Modified competing polymerase chain reaction primer for single tube quantitative PCR

Abstract

We developed a modified polymerase chain reaction (PCR) primer with 3′ phosphate instead of hydroxyl group for single-tube accurate transcript quantification. 18S ribosomal RNA (rRNA) reference gene-specific modified primer was used for precise single-tube quantification of two target transcripts, namely chymotrypsin and jhamt (juvenile hormone acid methyl transferase) of Helicoverpa armigera. A comparative study of 3′ phosphorylated primers, 3′ mismatched primers, and commercial Competimers revealed that 3′ phosphorylation was more efficient than the 3′ mismatch and was on par with Competimers in blocking the primer extension. Thus, the modified primers can be used in single-tube, economical, and accurate PCR quantification of the target gene using any assay-specific reference gene.