title: Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing.
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.
authors: Rutgers, The State University of New Jersey, Department of Plant Biology and Pathology, The Waksman Institute of Microbiology, Piscataway, NJ 08854, USA. lutzk@farmingdale.edu
authors: Kerry A
authors: KA
authors: Lutz
authors: Wenqin
authors: W
authors: Wang
authors: Anna
authors: A
authors: Zdepski
authors: Todd P
authors: TP
authors: Michael
references: Plant Methods. 2008 May 19;4:9
references: 18489765
references: Nat Methods. 2005 Mar;2(3):213-8
references: 16163802
references: PLoS One. 2007 Dec 19;2(12):e1326
references: 18094749
references: Science. 1999 Sep 3;285(5433):1579-82
references: 10477524
references: Methods Enzymol. 1974;31:528-44
references: 4418408
references: Am J Bot. 2011 Jan;98(1):e13-5
references: 21613076
references: Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):5934-8
references: 8327463
references: Plant Biol (Stuttg). 2006 Nov;8(6):770-7
references: 17203433
references: Plant Cell Physiol. 2006 May;47(5):601-12
references: 16524874
references: Mol Genet Genomics. 2010 Jan;283(1):35-47
references: 19911199
references: Proc Natl Acad Sci U S A. 2004 Aug 24;101(34):12759-64
references: 15310842
references: Biotechniques. 1992 Jul;13(1):52-4, 56
references: 1503775
references: Planta. 2008 May;227(6):1377-88
references: 18301915
references: Plant Physiol. 1990 Nov;94(3):1477-83
references: 16667857
references: Nature. 2010 Feb 11;463(7282):763-8
references: 20148030
references: Science. 1978 May 5;200(4341):561-3
references: 17839439
references: Plant Mol Biol. 1987 Mar;8(2):169-77
references: 24301052
references: BMC Plant Biol. 2009 Jan 07;9:3
references: 19128504
references: Genome Res. 2008 Dec;18(12):2024-33
references: 18818371
references: Ann Bot. 2007 Aug;100(2):195-203
references: 17550910
references: Biotechniques. 1991 Feb;10(2):162, 164, 166
references: 2059438
references: BMC Genomics. 2008 Oct 31;9:518
references: 18976483
references: Curr Genet. 2004 Sep;46(3):176-81
references: 15249983
references: Plant J. 2007 May;50(4):710-22
references: 17425718
references: Bioinformatics. 2010 Mar 15;26(6):849-50
references: 20207696
references: Biotechnol J. 2007 Mar;2(3):386-92
references: 17285676
references: Mol Phylogenet Evol. 2010 Aug;56(2):768-83
references: 20226867
references: Nat Genet. 2009 Dec;41(12):1275-81
references: 19881527
references: Nucleic Acids Res. 1980 Oct 10;8(19):4321-5
references: 7433111
references: Planta. 2006 Jun;224(1):72-82
references: 16362324