High sensitivity, high resolution physical mapping by fluorescence in situ hybridization on to individual straightened DNA molecules

Mei Wang, Thomas Duell, Joe W. Gray, Heinz Ulrich G. Weier

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

High resolution physical mapping of clonal DNA fragments with kilobase (kb) resolution can now be performed rapidly by fluorescence in situ hybridization (FISH) onto individual DNA molecules (DNA fibers). We developed a sensitive procedure termed 'quantitative DNA fiber mapping' which consists of three steps: preparation of DNA fibers, hybridization of non-isotopically labeled probes and determination of the relative mapping position by fluorescence image analysis. The DNA fibers are produced by binding linearized DNA molecules with one or both ends to a solid substrate followed by homogeneous stretching of the molecule by the action of a receding meniscus during drying ('molecular combing'). In a slight variation of this protocol, we deposit circular DNA molecules. Substrates for DNA immobilization are glass slides, coverslips or thin sheets of mica derivatized with amino-silane. Probes are prepared to counterstain the DNA fibers, from the clones to be mapped and for specific landmarks along linear or circular DNA molecules such as cloning vector sequences. Following hybridization and immunocytochemical detection of bound probes, images are analysed and relative distances are recorded for map assembly. Here, we describe our experience with substrate preparation, molecular combing and mapping of cloned or enzymatically synthesized probes ranging in size from 1.2 kb to 100 kb along DNA molecules that are between 17 kb and 1200 kb in size.

Original languageEnglish (US)
Pages (from-to)73-83
Number of pages11
JournalBioimaging
Volume4
Issue number2
DOIs
StatePublished - 1996

Keywords

  • DNA molecules
  • fluorescence in situ hybridization (FISH)
  • image analysis
  • molecular combin
  • physical mapping

ASJC Scopus subject areas

  • Biophysics
  • Radiology Nuclear Medicine and imaging

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