Mutation detection in rice waxy mutants by PCR-RF-SSCP

Y. Sato, T. Nishio

Research output: Contribution to journalArticlepeer-review

29 Citations (Scopus)

Abstract

PCR-RF-SSCP (PRS), which combines cleaved amplified polymorphic sequence (CAPS) and single-strand conformation polymorphism (SSCP), is expected to be a useful technique for DNA polymorphism analysis. We evaluated the ability of PRS to detect single nucleotide polymorphism (SNP) using the Waxy gene, Wx, of rice, and subsequently were able to identify point mutations in wx mutant lines. The approximately 6-kb Wx gene was divided into five regions for PCR amplification. Two regions, in which most of the point mutations of the wx mutants have been identified, were amplified by PCR and cloned into a vector, and those clones containing SNPs produced as a result of the inherent inaccuracy of PCR were used for the evaluation of PRS. The efficiency of PRS in the detection of SNPs of these clones was over 70%. PRS analysis of the wx genes in 18 waxy mutants was carried out in the five regions using two different restriction endonucleases and two gel conditions, with and without glycerol. Of the 18 lines tested, 17 showed band patterns different from that of the wild type. Most of the mutations identified in this study were nucleotide changes in exons, which result in amino acid changes. One mutation generated an in-frame stop codon, and another was a frame shift mutation by one-base deletion. Two mutations found at a splice site were considered to inhibit normal splicing of mRNA. These results show that PRS is a useful technique for detecting point mutations in large plant genes.

Original languageEnglish
Pages (from-to)560-567
Number of pages8
JournalTheoretical and Applied Genetics
Volume107
Issue number3
DOIs
Publication statusPublished - 2003 Aug 1

Keywords

  • DNA polymorphism
  • Point mutation
  • SNP
  • SSCP
  • Wx

ASJC Scopus subject areas

  • Biotechnology
  • Agronomy and Crop Science
  • Genetics

Fingerprint Dive into the research topics of 'Mutation detection in rice waxy mutants by PCR-RF-SSCP'. Together they form a unique fingerprint.

Cite this