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Tutorials : May 15, 2009 (Vol. 29, No. 10)

Obtaining Reliable PCR from Seed DNA

Epicentre Biotechnologies Launches DNA Extraction Tool for a Variety of Seed Types
  • Les Hoffman
  • ,
  • Shannon Krueger

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. 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 Biotechnologies’ 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 cetyltrimethyl-ammonium bromide that are commonly required for PCR-ready DNA isolation from seeds.

Using the QuickExtract Seed Solution, PCR-ready DNA can be isolated from 10 mg or less of seed material in under 10 minutes. The requirement for germinated seed-leaf DNA can often delay the sale or export of seed. QuickExtract 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 ensures reliable PCR of templates with up to 80% GC content. The combination of these two kits provides rapid DNA extraction and successful PCR from challenging plant seed samples.

Methods and Results

DNA was extracted from seed samples (ground, chipped, or fragmented) using the QuickExtract Seed Solution. The procedure consists of adding QuickExtract Seed Solution to the seed sample, followed by two incubation steps at 65°C and 98°C. PCR was performed using the PlantAmp PCR System with 35 cycles of amplification. PCR products were separated and visualized in 1% or 2% agarose gels. Primer sequences are available upon request from Epicentre.

DNA from Monocot Seeds

QuickExtract Seed Solution was used to screen monocot seeds, which often yield DNA that gives inconsistent amplification results by standard methods. Approximately 10 mg of a variety of ground or fragmented monocot crop seeds were processed using 100 µL of the solution for each sample. Aliquots of the undiluted extracts were amplified by PCR with conserved intron-spanning primers. The expected amplicon was produced in all cases (Figure 1).

Next, we used approximately 10 mg of corn flour in each conical well of a 96-well plate and extracted DNA with 100 µL of QuickExtract Seed Solution as before. The plate was centrifuged for five minutes at 3,000 rpm. A 0.5-µL aliquot of each extract was added to the PCR mixture in a separate 96-well plate containing the PlantAmp PCR PreMix and primers for a universal monocot gene. The amplified locus, BRSC3, spans an intron and is highly conserved among monocot plants. The expected band of around 250 bp was amplified in all 96 samples; representative examples are shown in Figure 2.

Seeds with High Polyphenolic Content

Cottonseed is known to be problematic for DNA isolations because of its polyphenolic content. We tested 10 mg samples of chipped cottonseed extracted with QuickExtract Seed Solution as before. Seed DNA was amplified with the PlantAmp PCR System using primers for a single-copy transcription factor gene, DREB1 (Figure 3).

Faithful amplification was seen with both 1:10 and 1:100 dilutions of cottonseed extracts. In addition, we have obtained consistent and reliable results from oil sunflower seed in both end-point and real-time PCR.

DNA from Other Plant Tissues

Use of the QuickExtract Plant DNA Solution for extraction of PCR-ready DNA from plant tissues, especially those with high polyphenolic content, is highly recommended. This solution has been used successfully with a variety of plants, including Arabidopsis, barley, maize, emmer, pepper, rice, spelt, spinach, soybeans, and wheat.

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 monocot and dicot seeds, even those containing polyphenols such as cottonseed and sunflower. The method is quick and effective, and avoids having to sprout seeds in order to use them for DNA analysis. The PlantAmp PCR System provides reliable PCR from seed DNA that contains PCR inhibitors.