title: Single-nucleotide polymorphism (SNP) genotyping using cationic conjugated polymers in homogeneous solution.
abstract: This protocol describes a simple, convenient and sensitive single-nucleotide polymorphism (SNP) genotyping method using an optically amplifying cationic conjugated polymer and single base primer extension reaction. The fluorescence resonance energy transfer (FRET) efficiency between the conjugated polymer (PFP, poly{(1,4-phenylene)-2,7-[9,9-bis(6'-N,N,N-trimethyl ammonium)-hexyl fluorene] dibromide}) and a fluorescein-labeled dNTP (dNTP-Fl) is correlated to the incorporation of the dNTP-Fl into an allele-specific primer; incorporation occurs by a single base extension reaction when the target DNA and the primer are complementary at the SNP site. By triggering the FRET from PFP to fluorescein and measuring the change in fluorescence intensity of samples, the SNP genotypes can be discriminated. In comparison with other SNP genotyping methods, this protocol simplifies procedures and improves sensitivity by eliminating the need for primer labeling, cumbersome workups, chemical/enzymatic coupling reactions and sophisticated instruments. The assay takes about 2 h for PCR amplification followed by 5.5-7.5 h to obtain the genotypes.
authors.affiliation: Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing, China.
authors.forename: Xinrui
authors.initials: X
authors.lastname: Duan
authors.forename: Wei
authors.initials: W
authors.lastname: Yue
authors.forename: Libing
authors.initials: L
authors.lastname: Liu
authors.forename: Zhengping
authors.initials: Z
authors.lastname: Li
authors.forename: Yuliang
authors.initials: Y
authors.lastname: Li
authors.forename: Fuchu
authors.initials: F
authors.lastname: He
authors.forename: Daoben
authors.initials: D
authors.lastname: Zhu
authors.forename: Gangqiao
authors.initials: G
authors.lastname: Zhou
authors.forename: Shu
authors.initials: S
authors.lastname: Wang
mesh_terms: D002412:Cations; D004247:DNA / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D023281:Genomics / Q000379:methods*; D005838:Genotype; D016133:Polymerase Chain Reaction / Q000379:methods; D011108:Polymers / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide
chemical_list: D002412:Cations; D011108:Polymers; D004247:DNA