Advertisement

Plant Molecular Biology Reporter

, Volume 19, Issue 2, pp 159–162 | Cite as

Detection of single nucleotide mutations in wheat using single strand conformation polymorphism gels

  • P. Martins-Lopes
  • H. Zhang
  • R. Koebner
Protocols

Abstract

We show that single strand conformation polymorphism (SSCP) analysis, using the mutation detection enhancement (MDETM) matrix, is efficient at detecting sequence polymorphisms in PCR amplicons. Four independent wheat genomic fragments were amplified from two contrasting templates and sequenced. The allelic fragments were differentiated at 1–6 single nucleotide positions, but MDE-SSCP was able to unequivocally distinguish each allelic pair. The approach is therefore considered a powerful way of identifying single nucleotide polymorphisms (SNPs) without extensive amplicon sequencing.

Key words

SNP SSCP wheat 

Abbreviations

MDE

mutation detection enhancement

SNP

single nucleotide polymorphism

SSCP

single strand conformation polymorphism

TBE

tris borate ethylene-diaminetetraacetic acid

TEMED

tetrametylethylenediamine

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bassam BJ, Caetano-Anolles G and Gresshoff PM (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 196: 80–83.PubMedCrossRefGoogle Scholar
  2. Livak KJ, Flood SJ, Marmaro J, Giusti W and Deetz K (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic-acid hybridization. PCR Methods Appl 4: 357–362.PubMedCrossRefGoogle Scholar
  3. Orita M, Iwahana H, Kanazana H, Hayashi K and Sekiya T (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 86: 2766–2770.PubMedCrossRefGoogle Scholar
  4. Sarkar G, Yoon HS and Sommer SS (1992) Screening for mutations by RNA single strand conformation (rSSCP): comparison with DNA-SSCP. Nucleic Acids Res 20: 871–878.PubMedCrossRefGoogle Scholar
  5. Sheffield VC, Beck JS, Kwitek AE, Sandstrom DW and Stone EW (1993) The sensitivity of single-strand conformation polymorphism analysis for the detection of single base substitutions. Genomics 16: 325–332.PubMedCrossRefGoogle Scholar
  6. Tyagi S, Bratu DP and Kramer FR (1998) Multicolor molecular beacons for allele discrimination. Nature Biotech 16: 49–53.CrossRefGoogle Scholar

Copyright information

© International Society for Plant Molecular Biology 2001

Authors and Affiliations

  • P. Martins-Lopes
    • 1
  • H. Zhang
    • 2
  • R. Koebner
    • 2
  1. 1.Departamento de Gen’etica e BiotecnologiaICETA-Universidade de Tr’as-os-Montes e Alto DouroVila RealPortugal
  2. 2.John Innes CentreNorwich Research ParkNorwichU.K.

Personalised recommendations