Earticle

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영어

We herein introduce the development of a novel multiplexed single-nucleotide polymorphisms (SNPs) genotyping method based on amplification of separated ligation-dependent probe (ASLP) and RecA assisted hybridization (RAH) technology. In the ASLP technique, allele-specific ligation reaction was first performed between annealed allele-specific oligonucleotide (ASO) probe and locus-specific oligonucleotide (LSO) probe containing the unique regions of homology to target genomic DNA. The ASO probe covering up to SNP site is modified with universal forward primer site at the 5’ end while the LSO probe is extended with universal separation (US) site and poly A tail at the 3’ end. The two probes would be ligated when they are hybridized to a correctly matching sequence at a specific SNP locus. The separation probe that consists of universal reverse primer site labeled with biotin at its 5’ end and complimentary sequence of US site at its 3’ end was then applied to the resulting ligated product. During the following extension reaction of the separation probe, the ligated probes were dissociated from target genomic DNA as a form of double-stranded DNA (dsDNA) with the strand extended from the separation probe. By using streptavidin-coated magnetic beads, the dsDNA containing ligated probes could be simply separated from the reaction mixture including genomic DNA and unligated probes. PCR amplification of the purified ligation products was then carried out using a primer pair of Cy3-labeled universal forward primer and universal reverse primer, followed by RAH of the PCR products on DNA microarrays. Using this strategy we successfully genotyped fifteen SNPs markers for the cultivar identification of pepper in a single tube reaction within 3 hours.

키워드

저자

• Sung Chul SHIN [ Dept. of Chemical and Biomolecular Engineering, KAIST, Daejeon. ]

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간행물

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• 간행물명

  한국생물공학회 학술대회

• 간기

  반년간

• 수록기간

  1985~2013

• 십진분류

  KDC 476 DDC 576