Homogeneous and one-step fluorescent allele-specific PCR for SNP genotyping assays using conjugated polyelectrolytes

A new label-free, homogenous, sensitive and economical one-step method to detect SNP genotyping of genomic DNA has been developed by combining allele-specific PCR technique with water-soluble cationic conjugated polyelectrolytes (CCP). The amplification of target DNA and fluorescence detection steps are combined into one-step. The target DNA fragment containing a G allele site acts as PCR template. For the G allele-specific forward primer whose 3'-terminal base is complementary to the G allele template, after the first step of reverse primer extension, G allele-specific primer perfectly anneals with newly formed strand and the extension reaction of forward primer starts. During the extension, dGTP-Fl and dUTP-Fl are incorporated into extension chain in the presence of Taq DNA polymerase and more fluorescein-labeled PCR amplicons are yielded. Upon adding the CCP, strong electrostatic interactions between DNA and CCP bring them close and efficient FRET from CCP to fluorescein occurs. For the C allele-specific forward primer, less fluorescein-labeled PCR amplicons are yielded and inefficient FRET occurs. By triggering the change of emission intensity of CCP and fluorescein, it is possible to assay the SNP genotypes. In contrast to previous reports, this method does not require designing dye-labeled primers, and gel electrophoresis and isolation step after PCR were avoided in this homogenous method. The genotyping of 50ng genomic DNA from human lung cancer cell is easily detected using our new method. These qualities will make the new detection system ideal for SNP genotyping.