Cationic conjugated polymers for optical detection of DNA methylation, lesions, and single nucleotide polymorphisms

Abstract

Simple, rapid, and sensitive technologies to detect nucleic acid modifications have important applications in genetic analysis, clinical diagnosis, and molecular biology. Because genetic modifications such as single nucleotide polymorphisms (SNP), DNA methylation, and other lesions can serve as hallmarks of human disease, interest in such methods has increased in recent years. This Account describes a new strategy for the optical detection of these DNA targets using cationic conjugated polymers (CCPs). Because of their unique signal amplification properties, researchers have extensively investigated conjugated polymers as optical transducers in highly sensitive biosensors. Recently, we have shown that cationic polyfluorene can detect SNPs within the DNA of clinical samples. When we incorporated deoxyguanosine triphosphate (dGTP-Fl) into the DNA chain at an SNP site where the target/probe pair is complementary, we observed higher fluorescence resonance energy transfer (FRET) efficiency between cationic polyfluorene and fluorescein label on the dGTP. By monitoring the change in emission intensity of cationic polyfluorene or fluorescein, we identified the homozygous or heterozygous SNP. The high sensitivity of this assay results from the 10-fold enhancement of fluorescein emission intensity by the FRET from polyfluorene. This method can detect allele frequencies (the proportion of all copies of a gene that is made up of a particular gene variant) as low as 2%. Using this novel method, we clearly discriminated among the SNP genotypes of 76 individuals of Chinese ancestry. Improving on this initial system, we designed a method for multicolor and one-tube SNP genotyping assays based on cationic polyfluorene using fluorescein-labeled deoxyuridine triphosphate (dUTP-Fl) and Cy3-labeled deoxycytidine triphosphate (dCTP-Cy3) in extension reactions. We also developed a one-step method for direct detection of SNP genotypes from genomic DNA by combining allele-specific PCR with CCPs. In 2008, we developed a new method for DNA methylation detection based on single base extension reaction and CCPs. Treatment of DNA with bisulfite followed by PCR amplification converts unmethylated DNA into a C/T polymorphism, which allows us to characterize the methylation status of the target DNA. Furthermore, we used CCPs to detect DNA lesions caused by ultraviolet light irradiation for the first time. By monitoring the color change of cationic polythiophene before and after DNA cleavage, we also detected oxidative damage to DNA by hydroxyl radical. These CCP-based new assays avoid primer labeling, cumbersome workups, and sophisticated instruments, leading to simpler procedures and improved sensitivity. We expect that these features could lead to major advances in human disease diagnostics and genomic study in the near future.