No abstract

No abstract

For the first time we used CuAAC click reaction for the synthesis of cyanine labeled qPCR probes. We performed the comparison of dyes and quenchers (Cy5 vs disulfo-Cy5 and BHQ2 vs BHQ3) in several TaqMan probes with different stability of the secondary structure. Based on the studies of thermal quenching of dyes, increase of fluorescence during probe melting and in the course of qPCR, we suggest that disulfo-Cy5-BHQ2 pair is a preferable one for molecular beacons due to the highest increase of fluorescence during melting. As Cy5-BHQ3 pair gave better Cq and maximal relative increase of fluorescence in qPCR in comparison to others we recommend this pair for Taqman style probe design.

An alkyl azide derivative of 1-phenylethynylpyrene (PEPy) dye was prepared and used in the functionalization of oligonucleotides via click chemistry. Spectral and photo-physical properties of the PEPy-modified oligonucleotides as a single strand, and in perfect or mismatched duplexes, have been studied. A series of PEPy-Dabcyl fluorogenic TaqMan probes were synthesized and tested in qPCR. PEPy proved to be a superior substitute for AMCA as a short wavelength fluorescent dye for qPCR probes. PEPy probes were shown to significantly reduce Cq (a fractional PCR cycle used for quantification) vs. AMCA labeled probes, thus improving on the reliability of detection. Moreover, a larger increase of fluorescence during amplification was observed in the case of PEPy probes that makes this dye very suitable for an end-point PCR technique. This study broadens the panel of fluorescent dyes suitable for the use in probes for quantitative real-time PCR.

No abstract