A sensitive method of Nested PCR for the detection of low copy number human telomerase reverse transcriptase mRNA

Lancer K. Brown, Penelope Burke, Larissa Tersteege, William H. Andrews, Jessica Wheeler, October Pawlik, and Christopher A. Foster
Sierra Sciences, LLC, Reno, NV

The human telomerase gene (hTERT) is repressed in normal human cells, resulting in telomere loss, and ultimately cellular aging. The identification of C0057684, a small molecule shown to activate hTERT expression in normal human fibroblast cells, has given us a valuable tool to optimize hTERT detection by RT-PCR in normal human cells. However, conventional TaqMan RT-PCR methods are not amenable to analyzing compounds that are weak inducers of hTERT. Nested PCR is commonly used to amplify low copy number targets where high levels of sensitivity and specificity are needed.

A major drawback to Nested PCR is the potential for interference of the inside (second) PCR reaction with primers from outside (first) PCR reaction. A common way to limit this interference is to dilute the first reaction for use in the second reaction, thus diluting primer concentrations, and subsequently diluting PCR product concentrations. This dilution requirement is not amenable to maximizing sensitivity of a PCR assay where every molecule counts. Sierra Sciences L.L.C. has developed a method of Nested PCR which avoids the need for sacrificing PCR signal to accommodate dilution. Our method uses Uracil-N-Glycosylase (UNG) present in the second PCR reaction to degrade the first primer set that was designed to have deoxyuracil (dUTP) in place of deoxythymidine (dTTP). UNG degradation of dUTP-containing DNA allows for the second PCR reaction cocktail to be added to the entire product of the first reaction (reducing contamination), eliminating the potential for interference between primer pairs, and maximizing the sensitivity of PCR.