A new technique of PCR hot-start using duplex primers has been developed which can decrease the undesirableproducts arising throughout PCR amplification thereby giving better results than a manual hot-start method.

A novel method for duplex probes is designed to simulate the TaqMan probe during polymerase chain reaction(PCR). In this method, two partly complementary single-labelled oligonucleotide probes labelled with afluorophore or a quencher, respectively, are used. At lower temperature the two probes can bind to each otherand form a mismatched duplex, in which the fluorophore and quencher are in close proximity and the sameenergy transfer mechanism as in molecular beacons may occur between them; thus, a quenching efficiencybetter than conventional TaqMan probes is acquired. In the anneal-extend step of PCR, one single-labelledprobe hybridises to the predetermined target and is cleaved by Taq DNA polymerase. Increased fluorescentsignal can be observed at lower temperature. The fluorescent data analysis demonstrated that a significantlyhigher level of fluorescent signal and hence higher sensitivity of detection is obtainable using our duplex probesin place of conventional TaqMan probes. Combined with real-time PCR instruments, the assay can be used toquantify the input target molecules and the dynamic linear range is of at least six orders of magnitude.

A novel method for duplex probes is designed to simulate theTaqMan probe during polymerase chain reaction (PCR); two single-labelled probes are used for this method, whichrelies on the 5'-exonuclease activity of nucleic acid polymerase.

A novel method for the detection of specific nucleic acids in homogenous solution was developed. The method is based onthe use of duplex probes in which fluorescent donor and quencher labeled on either oligonucleotide are held in close proximity, so that fluorescence is quenched. Amplification of the target sequence results in the cleavage of the probe and the resultingfluorescence can be detected. The fluorescent data analysis demonstrated that the duplex probes can specifically recognize thepresence of target, and a significantly higher lever of relative fluorescent signal than TaqMan probes is obtainable. Combinedwith real-time PCR instruments, the assay can be used to quantify the input target molecules. As few as five copies of initialtarget molecules can be detected, and a large dynamic linear ranger (five orders of magnitude) is obtained.

A modified molecular beacon that possesses a stem-hairpinstructure as seen in conventional molecular beacons and canbe cleaved during PCR is designed, and it can specificallyrecognize the presence of the target and was obviously moresensitive than conventional molecular beacons.