Download Rapid DNA Extraction from Plant Seeds for PCR

Rapid DNA Extraction from Plant Seeds for PCR
Les Hoffman and Shannon Krueger, EPICENTRE Biotechnologies
Introduction
Plants have evolved a variety of defense mechanisms against predators and environmental challenges. Among their
protective devices are a myriad of compounds, some very complex in structure, that allow plants to repel pests.1 These
protective compounds include polyphenols and tannins, sometimes found in seeds and seed coats. The presence of
storage carbohydrates and polyphenols can interfere with successful amplification of DNA prepared from seeds. Until
now, cumbersome preparation steps were needed to purify analytical amounts of seed DNA. EPICENTRE’s new
QuickExtract™ Seed DNA Extraction Solution facilitates the extraction of PCR-ready DNA from a wide variety
of seeds, both monocot and dicot. The kit eliminates the need for organic solvents or detergents such as cetyltrimethylammonium bromide (CTAB) that are commonly required for PCR-ready DNA isolation from seeds.
Using the QuickExtract Seed Solution, PCR-ready DNA can be extracted from around 10 mg of seed material in less
than 10 minutes. The simple procedure consists of adding QuickExtract Seed Solution to seed that is either ground or
fragmented, followed by two incubation steps at 65°C and 98°C (Fig. 1).
The procedure is ideal for 96-well DNA preparations, which are desirable for plant breeding and other molecular
screening applications. Techniques such as targeting induced local lesions in genomes (TILLING), sequencecharacterized amplified region (SCAR), and other methods for plant mutation and marker discovery normally depend
on the purification of leaf DNA templates for PCR. The time needed for genetic tests using DNA extracted from leaves
can often delay the sale or export of seed. The QuickExtract Seed DNA Extraction Solution saves the time and effort
needed to plant and sprout seeds for PCR-based analysis.
The PlantAmp™ PCR System is specially formulated for PCR from plant DNA that contains polyphenols and other
PCR inhibitors. In addition, the system contains EPICENTRE’s PCR Enhancer (with betaine*) that ensures reliable
PCR of templates with up to 80% GC content.2 The combination of these two kits provides rapid DNA extraction and
successful PCR from challenging plant seed samples.
*Covered by issued and/or pending patents; see www.EpiBio.com/legal.
Methods and Results
Seeds were obtained from various suppliers including
the Willy St. Grocery Co-op, Madison, WI and
Reimer Seeds, Mt. Holly, NC. Primer sequences are
available upon request from EPICENTRE. PCR was
performed using either the FailSafe™ PCR System
(EPICENTRE) or the PlantAmp PCR System with 35
cycles of amplification. PCR products were separated and
visualized in 1% or 2% agarose gels.
PCR of rice DNA
Rice is the most important grain crop worldwide. We
demonstrate the ability of the QuickExtract Seed Solution
to aid screening large numbers of rice seeds or fragments
of seeds (Fig. 2). Approximately 10-mg pieces of brown
rice were extracted with 100 µl of QuickExtract Seed
Solution, and the extracts were used directly in PCR of a
single-copy gene. The expected amplicon was produced
in all cases (Fig. 2).
PCR of DNA extracted from challenging seeds
Next, we used several dicot seeds known to be
problematic for DNA isolations because of their
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polyphenolic content. Oil sunflower seed and cottonseed
were chipped into 10-mg pieces and extracted as
before. Seed DNA was amplified with the PlantAmp
PCR System and either chloroplast gene (cotton) or
microsatellite single-copy gene primers (sunflower)
were used for PCR. For sunflower PCR, we used either a
standard FailSafe™ PCR PreMix (Fig. 3) or a PlantAmp
Up to 10 mg of seed
Take 1 µl for PCR
Add QuickExtract™
Seed DNA Extraction
Solution to sample
Heat at 65°C for 6 minutes
and 98°C for 2 minutes
PCR-ready
DNA
Figure 1. Overview of the QuickExtract™ Seed DNA extraction
procedure.
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13
Seed DNA Extraction
M
1
2
3
4
5
6
Figure 2. PCR of DNA extracted from
brown rice seeds. Samples (10 mg
each) of five different brown rice seeds
were used for DNA extraction, and a
1-μl aliquot of each was used for PCR
of the single-copy RIA gene. Lane M,
100-bp ladder; lane 1, no-template
control; lanes 2-6, PCR products from
five different seed samples.
1
2
3
4
5
6
M
Figure 3. PCR of DNA from
oil sunflower seed. DNA was
extracted from approximately
10 mg of sunflower seed as in
Fig. 1 and amplified using a set
of primers corresponding to SSR
locus G67-408. Amplifications
contained 1 µl of undiluted extract
in a FailSafe™ PCR. Lanes 1 and 2,
negative controls containing only
TE buffer or QuickExtract Seed
Solution, respectively; lanes 3-6,
PCR products from sunflower DNA
samples; lane M, 100-bp ladder.
PreMix. Although correct amplicons were synthesized with
either PreMix, notably more product was observed with the
PlantAmp PreMix.
Cottonseed contains a toxic terpenoid (gossypol),1 which is
highly inhibitory to PCR. We used the QuickExtract Seed
Solution to obtain DNA from 10-mg pieces of cottonseed,
which was amplified using primers for a single-copy
transcription factor gene, DREB1 (Fig. 4). Faithful
amplification was seen with both 1:10 and 1:100 dilutions of
cottonseed extracts.
]
Real-time PCR analysis
Real-time PCR was performed with oil sunflower seed using
QuickExtract Seed Solution for DNA extraction and the
PlantAmp PCR System with primers corresponding to SSR
locus G67-408. Tenfold dilutions (from 1:10 to 1:10,000)
of seed extract were amplified (Fig. 5). Reliable, specific
amplification was achieved.
Conclusions
The presence of storage carbohydrates and polyphenols can
interfere with successful amplification of DNA prepared
from seeds. The QuickExtract Seed DNA Extraction Solution
facilitates the extraction of PCR-ready DNA from a wide
variety of seeds, even those containing polyphenols such as
sunflower and cottonseed. The method is quick and effective,
and avoids having to sprout seeds in order to use them for
DNA preparations. The PlantAmp PCR System provides
reliable PCR from seed DNA that contain polyphenols and
other PCR inhibitors.
References
1. Sunilkumar, G. et al. (2006) Proc. Natl. Acad. Sci. USA 103, 18054.
2. Grunenwald, H. (2008) Gen. Eng. News 28(14), 28.
Cycle
Figure 5. Real-time PCR of DNA extracted from oil sunflower seed.
DNA was extracted and PCR was performed as described in Fig. 3. Tenfold
dilutions (from 1:10 to 1:10,000) of the extracts were amplified with the
PlantAmp™ PCR System using Green Dye 12.
Cat. #
Quantity QuickExtract™ Seed DNA Extraction Solution
QES08095T
5 ml (50 Extractions)
QES080950
50 ml (500 Extractions)
PlantAmp™ PCR System
PA0809100
PA08091K
14 www.EpiBio.com
Figure 4. PCR of DNA extracted from
cottonseed. Cottonseed (a green
fiber cultivar) was processed as in
Fig 1. Several dilutions of the extract
were tested in PCR using primers
for the transcription factor DREB1
and the PlantAmp™ PCR System
(EPICENTRE). Lane M, 100-bp ladder;
lanes 1-4, 1:10 dilutions of cottonseed
extracts; lanes 5-6, 1:100 dilutions of
cottonseed extracts; lanes 7 and 8,
negative controls containing only TE
buffer or QuickExtract Seed Solution,
respectively.
Relative Fluorescence Units
[
Faithful amplification was seen with both 1:10 and 1:100
dilutions of cottonseed extracts.
M 1 2 3 4 5 6 7 8
100 Units
1,000 Units
Volume 16-1 • February 2009