Characterization of solution-phase DNA hybridization by fluorescence correlation spectroscopy: Rapid genotyping of C677T from methylenetetrahydrofolate reductase gene

In this paper, fluorescence correlation spectroscopy (FCS) was applied to measure the hybridization fraction of the ssDNA probe with its perfectly matched 146 mer ssDNA and a base mismatched 146 mer ssDNA from human methylenetetrahydrofolate reductase (MTHFR) gene. The ssDNA fragments in this study were obtained by asymmetric PCR techniques. The measurements were performed on a laboratory-built FCS system based on the two components fitting procedure. The obtained results showed that FCS could discriminate the difference of thermal stability between perfectly matched and mismatched DNA duplex, and be used to characterize the genotype of C677T in MTHFR gene. Our data illustrated that FCS was a useful tool for rapid screening of single point genetic mutations/polymorphisms (SNP) combined with DNA hybridization.