Hybridization probe size control: optimized ‘oligolabelling’

Abstract

DNA hybridization probes can be radiolabelled internally with 32P either by nick-translation (1 2) or by oligonucleotide-priming (oligolabelling, 3-4).The latter method has the advantage that two sources of nuclease activity (DNAse I and 5'-3' exonuclease) are eliminated, allowing greater control over the size of the end product.

New Approaches to the Detection of Microbial Plant Pathogens

“…Nucleic acid probes may be labelled with a range of radioactive or non-radioactive markers (Cunningham and Mundy, 1987;Dattagupta et al, 1987;Hodgson and Fisk, 1987;Li etal., 1987;Seriwatana et al, 1987). Their comparative advantages and disadvantages have been reviewed extensively (Al-Hakim and Hull, 1986;Zwadyk, Cooksey and Thomsberry, 1986;Donovan etal., 1987;Mifflin et al, 1987;Tabares, 1987).…”

“…More recently, commercial kits based on multipriming of denatured ssDNA with oligonucleotide primers (random primers or hexamer primers) and extension using the Klenow fragment of DNA polymerase can produce a specific activity of 5 x 109 dpm 1.tg-DNA probe Vogelstein, 1983, 1984;Hodgson and Fisk, 1987). Another method is to use T4 DNA polymerase to digest one end of each strand of DNA in a dsDNA template from the 3' to 5' direction, followed by resynthesis of dsDNA from the 5' to 3' direction using radioactive precursors (Morris etal., 1979).…”

Isolation and characterization of sixteen novel p53 response genes

“…Poly(A)+ RNA was obtained by standard methods using oligo-dT cellulose chromatography (Sambrook et al, 1989). Formaldehyde gels (Lehrach et al, 1977) were transferred to nylon membranes Machery-Nagel (DuÈ ren, Germany) and hybridized (Church and Gilbert, 1984) with random primer labelled probes (Feinberg and Vogelstein, 1984;Hodgson and Fisk, 1987).…”

Expression and function of Drosophila cyclin a during embryonic cell cycle progression

“…RNA was resolved on formaldehyde-agarose gels, transferred to nylon membranes, and hybridized by standard procedures. Probes for northern and southern blots were prepared by random primer labeling (Hodgson and Fisk, 1987).…”