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Open Access Highly Accessed Methodology article

Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

Kerry A Lutz12*, Wenqin Wang1, Anna Zdepski1 and Todd P Michael3

Author Affiliations

1 Rutgers, The State University of New Jersey, Department of Plant Biology and Pathology, The Waksman Institute of Microbiology, Piscataway, NJ 08854, USA

2 Current Address: Farmingdale State College, Ward Hall, Room 307, 2350 Broadhollow Road, Farmingdale, NY 11735, USA

3 Monsanto Company, 800 North Lindbergh Blvd., Creve Coeur, Missouri 63167, USA

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BMC Biotechnology 2011, 11:54  doi:10.1186/1472-6750-11-54

Published: 20 May 2011

Abstract

Background

High throughput sequencing (HTS) technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination.

Results

We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR). We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment.

Conclusions

Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.

Keywords:
chloroplast DNA; high throughput sequencing; mitochondrial DNA; nuclear DNA isolation; plant genome sequencing; quantitative real-time PCR (qPCR)