A differential cloning procedure of complex genomic DNA fragments

Hiroshi Yokota, Shinya Amano, Tetsuo Yamane, Kazuto Ataka, Eriko Kikuya, Michio Oishi

研究成果: Article査読

9 被引用数 (Scopus)

抄録

We have developed a differential cloning procedure designed for cloning of anonymous restriction DNA fragments whose molecular sizes differ between two genomic DNA preparations from higher organisms. The procedure, which was extensively revised from the original one, consists of several steps as summarized below. (i) Digestion of two DNA preparations (target and reference DNA) with the same restriction enzyme (4 base cutter). (ii) Biotinylation of target DNA and conversion of reference DNA to nonamplifiable form by terminal dephosphorylation. (iii) Electrophoresis of the two DNA preparations through a synthetic gel with a large excess of reference DNA as a competitor. (iv) In-gel alkaline dissociation of DNA, followed by reassociation (in-gel competitive reassociation). (v) Elution of DNA from the gel and PCR after adapter ligation and adsorption of DNA onto streptavidin-coated matrix. By repeating these steps, we attained substantial enrichment (~10,000-fold) of DNA fragments which were originally present at one copy or less per complex mammalian genome. The details of the procedure and its unique characteristics in cloning of altered genomic DNA fragments, particularly from mammalian genome, are discussed.

本文言語English
ページ(範囲)131-138
ページ数8
ジャーナルAnalytical Biochemistry
219
1
DOI
出版ステータスPublished - 1994 5月 15
外部発表はい

ASJC Scopus subject areas

  • 生物理学
  • 生化学
  • 分子生物学
  • 細胞生物学

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