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Normalization Of Dna Using Duplex Specific Nuclease

1766 words - 7 pages

Normalization of genomic DNA using duplex-specific nuclease
Whole genome shotgun sequencing (WGS) is a potent method for the study of reference sequences in genomes. It generates several sequence data, which result in overlapping sequences eventually. The aligning DNA sequences achieved overlapping sequence reads assembly into contigs, which could read through the computer program. Due to the presence of redundant repetitive sequences in small genomes, the WGS method is not applicable1 (cited in 1). Several methods have proposed to eradicate redundancy in plant genomes which depends on the hypermethylation tendency of repetitive sequences. The use of enzymes or to establish a genomic library could modify the genome, but it is restricted to limited plant genomes 2-4 (cited in 1). Another method proposed in this article called ‘high-C0t DNA analysis’ which is based on DNA renaturation kinetics. In this technique, genomic DNA is denatured and reannealing slowly and then, hydroxyapatite chromatography used for separation of dsDNA. With the help of detailed knowledge of DNA reassociation kinetics and proficiency in spectrophotometry, high-C0t DNA analysis can be applied to any genome5-7 (cited in 1).
Shagina and others, 2010 has discovered the application of duplex-specific nuclease (DSN) normalization technology for eukaryotic genomic DNA. It is a simple and effective method, based on hybridization kinetics excluding physical separation of ssDNA and dsDNA both. After re-association, denatured dsDNA contained repetitive sequence which hydrolyzed by DSN and PCR used for amplification of ssDNA which contain low copy molecules8, 9(cited in 1). DSN enzyme has isolated from the Kamchatka crab which is thermostable and specific to dsDNA10 (cited in 1). DSN normalization is also used for post amplification of microbial genomes acquired from whole genome amplification methods 11 (cited in 1). In this report, the introduction is briefly summarized and stated the advances and significance of novel modification to existing methods of normalization of DNA. The flaw of talk took a different route in the introduction when the authors explained different methods instead of talking about their novel discovery. It increased my efforts towards to understand introductory information.
In material and methods the authors used 50μg sheared human genomic DNA which was 500kb mean length. Agarose gel electrophoresis was used to determined length of DNA. Then, phenol: chloroform extract used for extraction, precipitated with ethanol and redissolved in Tris HCl. An aliquot of sheared DNA was treated with T4DNA polymerase to create blunt ends. To make a concentration of 100ng/ µL, the above procedures of purification, precipitation, and redissolved in Tris HCl was followed. The phosphorylation occurred at 5’ end of DNA fragment due to incubating blunt ended DNA samples with ligase buffer, riboATP, polynucleotide kinase at 37º C for 15 min. After passing through...

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