DETECTION AND CONFIRMATION OF NUCLEIC ACID SEQUENCES BY USE OF OLIGONUCLEOTIDES COMPRISING A SUBSEQUENCE HYBRIDIZING EXACTLY TO A KNOWN TERMINAL SEQUENCE AND A SUBSEQUENCE HYBRIDIZING TO AN UNIDENTIFIED SEQUENCE

摘要

The present invention discloses a methodology which is directed to providingpositive confirmation that nucleic acids, possessing putatively identifiedsequences predicted to generate observed GeneCallingTM signals, are actuallypresent within the sample from which the signal was originally derived. Theputatively identified nucleic acid fragment within the sample possesses 3'-and 5'-ends with known terminal subsequences, said method comprising:contacting said nucleic acid fragments in said sample in amplifying conditionswith (i) a nucleic acid polymerase; (ii) "regular" primer oligonucleotideshaving sequences comprising hybridizable portions of said known terminalsubsequences; and (iii) a "poisoning" oligonucleotide primer, said poisoningprimer having a sequence comprising a first subsequence that is a portion ofthe sequence of one of said known terminal subsequences and a secondsubsequence that is a hybridizable portion of said putatively unidentifiedsequence which is adjacent to said one known terminal subsequence, whereinnucleic acids amplified with said poisoning primer are distinguishable upondetection from nucleic acids amplified with said nucleic acids amplified onlywith said regular primers; separating the products of the contacting step; andthe detecting sequence is confirmed if the nucleic acids amplified with saidpoisoning primer are detected.