Download A rapid one-tube genomic DNA extraction process

Nucleic Acids Research, 1995, Vol. 23, No. 13 2569-2570
A rapid one-tube genomic DNA extraction
for PCR and RAPD analyses
process
J. J. Steiner*, C. J. Poklemba, R. G. Fjellstrom and L. F. Elliott
National Forage Seed Production Research Center, USDA-ARS, 3450 SW Campus Way, Corvallis, OR 97331, USA
Received March 24, 1995; Revised and Accepted May 18, 1995
Although the polymerase chain reaction (PCR) is a powerful
genetic tool, its use in population analyses is limited by the
labor-intensive extraction of genomic DNA from large numbers of
samples. An ideal technique for DNA extraction should ni mize
the number of times a tissue sample is handled from collection to
analysis, optimize yield of DNA extracted from a sample, be
applicable to diverse organisms, be suited to mass handling of
samples while minimig labor and materials costs and not generate
hazardous waste that may negatively impact the environment. We
demonstrate a novel DNA extraction process performed in a single
tube from the time a tissue is collected until diluted aliquots of the
extractant are taken for PCR reactions. The process does not require
centrifugation, can prepare up to 6000 samples per day, results in
enough DNA to perform 4000 PCR amplifications per sample, and
can be used for plant, animal and microbial sources of DNA.
Our rapid extraction process consists of two parts: sample
preparation for extraction and a one-step DNA chemical extraction. To prepare samples for DNA extraction, fresh plant leaves
(-3 cm2) are collected into 1.1 ml tubes containing five 3.0 mm
diameter glass beads that are held in microtiter format racks. The
tube racks with samples are lyophilized until the leaf samples are
dry, resulting in -5 mg of dry leaf material per tube. The tubes are
then capped and the samples ground by shaking the tube racks for
20 min on a wrist-action shaker (Labline Instruments, Melrose
Park, USA). To conduct the DNA extraction, 200 jl of rapid
one-step extraction (ROSE) buffer containing 10 mM Tris-HCl,
pH 8.0; 312.5 mM EDTA, pH 8.0; 1% sodium lauryl sarkosyl;
and 1% polyvinylpolypyrrolidone (PVPP, water insoluble) is
added to the ground lyophilized tissue. The tubes and contents are
vortexed thoroughly and then incubated in a hybridization oven
at 90°C for 20 min and mixed constantly by attaching the tube
racks to the oven rotor. Alternatively the tubes can be set in a
90°C water bath and frequently inverted. The samples are placed
on ice for 5 min to allow the tissue and PVPP to settle before
aliquots of extract are taken for dilution and amplification by
PCR. The original tube with sample and extractant, or dilutions
of the extractant, can be stored for months at 4°C. For
amplification of ROSE-extracted DNA by PCR, 10 gl of the
original extract is diluted 170-fold with 1690 jl of water and then
2.5 gl of diluted extract is used in a 12.5 jl reaction mixture.
Initially, we isolated DNA using a modification of the widely
used procedure (1) using CTAB detergent, but found this method
too labor intensive for processing thousands of small samples
(Table 1). The CTAB method also generated significant amounts
*
To whom correspondence should be addressed
of hazardous waste and required the purchase of special
centrifuge equipment to utilize a 96-tube microriter format to
increase daily throughput. Our search of the literature revealed
that non-centrifugal methods could be used to extract DNA, such
as one method (2) that used proteinase K (ProK). This method
greatly reduced the number of steps required for DNA extraction
and did not use organic solvents. However, we observed DNA
degradation upon heat inactivation (100°C for 5 min) of the
proteinase K. A rapid alkaline extraction method (3) was also
used, however it yielded a predominace of low molecular weight
DNA that did not reliably produce PCR amplification products
>600 bp in length (data not shown).
Table 1. Comparison of three DNA extraction methods for use with PCR
amplification after leaf tissue samples are collected, dried, and ground
Optimized samples per day (number)
Number of chemical extraction stepsa
Hazardous waste per day (ml) (based
on optimized number)
Daily expendable costs (US $) (based
on optimized number)
Expenses per sample (US $):
expendables
laborb
total
Extraction method
ProK
CTAB
2016
384
2
7
0
600
ROSE
6144
1
0
180.00
621.00
1416.00
0.47
0.17
0.64
0.31
0.03
0.34
0.23
0.01
0.24
aAn extraction step is defined as any activity requiring the transfer of tube contents or the addition of reagents after the initial extraction buffer is added to
ground tissue.
bAssuming $8.00 h-1 and the optimized number of samples are processed in an
8 h day.
We have routinely recovered 0.5-5.0 jg of DNA using CTAB
and 0.8-1.2 ,ug using ROSE from 5.0 mg of lyophilized leaftissue
of Lotus corniculatus. The high molecular weight DNA exracted
by our ROSE method is of comparable quality with that of the
CTAB method with length . 40 kb (Fig. 1). We have obtained
identical RAPD (Fig. 2) and single gene (Fig. 3) PCR amplification products using the CTAB and ROSE extraction methods.
ROSE DNA samples incubated at 37°C for 64 h produce the
same RAPD PCR products as samples stored at 4°C (Fig. 2),
2570 Nucleic Acids Research, 1995, Vol. 23, No. 13
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Figure 1. Genomic gel comparing DNA quality from CTAB and ROSE
extracts from Lcorniculatus cvs. NC-83 (N) and G31276 (G). Undigested X
DNA, lane 1. CTAB extracts, lanes 2, 3, 6 and 7; ROSE extracts, lanes 4, 5, 8
and 9. Electrophoresis was performed in 0.5% SeaKem agarose (FMC,
Rockland, USA) lx Tris-acetate gels.
GI l
&oo
Figure 2. RAPD PCR products of CTAB and ROSE extracted DNA from
Lcomiculatus cvs. NC-83 (N) and G31276 (G). Size standard 100 bp ladder
(Gibco-BRL, Gaithersburg, USA), lane 1; CTAB extracts, lanes 2 and 6; ROSE
extracts diluted 170-fold, lanes 3 and 7; ROSE extracts diluted 170-fold and
incubated at 37°C for 64 h, lanes 4 and 8; ROSE extracts diluted 85-fold, lanes
5 and 9. RAPD reaction conditions: 0.2 ng gt-l CTAB or 0.014 ng d-1 of
170-fold diluted ROSE template DNA; 55.0 mM Tris-HCl, pH 9.0,45.0 mM
(NH4)2SO4, 1.5 mM MgC92, 100 jM dNTP, 0.2 jM primer OPB-08 (Operon,
Alameda, USA), and 0.04 U p1-1 Tfl DNA polymerase (Epicentre Technologies, Madison, USA); and overlaid with 50 g1 mineral oil. The thermal profile
for RAPD PCR was: 95°C initial denaturation for 2.5 min then 42 cycles of
94°C for 40 s, 46°C for 40 s, and 72°C for 1.0 min; and finally 72°C for 9.0
min. PCR was performed in a MJ Research (Watertown, USA) thermocycler
(PTC-100) using a 96-well microriter format. PCR products were separated by
electrophoresis in 1.75% NuSieve 3:1 agarose (FMC, Rockland, USA) lx
Tris-borate gels, stained with ethidium bromide, and visualized under UV light.
demonstrating the stability of ROSE-extracted DNA. Due to the
inhibitory effects of buffer components at their initial strength,
and perhaps other natural compounds in the extract, the ROSE
buffer extractant should be diluted 170-fold before the DNA is
PCR amplified (Fig. 2).
The general utility of the DNA extraction chemistry component
of the one-tube extraction process has been successfully performed
to give reproducible RAPD or single gene PCR products with
samples of the following species (data not shown): -100 mg fresh
weight or 5 mg dry weight of leaf tissue of Beckmannia grass, black
pine, cattail, cereal rye, Deschampsia grass, dwarfbanana, fan palm.
kiwi, orchardgrass, Pandanus, perennial ryegrass, Pienis, red clover,
sycamore, table beet and tall fescue; -100 mg fresh weight of fruit
pericarp tissue of banana and lime; whole body tissue from ladybird
beetle and brown garden slug, and 1 cm length of earthworm; -5 x
109 bacterial cells of Pseudononas sp.; 5 mg fungal uredospores of
Puccinia graminus; and 100 p1 whole human blood. Sample tissue
grinding was done en masse as described above or individually with
glass rods using liquid N2 in 2.0 ml microcentrifuige tubes for
Figure 3. Single gene PCR amplification products of the glutathione reductase
DNA sequence (5) of CTAB and ROSE extracted DNA from Lcorniculatus
cvs. NC-83 (N) and G31276 (G). Size standard 100 bp ladder, lanes 1 and 6;
CTAB extracts, lanes 2, 3, 6 and 7; ROSE extracts diluted 170-fold, lanes 4, 5,
8 and 9. PCR reaction mixtures were the same as Figure 2, but using 2.0 mM
MgCI2 and 0.2 FiM each of 24 base forward and reverse primers (Fjellstrom and
Steiner, unpublished data, 1994). The thermal profile for PCR was: 94°C initial
denaturation for 5.0 min; 42 cycles of 94'C for 1.0 min, 56°C for 1.0 min and
72°C for 1.5 min; and a final step at 72°C for 8.5 min. PCR products were
detected as in Figure 2.
lyophilized and fresh tissue, respectively. DNA from fresh or
previously frozen human blood was extrcted directly in 2.0 ml
microcentrifuge tubes using 400 p1 of buffer.
Our DNA extraction process has several advantages when
compared with the above-mentioned DNA extraction methods.
By lyophilizing and grinding the tissues with glass beads in a
96-tube microtiter format, sample preparation for DNA extraction is not a limiting factor of daily throughput (Table 1). The
96-tube format can also be used to prepare tissues for extraction
by the other two methods, but further transfers or additions are
needed before the extractant is ready for storage or dilution for
DNA amplification by PCR. In our method, the ROSE buffer is
added to the ground samples, the tubes are capped and incubated,
and no further transfer or addition steps are required. The
completely self-contained extraction environment also reduces
the chances of cross-sample contamination. Our process can also
be modified for DNA extraction directly from -3 cm2 of fresh
tissue (-100 mg) crushed in 200 p1 of buffer with a glass grinding
rod without need for lyophilization. For mass production, this
gready reduces the number of samples that can be processed in
one day, but readily lends it self to field sampling applications.
Unlike the CTAB method, the ROSE and ProK methods do not
require the use of a fume hood or produce any hazardous waste
because no organic solvents are used. Disposal of hazardous
waste adds to actual extraction costs, and mandated reductions in
hazardous waste generation necessitates the develoment of
reduced waste techniques (4). Considering all factors involved,
the ROSE method appears to be the most efficient and
cost-effective DNA extraction procedure of the three methods
tested, should provide an alternative method that does not
generate hazardous waste, and should readily lend itself to both
automation as well as field sample preparation.
REFERENCES
Doyle, J.J. and Doyle, J.L. (1990) Focus, 12, 13-15.
Guidet, F. (1994) Nucleic Acids Res. 22, 1772-1773.
Wang, H. et al. (1993) Nucleic Acids Res. 21, 4153-4154.
US Department of Agriculture (1995) ARS Safety, Health, and
Environmental Management Program, Manual 230.0.
S Creissen, G. et al. (1992) Plant J, 2, 129-131.
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