Synthesis of single-stranded hybridization probes from reusable DNA templates bound to solid support

Patricia L. Ashley, Raymond J. MacDonald

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

A convenient and rapid technique for preparing radiolabeled single-stranded DNA hybridization probes has been developed. Single-stranded recombinant M13 phage DNA containing the mRNA strand of a cloned cDNA is bound to diazobenzyloxymethyl-cellulose in a manner that permits the synthesis of a complementary DNA using reverse transcriptase and primed with either oligo(dT) or the M13 single-stranded primer. A procedural advantage is that after synthesis the unincorporated radiolabeled nucleotides are washed away easily, and the radiolabeled single-stranded DNA probe is eluted with formamide, ready for use. To limit the DNA copy to the insert, a preliminary synthesis reaction is performed with unlabeled nucleotides, primer, and enzyme, followed by digestion of the reaction mix with a restriction endonuclease that recognizes a unique site in the recombinant immediately upstream of the cDNA insert. After elution of the unlabeled synthesized complementary DNA, a second synthesis reaction yields highly radiolabeled single-stranded DNA that extends only the length of the mRNA insert. A major advantage is that the restriction enzyme-cleaved, cellulose-bound template can be stored and reused repeatedly.

Original languageEnglish (US)
Pages (from-to)95-103
Number of pages9
JournalAnalytical Biochemistry
Volume140
Issue number1
DOIs
StatePublished - Jul 1984

Keywords

  • DBM-cellulose
  • bacteriophage M13
  • complementary DNA
  • hybridization
  • mRNA quantitation
  • recombinant DNA

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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