Reagents and methods for PCR

摘要

Modified double-stranded oligonucleotides that have terminal regions on each of their strands, that have a hybrid length of 6-50 nucleotides long, that have a melting temperature Tm of at least 32° C., and that include 2-4 modifying groups, each covalently attached to a different terminal region, preferably to a terminal nucleotide, said modifying groups being polycyclic substituents that do not have bulky portions that are non-planar, said modified oligonucleotide being capable of binding to the 5′ ex-nuclease domains of DNA polymerases and, when included in a PCR or other primer-dependent DNA amplification reaction at a concentration, generally not more than 2000 nM, that is effective for at least one of the functions of suppressing mispriming, increasing polymerase selectivity against 3′ terminal mismatches, increasing polymerase selectivity against AT-rich 3′ ends, reducing scatter among replicates, suppressing polymerase 5′ exonuclease activity, and inhibiting polymerase activity; as well as amplification reaction mixtures containing such modified double-stranded oligonucleotides, and amplification reactions, amplification assays and kits that include such modified double-stranded oligonucleotides.

主权项

1. An amplification reaction mixture comprising:
(a) at least one primer pair, (b) a DNA polymerase, (c) dNTP's, and (d) at least one double-stranded oligonucleotide additive:
(i) comprising two separate nucleic acid strands, each of the strands 6-50 nucleotides in length,(ii) that is at least fifty percent double-stranded at 32° C.,(iii) having blunt ends or overhangs of 1-8 nucleotides,(iv) that has terminal regions on each of its strands and includes 1-4 modifying groups, each covalently attached to a different terminal region, said modifying groups being polycyclic moieties that do not have bulky portions that are non-planar, and(v) wherein said at least one double-stranded oligonucleotide additive is included in said reaction mixture at a concentration of 25-2000 nM.