A simple and efficient amplification method of DNA with unknown sequences and its application to microdissection/microcloning

Yoshlhlro Jinno*, Naoki Harada, Koh Ichiro Yoshiura, Tohru Ohta, Takaya Tohma, Tetsuya Hirota, Kazuhiro Tsukamoto, Han Xiang Deng, Mitsuo Oshimura, Norio Niikawa

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

An alternative method for amplification of DNA with unknown sequences was developed. This involves the direct ligation of a primer oligodeoxyribonucleotide itself to restricted DNA fragments with unknown sequences to be amplified by PCR. The oligonucleotide need not be phosphorylated and need not be annealed with its complementary oligonucleotide in advance for ligation. The ligation reaction seems to be independent of the concentration of unknown DNA, proceeds in short time, and is efficient. The ligation efficiency was more than 30% at a low concentration, 10fg/μl, of DNA. This method was applied to a microdissection/microcloning of the short arm of human chromosome 2. Of 66 clones screened for the highly repetitive sequences with total human genomic DNA, eleven (17%) were positive. Their inserts ranged in size from 160 to 1,200 bp (average, 460 bp). In Southern blot analysis, thirty consecutive clones all detected signals common to both total human genomic DNA and mouse-human hybrid cell DNA containing only chromosome 2 of human origin. Among them, 24 (80%) were unique sequences, and 6 (20%) were multi-copy (or intermediate-repeat) sequences. Thus, this method is simple and efficient, and provides an alternative way to amplify unknown DNA.

Original languageEnglish (US)
Pages (from-to)75-80
Number of pages6
JournalJournal of Biochemistry
Volume112
Issue number1
DOIs
StatePublished - Jul 1992

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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