title: Chemiluminescence detection of telomere DNA in human cells on a membrane by using fluorescein-5-isothiocyanate-labeled primers.
abstract: Telomere DNA is related to cell aging and cancer genesis because the telomeric region of DNA sequences at chromosome ends are shortened with cell divisions. Therefore, a sensitive and specific detection method is required for the telomere DNA. Here we propose a chemiluminescence (CL)-based method for the sensitive detection of telomere DNA in human cells. In this study, the telomere DNA was amplified by polymerase chain reaction (PCR) using special forward and reverse primers labeled with fluorescein-5-isothiocyanate (FITC) at the 5' end, and then the FITC-containing PCR products were detected by CL reaction with 3,4,5-trimethoxyphenylglyoxal (TMPG) after electrophoresis followed by Southern blot onto a nylon membrane. The TMPG reagent specifically reacted with guanine moiety in DNA at room temperature and provided CL intensities. The CL intensities from the PCR products could be enhanced approximately 10-fold using FITC-labeled primers as compared with those using nonlabeled primers. The detection limit of the PCR products with the proposed method was 0.3 ng on the membrane. The developed CL method could quantitatively determine the telomere DNA in a small number of human cells (∼350) and gave approximately 10 times higher sensitivity than a conventional fluorescence-based method.
authors.affiliation: Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852-8521, Japan.
authors.forename: Mutsumi
authors.initials: M
authors.lastname: Yamasuji
authors.forename: Takayuki
authors.initials: T
authors.lastname: Shibata
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Kabashima
authors.forename: Masaaki
authors.initials: M
authors.lastname: Kai
mesh_terms: D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis*; D017931:DNA Primers / Q000737:chemistry*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D009061:Mouth Mucosa / Q000166:cytology; D016133:Polymerase Chain Reaction / Q000379:methods; D016615:Telomere / Q000737:chemistry*
chemical_list: D017931:DNA Primers; D004247:DNA; D016650:Fluorescein-5-isothiocyanate