Generation of high efficiency ssDNA hybridization probes by linear polymerase chain reaction (LPCR).
The polymerase chain reaction (PCR) methodology can be employed to produce DNA hybridization probes. The major advantages of this paradigm over other techniques include superior specific activity of the probes, the versatility of sequence selection, the ability to produce short probes, and the simplicity of the procedure. We have further improved the efficiency of PCR probes by generating single stranded (ssDNA) probes that do not reanneal with themselves in solution, and hence, their availability for the interaction with the complementary sequences of the target is profoundly increased. Protocols for 32P-dCTP labeled and digoxigenin-dUTP labeled probes have been elaborated to maximize the incorporation rate of the label as well as to provide for the production of full-length probes. The ssDNA probes may be particularly suitable for nucleic acid detection in tissues by in situ hybridization.
Konat
G W
GW
Department of Anatomy, West Virginia University School of Medicine, Morgantown 26506-9128, USA. Gkonat@wvu.edu
D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000648:ultrastructure; D001923:Brain Chemistry; D015342:DNA Probes; D004277:DNA, Single-Stranded; D004076:Digoxigenin / Q000737:chemistry; D017403:In Situ Hybridization; D015336:Molecular Probe Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D012335:RNA, Ribosomal / Q000032:analysis; D051381:Rats
D015342:DNA Probes; D004277:DNA, Single-Stranded; D012335:RNA, Ribosomal; D004076:Digoxigenin