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Title 

Cloning of no t I-cleaved genomic DNA fragments appearing as spots in 2D gel electrophoresis

Authors 

Hisaki NagaiM. PongliktmongkolYong Sung KimHirohide YoshikawaKenichi Matsubara

Publisher 

Elsevier

Issue Date 

1995

Citation 

Biochemical and Biophysical Research Communications, vol. 213, no. 1, pp. 258-265

Keywords 

dna fragmentDNAdna cleavagedna sequenceelectrophoresistwo dimensional gel electrophoresis

Abstract 

RLGS (Restriction Landmark Genomic Scanning) is a simple and rapid scanning of genomic DNA in two-dimensional electrophoresis. Human genomic DNA is first cleaved by NotI, and the cleaved ends are radio-labeled and cleaved further by EcoRV, followed by size-fractionation by first dimensional electrophoresis. The sample is then cleaved in situ by the second enzyme HinfI and resolved by the second dimensional electrophoresis. Nearly 2,000 spots emerge with spot intensities reflecting the copy number in the genome. Because of the resolving power and capacity to scan the entire genome, RLGS has been used to monitor genomic aberrations and imprinting. Here, we report a means of cloning the DNA in spots. The DNA was eluted and ligated to biotinylated NotI and HinfI linkers followed by affinity separation using streptavidin. The ligated fragment was recovered by EcoRV cleavage, the target sequence of which was located in the NotI linker and amplified by PCR using a primer pair, the sequences of which lie in the linkers. The products were then cloned into a vector for further tests. An amplified spot in stomach cancer genomic DNA and a dwindling spot in liver cancer genomic DNA were taken as examples for cloning.

ISSN 

0006-291X

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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