A high throughput DNA extraction method with high yield and quality

Plant Methods

Junping Chen¹  Zhanguo Xin¹ 

[1] Plant Stress and Germplasm Development Unit, USDA-ARS, 3810 4th Street, Lubbock, TX, 79415, USA

关键词: MagAttract;    CTAB;    DNA extraction;   

Others  :  821994
DOI  :  10.1186/1746-4811-8-26

 received in 2012-05-24, accepted in 2012-07-20,  发布年份 2012

【 摘 要 】

Background

Preparation of large quantity and high quality genomic DNA from a large number of plant samples is a major bottleneck for most genetic and genomic analyses, such as, genetic mapping, TILLING (Targeting Induced Local Lesion IN Genome), and next-generation sequencing directly from sheared genomic DNA. A variety of DNA preparation methods and commercial kits are available. However, they are either low throughput, low yield, or costly. Here, we describe a method for high throughput genomic DNA isolation from sorghum [Sorghum bicolor (L.) Moench] leaves and dry seeds with high yield, high quality, and affordable cost.

Results

We developed a high throughput DNA isolation method by combining a high yield CTAB extraction method with an improved cleanup procedure based on MagAttract kit. The method yielded large quantity and high quality DNA from both lyophilized sorghum leaves and dry seeds. The DNA yield was improved by nearly 30 fold with 4 times less consumption of MagAttract beads. The method can also be used in other plant species, including cotton leaves and pine needles.

Conclusion

A high throughput system for DNA extraction from sorghum leaves and seeds was developed and validated. The main advantages of the method are low cost, high yield, high quality, and high throughput. One person can process two 96-well plates in a working day at a cost of $0.10 per sample of magnetic beads plus other consumables that other methods will also need.

【 授权许可】

   
2012 Xin and Chen; licensee BioMed Central Ltd.

【 图 表 】

Figure 1 .

Figure 3 .

Figure 4 .

【 参考文献 】

• [1]Amsterdam A, Hopkins N: Mutagenesis strategies in zebrafish for identifying genes involved in development and disease. Trends Genet 2006, 22(9):473-478.
• [2]Gilchrist EJ, O'Neil NJ, Rose AM, Zetka MC, Haughn GW: TILLING is an effective reverse genetics technique for Caenorhabditis elegans. BMC Genomics 2006, 7:262. BioMed Central Full Text
• [3]Henikoff S, Till BJ, Comai L: TILLING. Traditional mutagenesis meets functional genomics. Plant Physiol 2004, 135(2):630-636.
• [4]Slade AJ, Fuerstenberg SI, Loeffler D, Steine MN, Facciotti D: A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nat Biotechnol 2005, 23(1):75-81.
• [5]Till BJ, Reynolds SH, Weil C, Springer N, Burtner C, Young K, Bowers E, Codomo CA, Enns LC, Odden AR, et al.: Discovery of induced point mutations in maize genes by TILLING. BMC Plant Biol 2004, 4(1):12. BioMed Central Full Text
• [6]Dilworth E, Frey J: A Rapid Method for High Throughput DNA Extraction from Plant Material for PCR Amplification. Plant Mol Biol Rep 2000, 18:61-64.
• [7]Xin Z, Velten JP, Oliver MJ, Burke JJ: High-throughput DNA extraction method suitable for PCR. Biotechniques 2003, 34(4):820-826.
• [8]Geuna F, Hartings H, Scienza A: Plant DNA extraction based on grinding by reciprocal shaking of dried tissue. Anal Biochem 2000, 278(2):228-230.
• [9]Mace ES, Buhariwalla HK, Crouch JH: A high-throughput DNA extraction protocol for tropical molecular breeding programs. Plant Mol Biol Rep 2003, 21:459a-459h.
• [10]Flagel L, Christensen JR, Gustus CD, Smith KP, Olhoft PM, Somers DA, Matthews PD: Inexpensive, High Throughput Microplate Format for Plant Nucleic Acid Extraction: Suitable for Multiplex Southern Analyses of Transgenes. Crop Sci 2005, 45(5):1985-1989.
• [11]Xin Z, Chen J: DNA Sequencing II: Optimizing Preparation and Cleanup. In Extraction of Genomic DNA from Plant Tissue. Edited by Sudbury KJ. Jones and Bartlett Publishers, MA; 2006:47-59.
• [12]Bashalkhanov S, Rajora OP: Protocol: A high-throughput DNA extraction system suitable for conifers. Plant Methods 2008, 4:20. BioMed Central Full Text
• [13]Reynolds MM, Williams CG: Extracting DNA from submerged pine wood. Genome 2004, 47(5):994-997.
• [14]Abd-Elsalam KA, Asran-Amal A, El-Samawaty AMA: Isolation of high-quality DNA from cotton and its fungal pathogens. Journal of Plant Diseases and Protection 2007, 114:113-116.
• [15]Leitch IJ, Hanson L, Lim KY, Kovarik A, Chase MW, Clarkson JJ, Leitch AR: The ups and downs of genome size evolution in polyploid species of Nicotiana (Solanaceae). Ann Bot 2008, 101(6):805-814.
• [16]Tuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, Putnam N, Ralph S, Rombauts S, Salamov A, et al.: The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 2006, 313(5793):1596-1604.
• [17]Richards E, Reichardt M, Rogers S: Preparation of genomic DNA from plant tissue. Curr Protoc Mol Biol 1994, 1:2. 3.1 to 2.3.7
• [18]Michiels A, Van den Ende W, Tucker M, Van Riet L, Van Laere A: Extraction of high-quality genomic DNA from latex-containing plants. Anal Biochem 2003, 315:85-89.
• [19]Doyle JJ, Doyle JL: A rapid isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 1987, 19:11-15.
• [20]Dutta SK, Jones AS, Stacey M: The separation of desoxypentosenucleic acids and pentosenucleic acids. Biochim Biophys Acta 1953, 10:613-622.