Isolation of Y-chromosomal repetitive DNA sequences of Drosophila hydei via enrichment of chromosome-specific sequences by heterogeneous hybridization between female and male DNA.
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.
D000818:Animals; D002845:Chromatography / Q000379:methods; D004247:DNA / Q000302:isolation & purification; D004330:Drosophila / Q000235:genetics* / Q000648:ultrastructure; D005260:Female; D006882:Hydroxyapatites; D008297:Male; D009693:Nucleic Acid Hybridization; D009695:Nucleic Acid Renaturation; D012091:Repetitive Sequences, Nucleic Acid; D014998:Y Chromosome / Q000032:analysis*
D006882:Hydroxyapatites; D004247:DNA