Allele-specific hybridization using streptavidin-coated magnetic beads for species identification, S genotyping, and SNP analysis in plants

Kaoru Tonosaki, Junpei Kudo, Hiroyasu Kitashiba, Takeshi Nishio

研究成果: Article査読

9 被引用数 (Scopus)

抄録

Dot-blot hybridization has been successfully used for the construction of single nucleotide polymorphism (SNP)-based linkage maps, quantitative trait locus analysis, marker-assisted selection, and the identification of species and cultivars. This method is, however, time-consuming, even for a small number of plant samples. We propose a method in which streptavidin-coated magnetic beads replace the nylon membrane for immobilization of the PCR products and are hybridized with allele-specific oligonucleotide probes and a digoxigenin-labeled oligonucleotide hybridized with the allele-specific oligonucleotide probe. After amplification of plant DNA by PCR with the biotinylated primers, those oligonucleotide probes having species-specific or allele-specific sequences were mixed together with the digoxigenin-labeled oligonucleotide and the streptavidin-coated magnetic beads at a temperature suitable for each probe. Species-specific internal transcribed spacer 1 (ITS1) sequences and allele-specific sequences of the hypervariable region I of S-locus receptor kinase (SRK) specifically detected ITS1 sequences and SRK alleles in Brassica species, respectively. SNPs were also successfully analyzed by using allele-specific oligonucleotide probes and competitive oligonucleotides. In the SNP analysis, PCR products were indirectly captured by magnetic beads. SNP alleles of eight cultivars each of Brassica rapa and Raphanus sativus were analyzed using streptavidin-coated magnetic beads. The genotyping results corresponded well with those of dot-blot-SNP analysis. Although allele-specific hybridization using streptavidin-coated magnetic beads is somewhat costly, it is easier and more rapid than dot-blot hybridization. This method is suitable for the analysis of a small number of plant samples with a large number of DNA markers.

本文言語English
ページ(範囲)419-428
ページ数10
ジャーナルMolecular Breeding
31
2
DOI
出版ステータスPublished - 2013 2

ASJC Scopus subject areas

  • バイオテクノロジー
  • 分子生物学
  • 農業および作物学
  • 遺伝学
  • 植物科学

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