COMPARISON OF 3' AND 5' BIOTIN-LABELED OLIGONUCLEOTIDES FOR IN-SITU HYBRIDIZATION

Oligonucleotide probes enzymatically labelled at the 3'-end with bioti n have been used successfully to detect target RNA and DNA in combinat ion with in situ hybridisation. Addition of multiple biotin residues t o the 3'-end increases the hybridisation signals, but it is not known whether the same principle is applicable to the 5'-end. We have labell ed a 35-base oligonucleotide during synthesis with 1, 5 and 12 biotin molecules at the 5'-end and compared it to conventional 3'-labelling. In additional experiments the probes were labelled at both ends. Probe s were applied to histological sections obtained from paraffin-embedde d cell-clot-complexes that contain uninfected and Rhinoviral-infected cells, using a standard in situ hybridisation protocol with appropriat e controls. Hybridisation signals were compared for intensity of cytop lasmic signal and sensitivity as number of positive cells. Both parame ters increased in parallel with higher numbers of biotin residues atta ched to the 5'-end and 12 biotin residues were almost as effective as 3'-enzymatic tailing. The sensitivity could be increased above that of either 3'- or 5'-labelling by the addition of residues at both ends o f the probe. The 5'-attachment of biotin residues can extend the value of oligonucleotide probes employed for in situ hybridisation and yiel d increased sensitivity when combined with 3'-enzymatic labelling.