Division of Biotechnology, Indian Institute of Horticultural Research-IIHR, Hesaraghatta Lake-post, Bengaluru 560089, India.
Anal Biochem. 2012 Aug 15;427(2):175-7. doi: 10.1016/j.ab.2012.05.017. Epub 2012 May 26.
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.
我们开发了一种带有 3'磷酸而非羟基的改良聚合酶链反应 (PCR) 引物，用于单管准确转录定量。18S 核糖体 RNA (rRNA) 参考基因特异性修饰引物用于精确单管定量两种靶转录物，即棉铃虫的糜蛋白酶和 jhamt（保幼激素酸甲基转移酶）。对 3'磷酸化引物、3'错配引物和商业 Competimers 的比较研究表明，3'磷酸化比 3'错配更有效，并且与 Competimers 在阻止引物延伸方面相当。因此，修饰引物可用于单管、经济且准确的 PCR 定量分析任何特定于测定的参考基因的目标基因。
