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
Vol. 50 | No. 5 | 2011

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

325

(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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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
Vol. 50 | No. 5 | 2011

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,

327

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.

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Acknowledgments

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

subject to the Wellcome Trust Public

Access Policy.

Competing interests

The authors declare no competing

interests.

References

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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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