Normalization of Genomic DNA Using Duplex-Specific Nuclease Essay

Whole genome shotgun sequencing (WGS) is an effective method for the study of reference sequences in genomes. It generates several sequences data, which result in overlapping sequences eventually. The aligning DNA sequences achieved overlapping sequence assembly into contigs that could read through the computer program. The WGS method is not applicable when redundant repetitive sequences exist in large genomes1 (cited in 1). Several methods such as methylation-spanning linker libraries (MSLL), Methylation filtration (MF) and others have used eradicating redundancy in higher plant genomes that depended on the hypermethylation tendency of repetitive sequences. The use of enzymes or a genomic library set up could modify the genome, but it is applicable to limited plant genomes 2-4 (cited in 1). The authors proposed another method in this article called ‘high-C0t DNA analysis’ that followed DNA renaturation kinetics in which sheared, denatured, and gradually reanneled genomi c DNA is used. Then, hydroxyapatite chromatography is used for separation of repetitive sequences (dsDNA) from low-copy sequences (ssDNA). With the help of detailed knowledge of DNA reassociation kinetics and advance skills in spectrophotometry, high-C0t DNA analysis can be applied to any genome5-7 (cited in 1). Shagina and others (2010) has discovered duplex-specific nuclease (DSN) normalization technology for genomic DNA (1). It is a simple method that based on hybridization kinetics excluding separation of both ssDNA and dsDNA. The authors’ isolated DSN enzyme from the Kamchatka crab that is thermostable and specific to dsDNA8 (cited in 1). They first denatured dsDNA that contained repetitive sequence and hydrolyzed it by DSN and then ran PCR on ssDNA (low-... ...tion of normalized cDNA libraries enriched with full-length sequences. Bioorganic Khim. 31:170-177. 10Zhulidov PA, Bogdanova EA, Shcheglov AS, Vagner LL, Khaspekov GL, Kozhemyako VB, Matz MV, Meleshkevitch E. (2004). Simple cDNA normalization using Kamchatka crab duplex-specific nuclease. Nucleic Acids Res 32:e37. 11Rodrigue S, Malmstrom RR, Berlin AM, Birren BW, Henn MR, and Chisholm SW. (2009). Whole genome amplification and de novo assembly of single bacterial cells. PLoS One 4:e6864. 12Cheung F, Haas BJ, Goldberg SM, May GD, Xiao Y, and Town CD. (2006). Sequencing Medicago truncatula expressed sequenced tags using 454 Life Sciences technology. BMC Genomics 7:272. 13Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K. (2001). Initial sequencing and analysis of the human genome. Nature 409:860-921.