Genotyping of single nucleotide polymorphisms using base-quenched probe: A method does not invariably depend on the deoxyguanosine nucleotide
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1372005
- author
- Luo, Guanghua ; Zheng, Lu ; Zhang, Xiaoying ; Zhang, Jun ; Nilsson-Ehle, Peter LU and Xu, Ning LU
- organization
- publishing date
- 2009
- 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
- 2016-04-01 12:05:59
- date last changed
- 2022-01-26 22:46:21
@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 < 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}}, keywords = {{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}}, doi = {{10.1016/j.ab.2008.11.032}}, volume = {{386}}, year = {{2009}}, }