title: Genotyping of single nucleotide polymorphisms using base-quenched probe: a method does not invariably depend on the deoxyguanosine nucleotide.
abstract: Most available methods for detecting single nucleotide polymorphisms (SNPs) are based principally on the system that can produce an increased fluorescence signal during hybridization. In the current study, we demonstrate a method of base-quenched probe for polymerase chain reaction (PCR) genotyping that requires only a pair of primers and one fluorescent probe and does not invariably depend on the deoxyguanosine nucleotide. This method further exploits the phenomenon of fluorescence quenching of fluorescent-labeled probe during hybridization to its complementary target gene's sequence. 6-Carboxyfluorescein (FAM) can be directly conjugated to a base of either adenine (A), thymine (T), cytosine (C), or guanine (G), referred to as A-, T-, C-, or G-quenched probe, respectively, at either the 5' or 3' end. For describing the method in detail, we chose apolipoprotein M (apoM) as a target gene in the current study. DNA sequencing analyses validated that all four types of base-quenched probes could provide unbiased genotyping results (K = 1, P = 0.000), although the maximum speed of fluorescence increase, max(dF/dT), when using the G-quenched probe method, was approximately twofold lower than the others (P < 0.0001). Moreover, we applied this method to detect another seven SNPs in the genomes of phospholipase A2, monocyte chemoattractant protein 1 (MCP1), and l-ficolin, further confirming our method. It is concluded that this method is precise, simple, and economic as well as suitable for large-scale genotyping studies.
authors.affiliation: Comprehensive Laboratory, Third Affiliated Hospital, Suzhou University, Changzhou 213003, China.
authors.forename: Guanghua
authors.initials: G
authors.lastname: Luo
authors.forename: Lu
authors.initials: L
authors.lastname: Zheng
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Zhang
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Peter
authors.initials: P
authors.lastname: Nilsson-Ehle
authors.forename: Ning
authors.initials: N
authors.lastname: Xu
mesh_terms: D017931:DNA Primers / Q000737:chemistry*; D003849:Deoxyguanosine / Q000737:chemistry*; D005453:Fluorescence; D005838:Genotype; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D017931:DNA Primers; D003849:Deoxyguanosine