OLIGONUCLEOTIDE PRIMERS THAT DESTABILIZE NON-SPECIFIC DUPLEX FORMATION AND USES THEREOF

摘要

The present invention relates to the demonstration that a modification of ahomopolymeric stretch in an oligonucleotide, or primer, improves thediscrimination for binding of such a modified oligonucleotide or primer to itscomplementary homopolymeric target sequence, as compared to a non-homopolymeric sequence. More specifically, an oligo d(T) primer having two ofthe thymine bases substituted by 3-nitropyrrole were used in a poly A primedcDNA synthesis experiment to demonstrate an improvement in discriminationbetween the priming of cDNA synthesis from bona fide poly A sequence ascompared to internal A-rich sequences. The present invention relates tomodifications of homopolymeric sequences in oligos, decreasing the ridgingbonding capacity, in general, since other modifications, such as an oligo d(T)primer substituted with 2' deoxyinosine was also shown to improve thediscrimination between the binding to a bona fide poly A tail as compared to A-rich sequences. The present invention thus relates to universal primers whichreduce mispriming during cDNA library construction, thereby increasing theproportion of cDNA clones having been primed from the bona fide 3' poly Atail. The present invention further relates to the use of the discriminatingoligonucleotides of the present invention in other methods such as mRNApurification, PCR-based detection methods and sequencing.