HYBRIDIZATION AND MISMATCH DISCRIMINATION USING OLIGONUCLEOTIDES CONJUGATED TO MINOR GROOVE BINDERS

摘要

Conjugates between a minor groove binding molecule, such as the trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3), and anoligonucleotide form unusually stable hybrids with complementary targetsequences, in which the tethered CDPI3 group resides in the minor groove ofthe duplex. These conjugates can be used as probes and primers. Due to theirunusually high binding affinity, conjugates as short as 8-mers can be used asamplification primers with high specificity and efficiency. MGB conjugationalso increases the discriminatory power of short oligonucleotides, providingenhanced detection of nucleotide sequence mismatches by shortoligonucleotides. The MGB-conjugated probes and primers described hereinfacilitate various analytic and diagnostic procedures, such as amplificationreactions, PCR, detection of single-nucleotide polymorphisms, gene hunting,differential display, fluorescence energy transfer, hydrolyzable probe assaysand others; by allowing the use of shorter oligonucleotides, which have higherspecificity and better discriminatory power.