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 qua lity of the probes, which should be single-stranded when efficient com parison of signals obtained with antisense and control sense probes ar e needed. In this report, we describe an optimized synthesis of radioa ctive single-stranded DNA probes, without vector cloning and requiring a unique polymerization step. The sequence region selected as probe i s amplified by polymerase chain reaction in the presence of radiolabel led nucleotides. The sense and antisense probes are then yielded by th e action of the lambda bacteriophage exonuclease, which can specifical ly eliminate one out of the two strands of the amplified fragments. In this way, sense and antisense probes with identical length and specif ic activity can be generated by selecting the primer to be phosphoryla ted. We have verified the efficiency of our probes for in situ hybridi zation of the clusterin transcripts within the peripheral olfactory sy stem, after surgical lesion of its synaptic target.