title: Real-time fluorescence ligase chain reaction for sensitive detection of single nucleotide polymorphism based on fluorescence resonance energy transfer.
abstract: Most of practical methods for detection of single nucleotide polymorphism (SNP) need at least two steps: amplification (usually by PCR) and detection of SNP by using the amplification products. Ligase chain reaction (LCR) can integrate the amplification and allele discrimination in one step. However, the detection of LCR products still remains a great challenge for highly sensitive and quantitative SNP detection. Herein, a simple but robust strategy for real-time fluorescence LCR has been developed for highly sensitive and quantitative SNP detection. A pair of LCR probes are firstly labeled with a fluorophore and a quencher, respectively. When the pair of LCR probes are ligated in LCR, the fluorophore will be brought close to the quencher, and thus, the fluorescence will be specifically quenched by fluorescence resonance energy transfer (FRET). The decrease of fluorescence intensity resulted from FRET can be real-time monitored in the LCR process. With the proposed real-time fluorescence LCR assay, 10 aM DNA targets or 100 pg genomic DNA can be accurately determined and as low as 0.1% mutant DNA can be detected in the presence of a large excess of wild-type DNA, indicating the high sensitivity and specificity. The real-time measuring does not require the detection step after LCR and gives a wide dynamic range for detection of DNA targets (from 10 aM to 1 pM). As LCR has been widely used for detection of SNP, DNA methylation, mRNA and microRNA, the real-time fluorescence LCR assay shows great potential for various genetic analysis.
authors.affiliation: Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, Hebei Province, PR China.
authors.forename: Yueying
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, Hebei Province, PR China.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Lu
authors.affiliation: Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, Hebei Province, PR China.
authors.forename: Fengxia
authors.initials: F
authors.lastname: Su
authors.affiliation: Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, Hebei Province, PR China.
authors.forename: Limei
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, Shaanxi Province, PR China.
authors.forename: Chenghui
authors.initials: C
authors.lastname: Liu
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, Shaanxi Province, PR China.
authors.forename: Xinrui
authors.initials: X
authors.lastname: Duan
authors.affiliation: Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, Hebei Province, PR China; Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, Shaanxi Province, PR China. Electronic address: lzpbd@snnu.edu.cn.
authors.forename: Zhengping
authors.initials: Z
authors.lastname: Li
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation; D003199:Computer Systems; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation*; D022521:Ligase Chain Reaction / Q000295:instrumentation*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation*
chemical_list: D004247:DNA
keywords: Fluorescence resonance energy transfer; Ligase chain reaction; Real time; Single nucleotide polymorphism