An in vitro selection scheme for oligonucleotide probes to discriminate between closely related DNA sequences

Using an in vitro selection, we have obtained oligonucleotide probes with high discriminatory power against multiple, similar nucleic acid sequences, which is often required in diagnostic applications for simultaneous testing of such sequences. We have tested this approach, referred to as iterative hybridizations, by selecting probes against six 22-nt-long sequence variants representing human papillomavirus, (HPV). We have obtained probes that efficiently discriminate between HPV types that differ by 3-7 nt. The probes were found effective to recognize HPV sequences of the type 6, 11, 16, 18 and a pair of type 31 and 33, either when immobilized on a solid support or in a reverse configuration, as well to discriminate HPV types from the clinical samples. This methodology can be extended to generate diagnostic kits that rely on nucleic acid hybridization between closely related sequences. In this approach, instead of adjusting hybridization conditions to the intended set of probe-target pairs, we 'adjust', through in vitro selection, the probes to the conditions we have chosen. Importantly, these conditions have to be 'relaxed', allowing the formation of a variety of not fully complementary complexes from which those that efficiently recognize and discriminate intended from non-intended targets can be readily selected.