HYBRIDIZATION AND MISMATCH DISCRIMINATION USING OLIGONUCLEOTIDES CONJUGATED TO MINOR GROOVE BINDERS
申请人:Hedgpeth Joel
公开号:US20110275070A1
公开(公告)日:2011-11-10
Conjugates between a minor groove binding molecule, such as the trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-
e
]indole-7-carboxylate (CDPI
3
), and an oligonucleotide form unusually stable hybrids with complementary target sequences, in which the tethered CDPI
3
group resides in the minor groove of the duplex. These conjugates can be used as probes and primers. Due to their unusually high binding affinity, conjugates as short as 8-mers can be used as amplification primers with high specificity and efficiency. MGB conjugation also increases the discriminatory power of short oligonucleotides, providing enhanced detection of nucleotide sequence mismatches by short oligonucleotides. The MGB-conjugated probes and primers described herein facilitate various analytic and diagnostic procedures, such as amplification reactions, PCR, detection of single-nucleotide polymorphisms, gene hunting, differential display, fluorescence energy transfer, hydrolyzable probe assays and others; by allowing the use of shorter oligonucleotides, which have higher specificity and better discriminatory power.
一种小沟结合分子(例如1,2-二氢-(3H)-吡咯[3,2-e]吲哚-7-羧酸三聚体(CDPI3))与寡核苷酸之间的共轭体形成具有不寻常稳定性的杂交体,其中被拴系的CDPI3基团存在于双链的小沟中。这些共轭体可用作探针和引物。由于它们的结合亲和力异常高,因此即使是8个碱基的共轭体也可用作扩增引物,具有高特异性和高效性。MGB结合还增加了短寡核苷酸的鉴别能力,通过短寡核苷酸提高了核苷酸序列不匹配的检测能力。所述的MGB结合探针和引物促进了各种分析和诊断过程,例如扩增反应、PCR、单核苷酸多态性检测、基因猎取、差异显示、荧光能量转移、可水解探针测定等;通过允许使用更短的寡核苷酸,这些寡核苷酸具有更高的特异性和更好的鉴别能力。