Abstract
BackgroundReal-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule.ResultsWe describe a real-time PCR technique employing attached universal duplex probes (AUDP), which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP) and a complementary quenching probe (QP) lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT) and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO) quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods.ConclusionThe results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.
Highlights
Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule
PCR is a powerful tool for the amplification of minimal amounts of initial target genetic sequences [1,2], and it has been widely used for quantitative analysis of nucleic acids in medical diagnostics, drug discovery, virus detection, environmental monitoring, gene expression analysis and the detection of genetically modification organisms (GMO) [3,4,5]
A complete attached universal duplex probes (AUDP) PCR assay is composed of a target DNA-specific primer pair, with one primer containing an attached universal template (UT) sequence, and a set of universal duplex probes containing both fluorescent probe (FP) and quenching probe (QP) (Figure 1A)
Summary
Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. When the donor fluorophore and acceptor fluorophore are separated in the reaction by cleavage, the change in secondary structure or strand displacement of the fluorescent probe will result in increased fluorescence signal [23,24,25]. In these real-time PCR assays, either duallabeled probes or primers have to be synthesized, which limits the wide application of these methods due to incomplete quenching, complicated synthesis and labeling, and high costs [26]
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