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

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Ashley, PL; MacDonald, RJ

Published Date

  • July 1, 1984

Published In

Volume / Issue

  • 140 / 1

Start / End Page

  • 95 - 103

PubMed ID

  • 6207747

Pubmed Central ID

  • 6207747

International Standard Serial Number (ISSN)

  • 0003-2697

Digital Object Identifier (DOI)

  • 10.1016/0003-2697(84)90138-6

Language

  • eng

Conference Location

  • United States