Normalization of genomic DNA using duplex-specific nuclease.

An application of duplex-specific nuclease (DSN) normalization technology to whole-genome shotgun sequencing of genomes with a large proportion of repetitive DNA is described. The method uses a thermostable DSN from the Kamchatka crab that specifically hydrolyzes dsDNA. In model experiments on human genomic DNA, we demonstrated that DSN normalization of double-stranded DNA formed during C0t analysis is effective against abundant repetitive sequences with high sequence identity, while retaining highly divergent repeats and coding regions at base-line levels. Thus, DSN normalization applied to C0t analysis can be used to eliminate evolutionarily young repetitive elements from genomic DNA before sequencing, and should prove invaluable in studies of large eukaryotic genomes, such as those of higher plants.

[1]  Sallie W. Chisholm,et al.  Whole Genome Amplification and De novo Assembly of Single Bacterial Cells , 2009, PloS one.

[2]  S. Lukyanov,et al.  DSN Depletion is a Simple Method to Remove Selected Transcripts from cDNA Populations , 2009, Molecular biotechnology.

[3]  S. Lukyanov,et al.  Normalization of full-length enriched cDNA. , 2008, Molecular bioSystems.

[4]  B. Haas,et al.  Sequencing Medicago truncatula expressed sequenced tags using 454 Life Sciences technology , 2006, BMC Genomics.

[5]  J. Bennetzen,et al.  Gene enrichment in maize with hypomethylated partial restriction (HMPR) libraries. , 2005, Genome research.

[6]  S. Lukyanov,et al.  A method for the preparation of normalized cDNA libraries enriched with full-length sequences , 2005, Russian Journal of Bioorganic Chemistry.

[7]  S. Lukyanov,et al.  Simple cDNA normalization using kamchatka crab duplex-specific nuclease. , 2004, Nucleic acids research.

[8]  S. Dike,et al.  Maize Genome Sequencing by Methylation Filtration , 2003, Science.

[9]  Beat Keller,et al.  CACTA Transposons in Triticeae. A Diverse Family of High-Copy Repetitive Elements1 , 2003, Plant Physiology.

[10]  Yinan Yuan,et al.  High-Cot sequence analysis of the maize genome. , 2003, The Plant journal : for cell and molecular biology.

[11]  J. Bennetzen,et al.  Structural Analysis of the Maize Rp1 Complex Reveals Numerous Sites and Unexpected Mechanisms of Local Rearrangement Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.006338. , 2002, The Plant Cell Online.

[12]  S. Lukyanov,et al.  A novel method for SNP detection using a new duplex-specific nuclease from crab hepatopancreas. , 2002, Genome research.

[13]  J. Bennetzen,et al.  Methylation-spanning linker libraries link gene-rich regions and identify epigenetic boundaries in Zea mays. , 2002, Genome research.

[14]  Daniel G Peterson,et al.  Integration of Cot analysis, DNA cloning, and high-throughput sequencing facilitates genome characterization and gene discovery. , 2002, Genome research.

[15]  J. Bennetzen,et al.  Transposable elements, genes and recombination in a 215-kb contig from wheat chromosome 5Am , 2002, Functional & Integrative Genomics.

[16]  J. Bennetzen,et al.  National Science Foundation-sponsored workshop report. Maize genome sequencing project. , 2001, Plant physiology.

[17]  A. Nekrutenko,et al.  Transposable elements are found in a large number of human protein-coding genes. , 2001, Trends in genetics : TIG.

[18]  T. Wicker,et al.  Analysis of a contiguous 211 kb sequence in diploid wheat (Triticum monococcum L.) reveals multiple mechanisms of genome evolution. , 2001, The Plant journal : for cell and molecular biology.

[19]  P. Schulze-Lefert,et al.  A contiguous 66-kb barley DNA sequence provides evidence for reversible genome expansion. , 2000, Genome research.

[20]  Phillip SanMiguel,et al.  The paleontology of intergene retrotransposons of maize , 1998, Nature Genetics.

[21]  J. Bennetzen,et al.  Nested Retrotransposons in the Intergenic Regions of the Maize Genome , 1996, Science.

[22]  R. Flavell,et al.  Characterisation of the wheat genome by renaturation kinetics , 1975, Chromosoma.

[23]  K. Abromeit.  Music Received , 2023, Notes.

[24]  W. Barbazuk,et al.  Sequencing Genes and Gene Islands by Gene Enrichment , 2009 .

[25]  International Human Genome Sequencing Consortium.  Initial sequencing and analysis of the human genome , 2001, Nature.