Advertisement

Analysis of Mutation/Rearrangement Frequencies and Methylation Patterns at a Given DNA Locus Using Restriction Fragment Length Polymorphism

  • Alex BoykoEmail author
  • Igor Kovalchuk
Protocol
  • 1.6k Downloads
Part of the Methods in Molecular Biology™ book series (MIMB, volume 631)

Abstract

Restriction fragment length polymorphism (RFLP) is a difference in DNA sequences of organisms belonging to the same species. RFLPs are typically detected as DNA fragments of different lengths after digestion with various restriction endonucleases. The comparison of RFLPs allows investigators to analyze the frequency of occurrence of mutations, such as point mutations, deletions, insertions, and gross chromosomal rearrangements, in the progeny of stressed plants. The assay involves restriction enzyme digestion of DNA followed by hybridization of digested DNA using a radioactively or enzymatically labeled probe. Since DNA can be digested with methylation sensitive enzymes, the assay can also be used to analyze a methylation pattern of a particular locus. Here, we describe RFLP analysis using methylation-insensitive and methylation-sensitive enzymes.

Key words

Restriction fragment length polymorphism (RFLP) Genome stability Mutation frequency Locus-specific methylation pattern Methylation sensitive enzymes 

References

  1. 1.
    Pethe V, Lagu M, Chitnis PK, Gupta V, Ranjekar PK (1989) Restriction fragment length polymorphism: a recent approach in plant breeding. Indian J Biochem Biophys 26:285–288PubMedGoogle Scholar
  2. 2.
    Barnes SR (1991) RFLP analysis of complex traits in crop plants. Symp Soc Exp Biol 45:219–228PubMedGoogle Scholar
  3. 3.
    Todd R, Donoff RB, Kim Y, Wong DT (2001) From the chromosome to DNA: Restriction fragment length polymorphism analysis and its clinical application. J Oral Maxillofac Surg 59:660–667CrossRefPubMedGoogle Scholar
  4. 4.
    Gusella JF, Wexler NS, Conneally PM, Naylor SL, Anderson MA, Tanzi RE et al (1983) A polymorphic DNA marker genetically linked to Huntington’s disease. Nature 306:234–238CrossRefPubMedGoogle Scholar
  5. 5.
    Kochert G (1991) Restriction fragment length polymorphism in plants and its implications. Subcell Biochem 17:167–190PubMedGoogle Scholar
  6. 6.
    Nagamura Y, Antonio BA, Sasaki T (1997) Rice molecular genetic map using RFLPs and its applications. Plant Mol Biol 35:79–87CrossRefPubMedGoogle Scholar
  7. 7.
    Wu YY, Csako G (2006) Rapid and/or high-throughput genotyping for human red blood cell, platelet and leukocyte antigens, and forensic applications. Clin Chim Acta 363:165–176CrossRefPubMedGoogle Scholar
  8. 8.
    Agarwal M, Shrivastava N, Padh H (2008) Advances in molecular marker techniques and their applications in plant sciences. Plant Cell Rep 27:617–631CrossRefPubMedGoogle Scholar
  9. 9.
    Boyko A, Kathiria P, Zemp FJ, Yao Y, Pogribny I, Kovalchuk I (2007) Transgenerational changes in the genome stability and methylation in pathogen-infected plants: (virus-induced plant genome instability). Nucleic Acids Res 35:1714–1725CrossRefPubMedGoogle Scholar
  10. 10.
    Nelson M, Raschke E, McClelland M (1993) Effect of site-specific methylation on restriction endonucleases and DNA modification methyltransferases. Nucleic Acids Res 21:3139–3154CrossRefPubMedGoogle Scholar
  11. 11.
    McClelland M, Nelson M, Raschke E (1994) Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases. Nucleic Acids Res 22:3640–3659CrossRefPubMedGoogle Scholar
  12. 12.
    Gonzalgo ML, Jones PA (1997) Mutagenic and epigenetic effects of DNA methylation. Mutat Res 386:107–118CrossRefPubMedGoogle Scholar
  13. 13.
    Wijsman EM (1984) Optimizing selection of restriction enzymes in the search for DNA variants. Nucleic Acids Res 12:9209–9226CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Department of Biological SciencesUniversity of LethbridgeLethbridgeCanada

Personalised recommendations