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Epicentre Forum 5 (4)
Les Hoffman and Erich Moan, Epicentre Technologies
The yeast Saccharomyces cerevisiae is an important genetics
and molecular biological tool whose total genome has recently been sequenced.
The ability to isolate yeast nucleic acids including chromosomal DNA,
plasmids, and yeast artificial chromosomes (YACs), is essential
to many lines of research. For example, thousands of YACs must be extracted
from yeast cells for high-throughput gene mapping and positional cloning.1
Yeasts in general, including S. cerevisiae and Candida
albicans, present particular difficulties for the isolation of
genomic DNA. The yeast cell wall is resistant to lysis using common
methods for mammalian cell, viral, and bacterial DNA purification.
Traditionally, yeast cells have been lysed with various enzyme preparations.2 In
many protocols, the beta-1,3-glucanase known as lyticase or zymolase
is used to remove a portion of the yeast cell wall.2,3 Proteases
are a common contaminant of lyticase and other yeast lytic enzyme preparations,
and lysis efficacy varies from preparation to preparation. Detergent
lysis of the spheroplasts produced by lyticase action is then required,
followed by various precipitation steps, spin column steps, or both,
resulting in a very cumbersome process with variable DNA yields.
Here, we introduce the MasterPure™ Yeast DNA Purification
Kit, a nonenzymatic approach to yeast nucleic acid purification. With
the MasterPure procedure, we have eliminated the need for lyticase and
proteolytic enzymes. Using a simple, short protocol, we obtain DNA yields
consistently above those of other commercially available kits. The integrity
of the DNA obtained with the MasterPure Kit, as determined by pulsed
field gel electrophoresis (PFGE), is greater than that of DNA
purified using a kit from another supplier. In this article, we also
emphasize the importance of DNA quantification by fluorimetry rather
than by the more common method of A260 determinations.
DNA preparations
We purified yeast DNA from 1.5 ml cultures of S. cerevisiae, C.
albicans, and C. krusei using the MasterPure Kit and two
other commercially available yeast DNA purification kits (from
suppliers Q and F) according to the manufacturers' protocols. Supplier
F's kit was chosen to represent four suppliers' yeast DNA isolation
kits that have similar reagents, specifications, and protocols. The
kit from supplier Q was supplemented with lyticase from Oerskovia
xanthineolytica (Epicentre) according to supplier Q's recommendations.
Although Solution A provided in supplier F's kit was found to contain
RNase activity, no RNase was used in conjunction with the MasterPure
Kit or the supplier Q kit.
The protocol for extraction of yeast DNA with the MasterPure
Kit is shown in Table 1.
We then performed DNA purifications from 30 ml stationary
phase cultures of S. cerevisiae and C. albicans DNA using
the MasterPure Kit simply by using proportionally greater reagent volumes.
DNA was also isolated from single plate colonies. Yeast colonies from
one week to two months old were removed from YPD agar plates with a pipet
tip and resuspended in 300 µl of Yeast Lysis Solution. The samples
were then processed according to the protocol in Table 1.
- Centrifuge 1.5 ml of a stationary, overnight culture of yeast
(A600= 8-12) and resuspend thoroughly in 300 ml of Yeast Lysis
Solution.
- Heat 65°C for 15 minutes. Chill the tube on ice, add 150 ml
of Protein Precipitation Reagent, and spin in a microcentrifuge
for 10 minutes.
- Add 500 ml of isopropanol to the supernatant and microcentrifuge
as before.
- Wash the nucleic acid pellet with 70% ethanol and resupend
the pellet in 50 ml of TE Buffer.
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Quantification of DNA yields
The Hoefer DyNA Quant™ fluorimeter and Hoechst 33258
fluorescent dye were used to quantify yeast DNA yields according to the
manufacturer's specifications. Calf thymus DNA was used as a double-stranded
DNA standard. Pulsed field gel electrophoresis of yeast DNAs Five hundred
nanograms of each DNA isolated using the MasterPure Kit or supplier Q's
kit were resolved by electrophoresis in a 1% agarose gel in TAE buffer
with field alternation produced by a Hoefer SwitchBack™ Pulse Controller
Model PC500.
Yeast DNA amplification
PCR was chosen as a convenient method to evaluate DNA isolated
with the kits compared in this study. Amplification of the RNA guanine-7-methyltransferase
gene was performed with S. cerevisiae DNA. The primers used were: 5'-CGGCATCCAGGAGGAAAGTAGAATGTCAACCAAACCA-3'
and 5'-GGGGTACCTCAGTTGTTCTTTACGCTTTC-3'. Fifty microliter reactions contained
MasterAmp™ PCR Optimization Kit PreMix A (containing 1.5 mM
MgCl2, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, and 200 µM
each dNTP), 50 pmoles of each primer, 10 ng yeast DNA, and 1.25 units
of MasterAmp Taq DNA Polymerase (Epicentre). The expected PCR
product was 1.35 kb in length. Cycling conditions were: 95°C for 3 minutes;
then 4 cycles of 94°C for 30 seconds, 49°C for 10 seconds with a decrease
of 1.5°C per cycle, and 68°C for 1 minute; followed by 20 cycles of 94°C
for 10 seconds and 70°C for 1 minute, and completed by 72°C for 2 minutes.
The dUTP pyrophosphatase (DUT) locus was chosen for
amplification from C. albicans DNA. Amplification conditions were
the same as those used for S. cerevisiae DNA above. The DUT gene primers
were 5'-GTAAAGTACCAACCAAAGG-3' and 5'-AAGACTACTTTCACTTCACC-3'. The expected
PCR product was 230 bp in length. The reactions were heated at 95°C for
2 minutes, followed by 30 cycles of: 95°C for 30 seconds, 50°C for 30
seconds, and 70°C for 30 seconds, followed by 70°C for 2 minutes.
A haploid yeast cell contains about 70 times the mass of
RNA as DNA.3 The more abundant RNA absorbs at the same wavelength
as DNA in the ultraviolet, confounding the spectrophotometric determination
of yield. Even if yeast RNA is partially or mostly degraded by RNase,
its absorbance at 260 nm greatly overshadows that of DNA in total nucleic
acid preparations (data not shown). The Hoechst fluorescent dye
33258 binds to DNA very specifically and enables accurate fluorimetric
DNA concentration determinations without interference from RNA. Hoechst
33258 binds to RNA less than 4% as efficiently as it does to DNA.
Under usual growth conditions, S. cerevisiae strains reach
a maximum cell density of about 2 x 108 cells/ml.3 Yeast
cell DNA content is about 0.017 pg/cell, from which a maximum yield of
about 5 µg per 1.5 ml of saturated culture can be obtained. Yeast
DNA isolation kits from suppliers Q and F claim yields of 7.5-12.5 µg
DNA from 1.5 ml of haploid yeast culture4,5, more than the
total theoretical quantity of DNA in the cells. Their yields are determined
by A260 measurements.
Here, we have used the more accurate method, fluorimetry,
to measure DNA yields obtained using the MasterPure Kit and kits from
suppliers Q and F. In two separate isolation experiments with duplicate
samples for each experiment, the kit from supplier F averaged 250 ng
of DNA from 1.5 ml of S. cerevisiae, whereas the MasterPure Kit produced
an average of 2,940 ng, almost 12 times as much DNA (Table 2 and
Figure 1). The supplier Q kit produced 1,800 ng of S. cerevisiae DNA
from the same culture volume.
The results with C. albicans were even more striking.
Supplier F's kit yielded an average of 450 ng DNA, whereas the MasterPure
Kit gave an average of 7,630 ng DNA, almost 17 times more (Figure
1 and Table 2). The kit from supplier Q produced 2,900 ng of DNA
from 1.5 ml of culture. Lower DNA yields were obtained from C. krusei than C.
albicans, but the MasterPure Kit exceeded supplier Q's kit by almost
a factor of four and the supplier F kit by a factor of six (Table
2).
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Figure 1. DNA yields from 1.5 ml cultures of S. cerevisiae and
C. albicans using various yeast DNA purification kits. The
data represent the average of two experiments with duplicate extractions
in each experiment. |
| Supplier |
Avg. Yield (ng DNA) |
| F |
175 |
| " |
325 |
| Epicentre |
3,275 |
| " |
2,600 |
| Q |
1,800 |
| " |
1,800 |
| Supplier |
Avg. Yield (ng DNA) |
| F |
450 |
| Epicentre |
8,250 |
| " |
7,000 |
| Q |
3,000 |
| " |
2,800 |
| Supplier |
Avg. Yield (ng DNA) |
| Epicentre |
6,225 |
| Q |
1,600 |
| F |
1,000 |
We used the MasterPure Kit to isolate DNA from both lesser
and greater quantities of yeast cells than the standard 1.5 ml stationary
culture preparations. First, we increased the volume of the preparation
by 20-fold. The yields from the MasterPure purifications starting with
30 ml of culture were 77 µg for S. cerevisiae and 430 µg
of DNA for C. albicans, exceeding the anticipated quantities extrapolated
from the 1.5 ml yields. Next, DNA was purified from single plate colonies
using the same protocol as that described for a 1.5 ml culture volume.
The average yields of DNA per colony, measured by fluorimetry, for the
three yeast species were 688 ng for C. krusei, 538 ng for C.
albicans, and 188 ng for S. cerevisiae. Agarose gel electrophoresis
revealed approximately the same size of DNA from colonies as from cells
cultured in broth (data not shown).
To illustrate the large discrepancies in DNA yield calculated
by A260 versus fluorimetry, we compared yield values calculated
by both methods for S. cerevisiae and C. albicans DNA purified
using the supplier Q kit and the MasterPure Kit. As Table 3 shows, there
is almost 30 times more DNA measured by A260 with Saccharomyces DNA
purified using either kit, and 8-12 times more with Candida DNA. Interestingly,
the S. cerevisiae A260 yields from the supplier Q kit
are much larger than what is advertised in the kit literature, and far
in excess of the total available DNA in the yeast cells.
| Epicentre |
S. cerevisiae |
3.9 |
116 |
29.7 |
| Supplier Q |
S. cerevisiae |
1.6 |
45.6 |
28.5 |
| Epicentre |
C. albicans |
21.5 |
24.8 |
11.5 |
| Supplier Q |
C. albicans |
3.0 |
25.4 |
8.5 |
Using pulsed field gel electrophoresis, the size of the majority
of DNA isolated with the MasterPure Kit was estimated to be 40-50 kb,
while DNA of the same yeast species purified with kit from supplier Q
was mostly degraded to fragments smaller than 40 kb (Figure 2).
A similar size discrepancy was observed for Saccharomyces and Candida species
DNAs.
S. cerevisiae DNA was used as a template for PCR amplification
of the RNA guanine-7-methyltransferase gene as described. The results
are shown in Figure 3. DNA prepared with all three of the kits was successfully
used as a template for this amplification. C. albicans DNA was
used for amplification of the DUT gene. The smaller DUT amplicon was
not amplified using DNA purified with the kit from supplier F, which
contained RNase in one of the solutions. With C. albicans DNA
purified using the MasterPure Kit, the DUT gene was successfully amplified (Figure
3). We speculate that RNase may have variable effects upon the amplification
of DNA from different loci or from different species of yeast.
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Figure 2. Pulsed field gel electrophoresis of yeast DNA. Lanes
M, lambda DNA ladder; Lane 4, T7 DNA (40 kb). |
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Figure 3. Amplification of the RNA guanine-7-methyltransferase
gene from S. cerevisiae and the DUT gene from C. albicans. Ten
nanograms of DNA purified using each of the kits was amplified
as described in the text. Lanes 1, 8, and 15, 100 bp ladder. |
The new MasterPure Yeast DNA Purification Kit gave the highest
yields and the largest average size DNA from three yeast species compared
to the other yeast DNA purification kits tested. The yield of DNA from
S. cerevisiae using the MasterPure Kit was as much as 12 times that obtained
with the other kits, and for purification from C. albicans, the MasterPure
Kit yield was as high as 16 times greater. The average length of DNA
purified using the MasterPure Kit was considerably greater than the DNA
isolated with another supplier's kit, as was shown by pulsed field gel
electrophoresis. The MasterPure Kit can be used for purification from
midi-prep volumes for both S. cerevisiae and C. albicans and can be used
to isolate DNA from single yeast colonies. This method is both effective
and economical.
- Sears, P.P. et al. (1992) Proc. Natl. Acad. Sci. 89, 5296.
- Molecular Genetics of Yeast: A Practical Approach, Johnston, J.R.,
ed., IRL Press, Oxford, 1994.
- (1991) Methods in Enzymology 194, Guthrie, C. and Fink, G.R., eds.,
Academic Press, NY.
- PERFECT Yeast DNA™ Kit Protocol, 5 Prime-3 Prime, 1998. 5. QIAamp™ Blood
Kit and QIAamp™ Tissue Kit Handbook, Qiagen, 1997.
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