Improvement of the dot-blot-SNP technique for efficient and cost-effective genotyping

Sachiko Shiokai, Kenta Shirasawa, Yutaka Sato, Takeshi Nishio

    Research output: Contribution to journalArticlepeer-review

    27 Citations (Scopus)

    Abstract

    Although the dot-blot-SNP technique is a laborsaving, cost-effective method for SNP genotyping of a large number of plants, the synthesis of 5′-digoxigenin (DIG)-labeled oligonucleotides for use as probes is still costly. We developed two probe-labeling methods for this technique, one being digoxigenin labeling of oligonucleotides by PCR (PCR-DIG labeling) and the other being hybridization using a bridge probe and a 5′-DIG-labeled oligonucleotide (bridge hybridization). Bridge hybridization detected allele-specific signals under hybridization conditions similar to those for the 5′-DIG-labeled oligonucleotides and biotin-labeled oligonucleotides, while signals were detected only under a lower stringency condition by PCR-DIG labeling. As a method for genotyping using many markers at one time, two methods, i. e., PCR using mixed primer pairs and hybridization using mixed probes, were examined with successful results. Eighty-five SNP markers designed for genotyping of rice cultivars detected allele-specific signals, the genotyping results corresponding to the previously reported ones.

    Original languageEnglish
    Pages (from-to)179-185
    Number of pages7
    JournalMolecular Breeding
    Volume25
    Issue number1
    DOIs
    Publication statusPublished - 2010 Dec

    Keywords

    • Cultivar identification
    • Dot-blotting
    • Large-scale genotyping
    • SNP analysis

    ASJC Scopus subject areas

    • Biotechnology
    • Molecular Biology
    • Agronomy and Crop Science
    • Genetics
    • Plant Science

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