Oligodeoxynucleotides capable of binding to GC-rich regions in DNA are inappropriately synthesized during random hexanucleotide-primed labeling reactions.
Random hexanucleotide labeling is a commonly used and powerful technique for preparing radiolabeled nucleic acid probes. Because of the dependence of DNA polymerase upon single-stranded DNA as a template, the technique should theoretically also be useful for detecting small amounts of single-stranded DNA in the presence of a larger amount of double-stranded DNA. In the course of such an experiment, we identified an unexpected reaction that could contribute to background problems often encountered during Southern and Northern blotting. In the complete absence of template, random hexanucleotides appear capable of functioning both as template as well as primer, giving rise to labeled oligonucleotides as large as 20-mers. Some of these bind very tightly to GC-rich regions in target DNA immobilized on membranes, resisting high-stringency washing conditions. By choosing shorter incubation times and including sufficient single-stranded template, the problem may be minimized.
Chen
J H
JH
Ottawa Regional Cancer Centre, Ontario, Canada.
Birnboim
H C
HC
D016477:Artifacts; D001483:Base Sequence; D015152:Blotting, Northern / Q000379:methods*; D015139:Blotting, Southern / Q000379:methods*; D015342:DNA Probes; D004277:DNA, Single-Stranded / Q000032:analysis; D007553:Isotope Labeling; D008969:Molecular Sequence Data
D015342:DNA Probes; D004277:DNA, Single-Stranded