title: RNA-templated single-base mutation detection based on T4 DNA ligase and reverse molecular beacon.
abstract: A novel RNA-templated single-base mutation detection method based on T4 DNA ligase and reverse molecular beacon (rMB) has been developed and successfully applied to identification of single-base mutation in codon 273 of the p53 gene. The discrimination was carried out using allele-specific primers, which flanked the variable position in the target RNA and was ligated using T4 DNA ligase only when the primers perfectly matched the RNA template. The allele-specific primers also carried complementary stem structures with end-labels (fluorophore TAMRA, quencher DABCYL), which formed a molecular beacon after RNase H digestion. One-base mismatch can be discriminated by analyzing the change of fluorescence intensity before and after RNase H digestion. This method has several advantages for practical applications, such as direct discrimination of single-base mismatch of the RNA extracted from cell; no requirement of PCR amplification; performance of homogeneous detection; and easily design of detection probes.
authors.affiliation: Engineering Center for Biomedicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
authors.forename: Hongxing
authors.initials: H
authors.lastname: Tang
authors.forename: Xiaohai
authors.initials: X
authors.lastname: Yang
authors.forename: Kemin
authors.initials: K
authors.lastname: Wang
authors.forename: Weihong
authors.initials: W
authors.lastname: Tan
authors.forename: Huimin
authors.initials: H
authors.lastname: Li
authors.forename: Lifang
authors.initials: L
authors.lastname: He
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
mesh_terms: D000483:Alleles; D020137:Base Pair Mismatch; D011088:DNA Ligases / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D005456:Fluorescent Dyes; D011905:Genes, ras / Q000235:genetics; D009154:Mutation; D036103:Nanotechnology / Q000379:methods*; D012313:RNA / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016914:Ribonuclease H / Q000378:metabolism; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D013698:Templates, Genetic
chemical_list: D017931:DNA Primers; D005456:Fluorescent Dyes; D012313:RNA; D016914:Ribonuclease H; D011088:DNA Ligases