FLUORESCENCE POLARIZATION-BASED DETECTION OF SINGLE NUCLEOTIDE POLYMORPHISM IN HUMAN ANGIOGENIN USING PROPYNE-MODIFIED PROBES.

Detection of single nucleotide polymorphism (SNP) relies on specific hybridization of a fluorescently-labelled probe to its complementary target DNA sequence followed by measurement of change in probe-specific fluorescence intensity (FI). Structurally, the SNP probes contain a 5’ fluorophore and a 3’ no fluorescence quencher (NFQ). For enhanced thermal stability, a minor groove binder (MGB) in case of TaqMan probes is also required which makes synthesis of such probes complicated. In the present work, we report fluorescence polarization (FP)-based detection of SNP in human angiogenin by propyne- modified probes that contain only 5’ fluorophore but no NFQ and MGB on the 3’end. Our assay relied on the 5’ nuclease activity-based reporter fluorophore excision by Taq polymerase from two differently-labeled propyne-modified probes, one specific for the wild type (WT) and the other for K84E (Lysine to Glutamic acid) SNP and measurement of change in their FP value. The probe-specific reduction in FP after 40 cycles of PCR at 20 ng starting template were comparable to standard TaqMan NFQ-MGB probes suggesting that FP-based detection of SNP is possible using probes without NFQ on the 3’ end. Since synthesis of propyne-modified probes is simpler such probes cost significantly less compared to the standard TaqMan SNP probes and may be useful in high throughput FP-based detection of SNPs in clinical samples.