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

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.

Duke Scholars

Author Patricia Lynn Ashley Pediatrics, Neonatology