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Related Experiment Videos

RAPD-based screening of genomic libraries for positional cloning

W Dioh1, D Tharreau, M H Lebrun

  • 1Génétique Moléculaire des Champignons Phytopathogènes, Institut de Génétique et Microbiologie, CNRS-URA 2255, Bâtiment 400, Université Paris-Sud, 91405 Orsay, France.

Nucleic Acids Research
|February 28, 1998
PubMed
Summary

We developed a simple method using Random Amplified Polymorphic DNA (RAPD) analysis to identify specific DNA fragments in genomic libraries. This approach aids in positional cloning and mapping genes, even with repetitive DNA sequences.

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Area of Science:

  • Genetics
  • Molecular Biology
  • Plant Pathology

Background:

  • Random Amplified Polymorphic DNA (RAPD) markers are crucial for positional cloning.
  • Challenges exist in screening genomic libraries with RAPD markers due to repetitive sequences, hindering hybridization.

Purpose of the Study:

  • To develop a simplified RAPD analysis method for identifying specific DNA sequences within genomic libraries.
  • To overcome limitations of hybridization-based screening for RAPD markers in genomic library analysis.

Main Methods:

  • Developed a strategy based on identifying cosmid pools and clones that amplify the target RAPD marker.
  • Utilized a straightforward RAPD protocol for analyzing cosmid pools and individual clones.
  • Applied the method to four RAPD markers linked to avirulence genes in *Magnaporthe grisea*.

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Main Results:

  • Successfully identified cosmids containing the targeted RAPD markers without prior cloning or characterization of the marker itself.
  • The method efficiently screened cosmid pools and clones, enabling rapid identification.
  • Facilitated the construction of physical contigs at specific genetic loci.

Conclusions:

  • The developed RAPD analysis of genomic libraries is a simple, rapid, and effective method for identifying DNA markers.
  • This technique is valuable for positional cloning and gene mapping, particularly for repetitive sequences.
  • Enables efficient physical contig construction for genes of interest, such as avirulence genes in *Magnaporthe grisea*.