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Genotyping of single nucleotide polymorphisms using base-quenched probe: A method does not invariably depend on the deoxyguanosine nucleotide

Luo, Guanghua; Zheng, Lu; Zhang, Xiaoying; Zhang, Jun; Nilsson-Ehle, Peter LU and Xu, Ning LU (2009) In Analytical Biochemistry 386(2). p.161-166
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-,... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Polymerase chain reaction, Fluorescence quenching, Single nucleotide, polymorphism
in
Analytical Biochemistry
volume
386
issue
2
pages
161 - 166
publisher
Elsevier
external identifiers
  • wos:000263789500004
  • scopus:59749091237
ISSN
1096-0309
DOI
10.1016/j.ab.2008.11.032
language
English
LU publication?
yes
id
f1703436-deaa-4f06-a9c6-a8cdbe970664 (old id 1372005)
date added to LUP
2009-05-08 14:12:49
date last changed
2017-11-05 03:38:48
@article{f1703436-deaa-4f06-a9c6-a8cdbe970664,
  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 &lt; 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.},
  author       = {Luo, Guanghua and Zheng, Lu and Zhang, Xiaoying and Zhang, Jun and Nilsson-Ehle, Peter and Xu, Ning},
  issn         = {1096-0309},
  keyword      = {Polymerase chain reaction,Fluorescence quenching,Single nucleotide,polymorphism},
  language     = {eng},
  number       = {2},
  pages        = {161--166},
  publisher    = {Elsevier},
  series       = {Analytical Biochemistry},
  title        = {Genotyping of single nucleotide polymorphisms using base-quenched probe: A method does not invariably depend on the deoxyguanosine nucleotide},
  url          = {http://dx.doi.org/10.1016/j.ab.2008.11.032},
  volume       = {386},
  year         = {2009},
}