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Related Experiment Videos

Isolation of YAC insert sequences by representational difference analysis

M Schutte1, L T da Costa, C A Moskaluk

  • 1Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD 21205-2196, USA.

Nucleic Acids Research
|October 25, 1995
PubMed
Summary
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This study introduces a new method using representational difference analysis (RDA) to isolate yeast artificial chromosome (YAC) insert sequences. The technique efficiently identifies YAC DNA, enabling the discovery of genetic markers and probes.

Area of Science:

  • Genomics
  • Molecular Biology
  • Biotechnology

Background:

  • Yeast artificial chromosomes (YACs) are crucial for cloning large DNA fragments.
  • Isolating specific YAC insert sequences presents technical challenges.
  • Efficient methods are needed for YAC sequence characterization and application.

Purpose of the Study:

  • To develop and validate a method for isolating YAC insert sequences.
  • To assess the efficiency and utility of the developed technique.
  • To enable downstream applications using isolated YAC sequences.

Main Methods:

  • Representational Difference Analysis (RDA) was employed for sequence isolation.
  • Amplicons were generated from MboI digestion products for maximal sequence representation.

Related Experiment Videos

  • RDA was performed on a 970 kb insert YAC clone over two rounds of selective amplification.

    • Main Results:

    • 92% of the resulting difference product comprised sequences derived from the YAC insert.
    • Twenty insert-specific sequence-tagged sites (STSs) were successfully identified.
    • The method facilitated the isolation of microsatellite markers and human PAC library clones.

    Conclusions:

    • Representational Difference Analysis is an effective method for isolating YAC insert sequences.
    • The identified sequences are valuable for genetic marker development.
    • The technique is versatile, supporting applications like chromosome painting via fluorescence in situ hybridization (FISH).