Single point mutation analysis using magnetic beads coated with streptavidin

DOI IR Web Site Open Access

Bibliographic Information

Other Title
  • ストレプトアビジン修飾磁性微粒子を利用したDNA点突然変異の解析
  • ストレプトアビジン シュウショク ジセイ ビリュウシ オ リヨウ シタ DNA テントツゼン ヘンイ ノ カイセキ

Search this article

Abstract

One of the promising techniques that can distinguish low-abundant mutant DNA from wild-type DNA is the ligase detection reaction (LDR) coupled to a primary polymerase chain reaction (PCR). In the present PCR/LDR assay, we used discriminating primers that were labeled with different fluorescent dyes (ROX, HEX, TET, & TAMRA) at their 5'-ends and can discriminate a single nucleotide difference at the mutation site of the target sequence at their 3'-ends by the allele-specific ligation with the common primer which was labeled with biotin at its 3'-end. Following the PCR/LDR processing, we immobilized the resultant LDR products on streptavidin-coated magnetic beads via biotin-streptavidin bonding and purified the product by magnetically collecting and rinsing the beads. Then, the purified LDR products were released in a solvent of formamide containing 10 mM EDTA by thermally cleaving the streptavidin-biotin bonding at 75°C for 5 min. Fluorescent signatures could be acquired from the recovered LDR products by fluorescence monochrometer measurements, enabling rapid and simple determination of single nucleotide substitutions on codon 12.2 of exon 1 of K-ras oncogene based on the difference in fluorescence spectra of the labeled dyes.

Journal

Keywords

Details 詳細情報について

Report a problem

Back to top