Heterochronous multiplex real-time PCR with intercalating dye using uracil-DNA N-glycosylase (UNG) and multiple primer pairs to revaluate post PCR product

Abstract

Real-time PCR with intercalating dyes can only be performed once. The expensive fluorescent hydrolysis probes are target specific and are suitable to detect multiplex targets. Uracil-DNA N-glycosylase (UNG), which specifically hydrolyzes and degrades any uracil-containing PCR products, is often applied before PCR to reduce carryover contamination. We developed an optimized protocol for recovering DNA from PCR products and revaluating by real-time PCR with intercalating dye using UNG processing, which is particularly useful when the sample volume is very small and insufficient for multiple assays of real-time PCR.•A real-time PCR master mix with dUTP instead of dTTP was used.•UNG at 1 % and 10 % concentrations of PCR product volumes were used for the first and second processing.•The second real-time PCR was performed with different primer pairs than the first real-time PCR.

Keywords: Heterochronous real-time PCR with intercalating dye using UNG; Multiplex real-time PCR; SYBR green; Uracil-DNA N-glycosylase (UNG).

Graphical abstract

Fig. 1

The digestion of PCR product shown in the electrophoresis. A: PCR product after the first real-time PCR of Lpar1 pair 3. B: PCR product with UNG processing. C: The purified PCR product. D: The purified PCR product with UNG processing. Ladder: DNA ladder showing 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 and 1500 bp from bottom to top.

Fig. 2

The digestion of PCR product shown in the electrophoresis. A: PCR product after the first real-time PCR of Gapdh pair 3. B: PCR product with UNG processing. C: The purified PCR product. D: The purified PCR product with UNG processing. Ladder: DNA ladder showing 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 and 1500 bp from bottom to top.

Fig. 3

Dissociation curves of the second real-time PCR. Dissociation curves showed single peaks, indicating complete digestion of the PCR product.

Fig. 4

Dissociation curves of the second real-time PCR using the same primer pairs as the first real-time PCR after digestion with 0.1 % UNG. The dissociation curves of the second real-time PCR using Lpar1 pair 3 showed multiple peaks when the DNA recovery was performed with 0.1 % UNG, indicating indigestion and contamination of the PCR product.