Dideoxy sequencing method using denatured plasmid templates

Masahira Hattori, Yoshiyuki Sakaki

研究成果: Article

1039 引用 (Scopus)

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The dideoxy sequencing method in which denatured plasmid DNA is used as a template was improved. The method is simple and rapid: the recombinant plasmid DNA is extracted and purified by rapid alkaline lysis followed by ribonuclease treatment. The plasmid DNA is then immediately denatured with alkali and subjected to a sequencing reaction utilizing synthetic oligonucleotide primers. It takes only several hours from the start of the plasmid extraction to the end of the sequencing reaction. We examined each step of the procedure, and several points were found to be crucial for making the method reproducible and powerful: (i) the plasmid DNA should be free from RNA and open circular (or linear) DNA; (ii) a heptadecamer rather than a pentadecamer is recommended as a primer; and (iii) the sequencing reaction should be done at 37°C or higher rather than at room temperature. The method enabled us to determine the sequence of more than a thousand nucleotides from a single template DNA.

元の言語English
ページ(範囲)232-238
ページ数7
ジャーナルAnalytical Biochemistry
152
発行部数2
DOI
出版物ステータスPublished - 1986 2 1
外部発表Yes

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ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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