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Related Experiment Videos

A method for difference cloning: gene amplification following subtractive hybridization.

I Wieland1, G Bolger, G Asouline

  • 1Cold Spring Harbor Laboratory, NY 11724.

Proceedings of the National Academy of Sciences of the United States of America
|April 1, 1990
PubMed
Summary

Genomic difference cloning isolates unique DNA sequences by subtracting common DNA. This method enriches target sequences 100- to 700-fold for cloning unique genomic DNA.

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Area of Science:

  • Genomics
  • Molecular Biology
  • Biotechnology

Background:

  • Identifying unique DNA sequences between two populations is crucial for understanding genetic variation.
  • Existing methods for isolating differential DNA can be inefficient or lack specificity.

Purpose of the Study:

  • To develop and validate a novel method, genomic difference cloning, for efficient isolation of sequences present in a "tester" DNA population but absent in a "driver" DNA population.
  • To demonstrate the method's applicability in scenarios involving sequence gain (e.g., pathogen infection) and sequence loss (e.g., deletions).

Main Methods:

  • Genomic DNA populations (tester and driver) were subjected to subtractive hybridization using biotinylated tester DNA and a large excess of driver DNA.
  • Repeated cycles of subtractive hybridization enriched for tester-specific sequences.

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  • Avidin/biotin affinity chromatography separated enriched tester DNA from the driver DNA.
  • Single-stranded target DNA was amplified using polymerase chain reaction (PCR) to generate double-stranded, clonable DNA.

    • Main Results:

    • The genomic difference cloning procedure successfully enriched target sequences unique to the tester population.
    • Enrichment factors ranged from 100- to 700-fold.
    • The method was successfully modeled for both the gain of sequences (pathogen infection) and the loss of sequences (hemizygous deletion).

    Conclusions:

    • Genomic difference cloning is an effective method for isolating unique genomic sequences.
    • The technique offers significant enrichment, making it valuable for comparative genomics and genetic variation studies.
    • This method provides a powerful tool for identifying genetic differences, such as those arising from infections or deletions.