A Rapid DNA Extraction Method for RFLP and PCR Analysis from a Single Dry Seed

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1 Plant Molecular Biology Reporter 16: 1 9, Kluwer Academic Publishers. Printed in the Netherlands. Publish by Abstract A Rapid DNA Extraction Method for RFLP and PCR Analysis from a Single Dry Seed HEE WAN KANG, YONG GU CHO, UNG HAN YOON and MOO YOUNG EUN National Institute of Agricultural Science & Technology, Suwon , Korea Abstract. A single-seed DNA extraction method was developed for rapid identification of plant genotype. The method was applied to 12 plant species, including the oil seeds sesame and soybean. The results were comparable to those obtained for oil-less seeds such as rice. This method will be useful for genotypic selection that requires rapid screening of large populations. It can also be used to identify varietal purity of seed stocks by PCR and RFLP analysis. The method includes two major steps, (i) treatment by proteinase K in an SDS extraction buffer, and (ii) grinding of a single half seed in the buffer after incubation. About µg of DNA per half seed (the endosperm part) of rice was obtained and more than 200 half seed samples could be handled by one person in a day. The DNA could be used for fingerprinting and detection of target genes in a transgenic plant by PCR. The amplified PCR products from the half seed DNA showed the same banding patterns as those from leaf DNA. Yield and quality of DNA extracted from half seeds of rice was also sufficient for RFLP analysis. The remnant half seeds containing the embryo can be maintained for later germination of selected genotypes. Key words: DNA extraction, DNA fingerprint, half seed, PCR, RFLP, target gene Abbreviations: AFLP, amplified fragment length polymorphism; CAPS, cleaved amplified polymorphic sequence; RAPD, randomly amplified polymorphic DNA; STS, sequence tagged sites. Introduction PCR techniques such as RAPD (Williams et al., 1990), CAPS (Konieczny et al., 1993), STSs (Mazur et al., 1995), microsatellite (Panaud et al., 1996), and AFLP (Vos et al., 1995), have been used in plants for molecular mapping, identification of genotypes associated with genes of interest, and genetic diversity studies. A unique advantage of these PCR techniques is the rapid DNA 1 Author for correspondence. hwkang@niast.go.kr.

2 2 analysis of many plant samples using small quantities of DNA. Thus, a simple and rapid DNA extraction method is needed for studies, such as genetic analysis, that require large populations. Several methods for minimizing the DNA extraction steps have been reported (Berthomieu et al., 1991; Edwards et al., 1991; Tomas et al., 1989), but they require a large amount of plant tissue and grinding of plant tissues in liquid nitrogen. In addition, growth and management of plants and storage of the plant samples in freezers is often difficult due to space constraints. To overcome these problems, Chunwongse et al. (1993) developed a DNA extraction method using the dry half seeds of rice and wheat. However, the method has not been used extensively for other plant seeds, especially the oil seeds, and would only be used for PCR analysis. We describe here an efficient DNA extraction method for single dry seeds of rice. The DNA extracted by the method can be used for DNA fingerprinting and specific detection of target genes of transgenic plants by PCR. In addition, the quality and yield of the DNA is appropriate for RFLP analysis. The extraction method also produced microgram quantities of DNA from several other plant species including oilseeds. Material and Methods Dry seeds of plant species Rice, wheat, barley, soybean, pumpkin, cucumber, tomato, chinese cabbage, raphnus, pepper, and sesame seeds were used to test the following DNA extraction method. Transgenic rice seeds containing the phosphinothricin acetyltransferase (bar) gene, which gives resistance to the herbicide bialaphos, were used to test for detection of bar by PCR and RFLP. Primers and probe Random primer URP-6 (5 -GGCAAGCTGGGAGGTAC-3 ) was used for DNA fingerprinting by PCR amplification. Primers BL and BR were designed from the sequence of the bar gene. The following are the primer sequences: BL 5 -GATCTCGGTGACGGGCAGGA-3, BR 5-GGCGGTCTGCACC- ATCGTCAA-3. The rice genomic clone, RG220, a molecular marker associated with the semidwarf (sd-1) gene (Cho et al., 1994), was used for RFLP analysis.

3 Procedures for DNA extraction from a half seed of rice and other plant seeds The whole seeds of plants with small size, such as tomato, chinese cabbage, sesame, etc., were used for DNA extraction since they are not well restricted between embryo and endosperm parts and are difficult to cut in half. The plant seeds such as rice, wheat, barley, etc., which were well restricted between the parts, were cut in half and the half seeds containing the storage tissue (endosperm or cotyledon parts) were used for the following DNA extraction steps: Remove the seed coat and cut the seed in half. Place the seed half containing the storage tissue in a microcentrifuge tube (1.5 ml). Add 400 µl of extraction buffer 1 containing proteinase K (50 µg), incubate at 37 Cfor1h. Grind the seed in the buffer with a glass rod. Add 400 µl of CTAB solution (2%) 2. Gently extract using chloroform:isoamyl alcohol (24:1) with 5% phenol. Centrifuge at 12,000 rpm in microcentrifuge at 4 C for 10 min and transfer the supernatant to new tubes. Add 2/3 volume isopropanol and incubate the tube at room temperature for 10 min to precipitate DNA. Centrifuge at 12,000 rpm for 5 min, remove supernatant, and wash the DNA pellet with 70% Ethanol, air dry, and resuspend in 50 µl ofte buffer. Remove RNA by adding 1 µl of RNase (10 mg/ml). Notes 1. Extraction buffer: 200 mm Tris-HCl (ph 8.0), 200 mm NaCl, 25 mm EDTA, 0.5% SDS CTAB (cetyltrimethylammonium bromide) solution: 2% CTAB(w/v), 100 mm Tris- HCl (ph 8.0), 20 mm EDTA (ph 8.0), 1.4 M NaCl, 1% PVP (polyvinylpyrrolidone) Mr 40,000. PCR amplification of extracted DNA from a half seed Reaction mixture (50 µl): 5.0 µl 10 buffer, 3.0 µl 25mMMgCl 2, 4.0 µl dntp stock with 2.5 mm each of dntp, 10 pmol primer, 0.5 µl Taq DNA polymerase (Promega, 5 units/µl), 50 ng template DNA. Add 25 µl mineral oil gently onto the reaction mixture. PCR reaction performed in a Perkin Elmer Cetus Thermocycler model 480 using the following profile: 1cycle:94 C for 4 min (denaturing) 35 cycles: 94 C for 1 min (denaturing) 3

4 4 55 C for 1 min (annealing) 72 C for 2 min (extension). Amplified PCR products were electrophoresed on an agarose gel (1.7%) in TAE buffer and visualized by staining with ethidium bromide. Southern blot analysis using seed DNA To obtain sufficient DNA for Southern analysis, 5 half seeds were used for DNA extraction by the method mentioned above. The isolated DNA was digested with restriction enzyme XbaI and electrophoresed on an agarose gel (0.9%) in TBE buffer and then blotted onto a nylon membrane (Hybond N +). The DNA was hybridized with 32 P-labeled probe, RG220, and was detected by exposing to an X-ray film for 16 h at 70 C. Germination of remnant half seed One hundred rice half seeds containing the embryo were germinated after maintaining at 4 C for 8 months. The half seeds were treated with sodium hypochlorite solution (1%) to protect against contaminants on the seed surface, such as pathogens, and planted on 0.8% agar plates. The germination rate of the half seeds was compared with whole seeds of rice after maintaining at 30 Cfor7days. Results and Discussion To test the effect of various modifications to our DNA extraction protocol, we used single half seeds of rice. We first investigated the effect of detergents in the DNA extraction buffer. Detergents, SDS, CTAB and TritonX-100, were added to the solution containing 200 mm Tris-HCl ph 8.0, 200 mm NaCl, 25 mm EDTA, and proteinase K (50 µg). As shown in Figure 1, DNA could only be extracted with the solution containing SDS. The addition of proteinase K to the SDS extraction buffer prior to incubation is also a critical factor; DNA was not observed on the agarose gel when proteinase K was excluded in SDS buffer (Figure 1). Proteinase K worked the best when the concentrations ranged from 10 µgto50µg. It had no effect when concentrations were greater than 50 µg/400 µl buffer. Moreover, preliminary grinding of a half seed resulted in distinct DNA degradation. Thus, grinding of the dry seed after incubation in the buffer was also revealed as an important factor. During incubation of seeds in the buffer, the dry seeds having hard tissue

5 5 Figure 1. Agarose gel electrophoregram of DNA extracted from a single half seed of rice. DNA extraction from half seeds of rice was done to investigate effects of proteinase K (50 µg) and detergents (0.5%) in the extraction buffer. The DNAs extracted by different conditions were dissolved in 50 µl of TE buffer and then 10 µl of the DNA solution was electrophoresed on an agarose gel (0.9%). Lane 1, Proteinase K + extraction buffer without detergent; lane 2, Proteinase K + extraction buffer with CTAB; lane 3, Proteinase K + extraction buffer with Triton X-100; lane 4, Extraction buffer with SDS (proteinase K was not added); lane 5, Proteinase K + extraction buffer with SDS; lane 6, DNA (300 ng) extracted from leaf tissues of rice by standard method (Rogers et al., 1988). were softened by uptake of the buffer. This allowed the grinding to proceed more easily. These results indicate that the DNA extraction procedure should include a treatment with proteinase K in an SDS buffer and grinding of half seeds after incubation. Using this new method, we obtained DNA yields of µg per single half seed of rice. The quality and quantity of DNA obtained with this method was as good as leaf DNA purified with normal methods (Figure 1). More than 200 half seed samples could be handled by one person in a day and thus the method is useful for rapid DNA analysis of many genotypes. The DNA extracts could be maintained at 20 Cformore than one year without deleterious effects. In order to check the efficiency and reliability of the method, we first amplified the seed DNA of rice varieties using the primer, URP-6. The amplified PCR products of seed DNA showed identical band patterns and similar intensity to that of leaf tissue. However, different PCR patterns were obtained between indica and japonica rice varieties (Figure 2). We further tested direct use of rice seed DNA for detection of plants with the transformed bar gene. Using the primer set of BL and BR flanking the

6 6 Figure 2. PCR amplification of DNA extracted from half seeds of rice varieties using random primer URP-6. The DNA extraction was done using single half seeds and leaf tissue of each rice variety and about 50 ng of DNA was used for the PCR reaction. Amplified PCR products were electrophoresed on an agarose gel (1.7%). Lane 1, IR36; lane 2, Milyang 23; lane 3, Gihobyeo; lane 4, Mankeum; lane 5, Ilpoom; lane 6, Youngnam; lane 7, Jinbu; lane 8, Palgong. bar gene, we were able to obtain a single band (0.5 kb) of the expected size corresponding to the gene from the half seed DNA (Figure 3). As a positive control, we used the genomic DNA isolated from leaf tissue of the transformed plant by standard method (Rogers et al., 1988) and observed the same sized PCR product (Figure 3). The half seed DNA method can be directly used to detect target genes in transgenic plant by PCR amplification. We obtained 2 µg of DNA per a single half seed of rice. Thus, the amount of DNA extracted from four half seeds would be a sufficient quantity for RFLP analysis. RFLP analysis was done with the DNA isolated from four half seeds of Milyang 23, Gihobyeo, and Milyang/Gihobyeo F6 recombinant inbred lines (RILs). As expected, the banding pattern and intensity observed on seed DNA and on leaf DNA samples were the same (Figure 4). Results from these experiments indicate that the yield and purity of seed DNA is sufficient for application of RFLP as well as PCR analysis. Genetic analysis using half seed DNA is useful for a very large number of genotypes and the selected remnant half seeds containing the embryo parts can be germinated later (Chuwongse et al., 1993). The half seeds had similar germination rates

7 7 Figure 3. PCR amplification for specific detection of a target gene using half seed DNA of a rice plant transformed with the bar gene. The primer set of BL 5 -GATCTCGGTGACGGGCAGGA-3 and BR 5 -GGCGGTCTGCACCATCGTCAA-3 were used in PCR reaction for specific detection of the bar gene in the genomic DNA of half seeds. Lane 1, template DNA isolated from a non-transformed half seed; lane 2, template DNA isolated from a transformed half seed; lane 3, template DNA isolated form transformed leaf tissue; lane 4, template DNA isolated from plasmid pdm 320 harboring the bar gene. to the control uncut seeds on agar plates and the seedlings derived from the half seeds survived well after transplanting to soil. Genotyping by PCR analysis of half seed DNA from wheat and rice was reported by Chuwongse et al. (1993). They tested direct PCR amplification after a boiling step combined with addition of proteinase K or a chelate resin in the PCR buffer. We have tested our method on the dry seeds of several additional plant species: barley, soybean, pumpkin, cucumber, tomato, chinese cabbage, raphnus, pepper, and sesame. The amount of DNA extracted from a seed ranged from 0.5 µg to10µg per single seed and the difference in DNA yield seemed to be due to size and other morphological characteristics of the seeds. For example, large amounts of DNA (about 10 µg) were extracted using only 1/8 piece of single seed of soybean, whereas about 1 µg ofdna was obtained from the small-sized seeds of Brassica and Solanaceous species (data not shown). The results indicate that this method can extract DNA from the seeds of various plant species. In conclusion, a new method for direct DNA extraction from single dry seeds of rice was developed for PCR analysis. The amount and quality of DNA is also sufficient for RFLP analysis when more than one seed is used.

8 8 Figure 4. Southern blot analysis using DNA extracted from half seeds. DNA extraction from four half seeds of rice was done by the method described. The extracted DNA was digested with XbaI and then blotted onto Hybond-N + membrane. The membrane was hybridized with 32 P-labeled probe, RG220, and the signal was detected by exposing to X-ray film for 16 h at 70 C. Lane 1, leaf DNA (Milyang 23); lane 2, leaf DNA (Gihobyeo); lane 3, half seed DNA (Milyang 23); lane 4, half seed DNA (Gihobyeo); lanes 5 12, half seed DNAs of Milyang 23/Gihobyeo F6 RI population. The DNA is as reliable as DNA isolated by standard methods from other tissues such as leaf. Therefore, this method will be useful for rapid genotypic screening of many individuals and molecular confirmation of transgenic plants by using a dry half seed. The method could also be used to extract DNA from the dry seeds of several plant species in addition to rice. Acknowledgements We acknowledge Drs Young Tae Lee and Suk Young Lee for providing the rice and other plant seeds, and Dr Darlene M. Lawson for critical review of the manuscript. References Berthomieu P and Meyer C (1991) Direct amplification of plant genomic DNA from leaf and root pieces using PCR. Plant Mol Biol 17: Cho YG, Eun MY, McCouch SR and Chae YA (1994) The semidwarf gene, sd-1, of rice (Oryza sativa L.). II. Molecular mapping and marker-assisted selection. Theor Appl Genet 89: Chunwongse J, Martin GB and Tanksley SD (1993) Pre-germination genotypic screening using PCR amplification of half seeds. Theor Appl Genet 86:

9 Edwards K, Johnstone C and Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19: Konieczny A and Ausubel FA (1993) A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. The Plant J 4: Li QB, Chai Q and Guy CL (1994) A DNA extraction method for RAPD analysis from plants rich in soluble polysaccharides. Plant Mol Biol Rep 12: Maquire TL, Collins GG and Sedgley M (1994) A modified TAB DNA extraction procedure for plants belonging to the family proteaceae. Plant Mol Biol Rep 12: Mazur BJ and Tingey SV (1995) Genetic mapping and introgression of genes of agronomic importance. Curr Opinion Biotech 6: Panaud O, Chen X and McCouch SR (1996) Development of microsatellite markers and characterization of single sequence length polymorphism (SSLP) in rice (Oryza sativa L.). Mol Gen Genet 252: Rogers S and Bendich AJ (1988) Extraction of DNA from plant tissues. In: Gelvin SB, et al. (eds), Plant Molecular Biology Manual A6: Tomas HT and Tanksley SD (1989) A rapid and inexpensive method for isolation of total DNA from dehydrated plant tissue. Plant Mol Biol Rep 12: Vos P, Hoger R, Bleeker M, Reijans M, Lee TV, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M and Zabeau M (1995) AFLP: A new technique for DNA fingerprinting. Nucleic Acids Res 23: Williams JGK, Kubelic AR, Livak KJ, Rafalski JA and Tingey SV (1990) DNA polymorphisms amplified by arbitray primers are useful as genetic markers. Nucleic Acids Res 18:

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