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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR AND GENETIC ENHANCEMENT OF ABIOTIC STRESS TOLERANCE IN SORGHUM

Location: Plant Stress and Germplasm Development Research

Title: A high throughput DNA extraction method with high yield and quality

Authors
item Xin, Zhanguo
item Chen, Junping

Submitted to: Plant Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 20, 2012
Publication Date: July 28, 2012
Citation: Xin, Z., Chen, J. 2012. A high throughput DNA extraction method with high yield and quality. Plant Methods. 8:26.

Interpretive Summary: A high throughput, high yield, and high quality genomic DNA extraction method is developed. 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 very cost-inhibitive. We developed a high throughput DNA isolation method by combining a high DNA yield CTAB extraction method with an improved DNA cleanup procedure based on MagAttract kit. The method yielded large quantity and high quality DNA from both lyophilized sorghum (Sorghum bicolor L. Moench) leaves and dry seeds. The DNA yield was improved by nearly 30 fold with four times less consumption of MagAttract beads. Based on the absorption spectrum from 220 to 400 nm and digestion by DNA restriction enzymes, the quality of DNA extracted with this method is high and consistent from sample to sample. The method has also been used successfully for isolating DNA from cotton, peanut, maize, and Arabidopsis leaves.

Technical Abstract: 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 very 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 DNA yield CTAB extraction method with an improved DNA 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. Conclusion: A high throughput system for DNA extraction from sorghum leaves and seeds was developed and validated. Based on the absorption spectrum from 220 to 400 nm and digestion by DNA restriction enzymes, the quality of DNA extracted with this method is high and consistent from sample to sample. The method has also been used successfully for isolating DNA from cotton, peanut, maize, and Arabidopsis leaves.

Last Modified: 7/27/2014
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