Reagents and methods for improving reproducibility and reducing mispriming in PCR amplification

摘要

Provided is a reagent capable of preventing at least one manifestation of mispriming in a polymerase chain reaction (PCR) amplification to produce at least one amplified DNA product when added at a concentration of not more than 650 nM to a PCR amplification mixture that includes 1.25 units of a thermostable DNA polymerase per 25 pA of reaction mixture, said reagent being an oligonucleotide that has a 3' end and a stem-loop structure having a stem comprising a double-stranded region that has a length is greater than six nucleotides and a terminus away from the loop comprising a 3' nucleotide and a 5' nucleotide, said stem having a calculated stem melting temperature (Tm) below 94øC, wherein (a) the 3' end is non-extendable by said DNA polymerase, (b) the oligonucleotide is not fluorescently labelled and does not contribute background fluorescence, and (c) said stem terminus is stabilized by means selected from the group consisting of non-fluorescent fluorophore-quenching moieties covalently attached to the 3' and 5' nucleotides of said stem terminus and pairs of non-natural nucleotides that bind more strongly than a natural DNA-DNA hybrid and that include each of the 3' and 5' nucleotides of said stem terminus. The reagent is useful in improving PCR quality.