Isolation of Y-chromosomal repetitive DNA sequences of Drosophila hydei via enrichment of chromosome-specific sequences by heterogeneous hybridization between female and male DNA

J Biochem Biophys Methods. 1986 Jan;12(1-2):37-50. doi: 10.1016/0165-022x(86)90049-7.

Abstract

Male or female DNA of Drosophila hydei was sheared by sonication, denatured, reannealed to different C0t-values and fractionated by hydroxyapatite. The highly repetitive, moderately repetitive and unique fractions of female DNA were denatured again and coupled via diazotization or cyanogen bromide to macroporous Sephacryl S-500. Enrichment of Y-chromosomal sequences was achieved by cycling each of the different fractions of male DNA under optimized hybridization conditions over a column with a manifold excess of immobilized female DNA of the corresponding complexity. Thereby, Y-chromosomal sequences of D. hydei could be enriched about 100-fold for highly and moderately repetitive DNA and about 10-fold for unique DNA. When a library of male D. hydei DNA was screened with Y-enriched highly repetitive DNA, more than 98% of all hybridizing phages contained inserts of repetitive Y-chromosomal DNA of at least four different sequence families.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromatography / methods
  • DNA / isolation & purification
  • Drosophila / genetics*
  • Drosophila / ultrastructure
  • Female
  • Hydroxyapatites
  • Male
  • Nucleic Acid Hybridization
  • Nucleic Acid Renaturation
  • Repetitive Sequences, Nucleic Acid*
  • Y Chromosome / analysis*

Substances

  • Hydroxyapatites
  • DNA