Optimization of PCR/lambda exonuclease-mediated synthesis of sense and antisense DNA probes for in situ hybridization.

摘要: In situ hybridization experiments are stringently dependent on the quality of probes, which should be single-stranded when efficient comparison signals obtained with antisense and control sense probes needed. this report, we describe an optimized synthesis radioactive DNA without vector cloning requiring a unique polymerization step. The sequence region selected as probe is amplified by polymerase chain reaction in presence radiolabelled nucleotides. then yielded action λ bacteriophage exonuclease, can specifically eliminate one out two strands fragments. way, identical length specific activity generated selecting primer to phosphorylated. We have verified efficiency our for clusterin transcripts within peripheral olfactory system, after surgical lesion its synaptic target.