High fidelity SNP genotyping using sequence-specific primer elongation and fluorescence correlation spectroscopy

Reliable, efficient and cost-effective modalities are urgently needed for mass screening of gene mutations. Previous reports have shown that SSCP or genechip methods require substantial time and monetary costs, thus limiting their appeal. Sequence Specific Primer Polymerase Chain Reaction (SSP-PCR) is a reliable and cost-effective method that utilizes the 3'-end discrimination properties of polymerase. However, the applicability of conventional SSP-PCR is limited due to the difficulties associated with determining optimal conditions and because mis-matched primers are amplified, resulting in signal noise during end-point assay. To overcome this problem, we eliminated the reverse primers from SSP-PCR, thus preventing amplification of mis-matched primers. We designated this method Sequence-Specific Primer Cycle Elongation (SSPCE). However, the detection of elongated sequence specific primers was difficult using conventional electrophoresis due to the small amounts of amplification product present. We therefore combined SSPCE and Fluorescence Correlation Spectroscopy, which is a novel technique used to determine the number and size of fluorophores at nano-molar concentrations, and designated the method SSPCE-FCS. We compared conventional SSP-PCR and SSPCE-FCS with regard to determining optimal conditions using two Mitochondrial SNPs (G --> A at position 1598, G --> A at position 12192). We were able to determine the optimal conditions for the SNP at position 1598 using either method. However, optimal conditions could only be determined for SSPCE-FCS with the 12192 mutation because non-specific amplification was observed at a wide range of annealing temperatures in SSP-PCR. We then applied this method to three other SNPs and the results were consistent with the results of sequencing data.