Genotyping of single nucleotide polymorphisms using base-quenched probe: A method does not invariably depend on the deoxyguanosine nucleotide
Författare
Summary, in English
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 I (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. (C) 2008 Elsevier Inc. All rights reserved.
Avdelning/ar
Publiceringsår
2009
Språk
Engelska
Sidor
161-166
Publikation/Tidskrift/Serie
Analytical Biochemistry
Volym
386
Issue
2
Dokumenttyp
Artikel i tidskrift
Förlag
Elsevier
Ämne
- Pharmacology and Toxicology
- Medicinal Chemistry
Nyckelord
- Polymerase chain reaction
- Fluorescence quenching
- Single nucleotide
- polymorphism
Status
Published
ISBN/ISSN/Övrigt
- ISSN: 1096-0309