RNA-templated single-base mutation detection based on T4 DNA ligase and reverse molecular beacon.
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.
Tang
Hongxing
H
Engineering Center for Biomedicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
Yang
Xiaohai
X
Wang
Kemin
K
Tan
Weihong
W
Li
Huimin
H
He
Lifang
L
Liu
Bin
B
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
D017931:DNA Primers; D005456:Fluorescent Dyes; D012313:RNA; D016914:Ribonuclease H; D011088:DNA Ligases