Optimization of PCR/lambda exonuclease-mediated synthesis of sense and antisense DNA probes for in situ hybridization.
In situ hybridization experiments are stringently dependent on the quality of the probes, which should be single-stranded when efficient comparison of signals obtained with antisense and control sense probes are needed. In this report, we describe an optimized synthesis of radioactive single-stranded DNA probes, without vector cloning and requiring a unique polymerization step. The sequence region selected as probe is amplified by polymerase chain reaction in the presence of radiolabelled nucleotides. The sense and antisense probes are then yielded by the action of the lambda bacteriophage exonuclease, which can specifically eliminate one out of the two strands of the amplified fragments. In this way, sense and antisense probes with identical length and specific activity can be generated by selecting the primer to be phosphorylated. We have verified the efficiency of our probes for in situ hybridization of the clusterin transcripts within the peripheral olfactory system, after surgical lesion of its synaptic target.
Michel
D
D
Laboratoire de Biologie Moléculaire et Cellulaire, ENS de Lyon, France.
Trembleau
A
A
Moyse
E
E
Brun
G
G
D000818:Animals; D010582:Bacteriophage lambda / Q000201:enzymology; D051152:Clusterin; D015342:DNA Probes / Q000096:biosynthesis*; D016373:DNA, Antisense; D004277:DNA, Single-Stranded; D005092:Exonucleases; D006023:Glycoproteins / Q000235:genetics; D017403:In Situ Hybridization; D051379:Mice; D008810:Mice, Inbred C57BL; D018832:Molecular Chaperones; D009297:Nasal Mucosa / Q000378:metabolism; D009830:Olfactory Bulb / Q000601:surgery; D016376:Oligonucleotides, Antisense / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D014158:Transcription, Genetic / Q000235:genetics
C495243:Clu protein, mouse; D051152:Clusterin; D015342:DNA Probes; D016373:DNA, Antisense; D004277:DNA, Single-Stranded; D006023:Glycoproteins; D018832:Molecular Chaperones; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D005092:Exonucleases