summary of the protocol in the Supplementary Material). Next, we determined the maximal length of PCR products that can be amplified from DNA obtained by the LiOAc-SDS method. One hundred microliter aliquots of cells from mid-log phase liquid culture were lysed with LiOAc-SDS (Method A) and for reference, DNA was also prepared by glass bead beating (2) (Method C) and by zymolyaseSDS treatment followed by phenolchloroform extraction (7) (Method D). As shown in Figure 2A, amplification of DNA fragments 3505 bp was successful regardless of the method used. However,

amplification of larger DNA fragments (4449 bp and 5920 bp) required delicately handled and purified DNA (Method D). Based on these results, we recommend the use of LiOAc-SDS DNA extraction method if the desired PCR amplicon does not exceed 3500 bp. We also confirmed that our method is suitable for direct gDNA extraction from other yeasts. Altogether six yeast species (K. lactis, H. polymorpha, S. pombe, C. albicans, P. pastoris, and S. cerevisiae) were tested. One colony from each YPD plate was lysed by LiOAc-SDS method. The lysis of cells from all selected species was successful as DNA was amplified from
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all samples (Figure 2B), indicating that the LiOAc-SDS method can be used for DNA extraction from many different yeast species. In addition to simple PCR-based genotyping, we have used LiOAc-SDS extracted and Pfu DNA polymerase amplified S. cerevisiae gDNA for BigDye v3.1based sequencing and we obtained DNA sequence readout of an entire 850-bp PCR fragment (data not shown). We have also used LiOAc-SDSextracted gDNA directly in real-time qPCR reactions for quick genotyping of yeast colonies (data not shown). However, although this method is suitable for gDNA extraction for a variety of downstream PCR applications, we do not recommend it for direct use in Southern blot analysis or nextgeneration sequencing, as the presence of RNA (Figure 1B) and proteins in the lysate might affect the critical steps in these protocols. In summary, we have developed a quick and reliable method for gDNA extraction from yeasts that is suitable for PCR amplification of DNA fragments 3500 bp. The protocol can be carried out in a single test tube 15 min and cells from both liquid and solid media can be used. The method is suitable for routine genotyping of yeasts either by simple detection of PCR products or for initial amplification of genomic DNA for sequencing, procedures that are widely used for analysis of scientific, environmental, industrial and clinical samples.

subject to the Wellcome Trust Public Access Policy.

Competing interests References

The authors declare no competing interests.

Acknowledgments

1.  L ing, M., F. Merante, and B.H. Robinson. 1995. A rapid and reliable DNA preparation method for screening a large number of yeast clones by polymerase chain reaction. Nucleic Acids Res. 23 :4924-4925. 2.  A mberg, D.C., D.J. Burke, and J.N. Strathern. 20 05. Methods in Yeast Genet ics: A Cold Spr i ng Ha rbor Laboratory Course Manual. 2005 ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 3. H  arju, S., H. Fedosyuk, and K.R. Peterson. 2004. Rapid isolation of yeast genomic DNA: Bust n Grab. BMC Biotechnol. 4 :8. 4.  A k ad a , R ., T. Mu ra k a ne, a nd Y. Nish izawa. 2 0 0 0 . DNA ex t ract ion method for screening yeast clones by PCR. Biotechniques 28 :668-670, 672, 674. 5.  I to, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153 :163-168. 6.  Thompson, J.R., E. Register, J. Curotto, M. Kurtz, and R. Kelly. 1998. An improved protocol for the preparation of yeast cells for transformation by electroporation. Yeast 14 :565-571. 7.  S edman, T., P. Jers, S. Kuusk, and J. Sedman. 2005. Helicase Hmi1 stimulates the synthesis of concatemeric mitochondrial DNA molecules in yeast Saccharomyces cerevisiae. Curr. Genet. 47:213-222.

We thank Tiina Tamm, Tiina Alame, and Signe Vrv for critical reading of the manuscript and providing the strains of different yeast species. This work was supported by the Wellcome Trust International Senior Research Fellowship (grant no. 081756) and EMBO Installation grant no. 1454. This paper is

Received 2 December 2010; accepted 16 March 2011. Address correspondence to Arnold Kristjuhan, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, Tartu 51010, Estonia. e-mail: arnoldk@ut.ee To purchase reprints of this article, contact: biotechniques@fosterprinting.com

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