Irina Shagina1, Ekaterina Bogdanova2, Ilgar Z. Mamedov2, Yury Lebedev2, Sergey Lukyanov2, Dmitry Shagin1,2
1Evrogen JSC, Moscow, Russia and 2Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russia
BioTechniques 48:455-459 ( June 2010) doi 10.2144/000113422
Keywords: genomic DNA; DSN normalization; duplex-specific nuclease
Supplementary material for this article is available at .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 demon- strated 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.
1Evrogen JSC, Moscow, Russia and 2Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russia
BioTechniques 48:455-459 ( June 2010) doi 10.2144/000113422
Keywords: genomic DNA; DSN normalization; duplex-specific nuclease
Supplementary material for this article is available at .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 demon- strated 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.
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