Dideoxy sequencing method using denatured plasmid templates

Masahira Hattori, Yoshiyuki Sakaki

Research output: Contribution to journalArticle

1037 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)232-238
Number of pages7
JournalAnalytical Biochemistry
Volume152
Issue number2
DOIs
Publication statusPublished - 1986 Feb 1
Externally publishedYes

Fingerprint

Plasmids
DNA
DNA Primers
Recombinant DNA
Alkalies
Ribonucleases
Nucleotides
RNA
Temperature

Keywords

  • alkaline denaturation
  • autoradiography
  • DNA sequencing
  • gel electrophoresis,nucleic acids
  • pUC plasmid
  • recombinant DNA

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Dideoxy sequencing method using denatured plasmid templates. / Hattori, Masahira; Sakaki, Yoshiyuki.

In: Analytical Biochemistry, Vol. 152, No. 2, 01.02.1986, p. 232-238.

Research output: Contribution to journalArticle

Hattori, Masahira ; Sakaki, Yoshiyuki. / Dideoxy sequencing method using denatured plasmid templates. In: Analytical Biochemistry. 1986 ; Vol. 152, No. 2. pp. 232-238.
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