title: An improved fluorescence polarization assay in 5'-nuclease reaction for gene promoter methylation detection.
abstract: The detection of gene promoter methylation plays increasing roles in personalized medicine. In this study, an improved gene promoter methylation assay based on fluorescence polarization in 5'-nuclease reaction was developed. The novel assay offered a homogeneous annealing and cleavage reaction fully integrated with PCR which used a probe labeled with fluorescence without quencher to obtain the decreased fluorescence polarization values. In this platform, gene promoter methylated and unmethylated alleles were detected simultaneously in a tube. O(6)-methylguanine-DNA methyltransferase gene promoter methylation in 103 glioma tissue samples and epidermal growth factor receptor gene promoter methylation in 116 primary non-small-cell lung carcinoma tissue samples were detected by the novel assay and sequencing, absolute quantitative analysis of methylated allele in parallel. The accuracy of the results measured by the improved fluorescence polarization assay was evaluated using the paired-samples t test. No significant difference was found ( P>0.05). Therefore, the improved fluorescence polarization assay in 5'-nuclease reaction demonstrated a homogeneous, reliable and cost-effective method for gene promoter methylation analysis in clinic. That would provide a scientific basis for applying a reasonable therapeutic regimen in future treatment.
authors.affiliation: Department of Oncology, Tangdu Hospital, The Fourth Military Medical University, Xian, Shaanxi 710038, China.
authors.forename: Haichuan
authors.initials: H
authors.lastname: Su
authors.affiliation: Department of Neurology, Xijing Hospital, The Fourth Military Medical University, Xian, Shaanxi 710033, China.
authors.forename: Zhongliang
authors.initials: Z
authors.lastname: Wu
authors.affiliation: Institute of Gene Diagnosis, State Key Laboratory of Cancer Biology, School of Pharmacology,The Fourth Military Medical University, Xian, Shaanxi 710032, China.
authors.forename: Yukun
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Institute of Gene Diagnosis, State Key Laboratory of Cancer Biology, School of Pharmacology,The Fourth Military Medical University, Xian, Shaanxi 710032, China.
authors.forename: Yinghao
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Department of Oncology, Xijing Hospital, The Fourth Military Medical University, Xian, Shaanxi 710032, China.
authors.forename: Shaoying
authors.initials: S
authors.lastname: Qiang
authors.affiliation: Department of Pathology, Xijing Hospital, The Fourth Military Medical University, Xian, Shaanxi 710032, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Cheng
authors.affiliation: Department of Oncology, Xijing Hospital, The Fourth Military Medical University, Xian, Shaanxi 710032, China. Electronic address: liuch@fmmu.edu.cn.
authors.forename: Wenchao
authors.initials: W
authors.lastname: Liu
authors.affiliation: Institute of Gene Diagnosis, State Key Laboratory of Cancer Biology, School of Pharmacology,The Fourth Military Medical University, Xian, Shaanxi 710032, China. Electronic address: jianzhong@fmmu.edu.cn.
authors.forename: Ju
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000483:Alleles; D019175:DNA Methylation / Q000235:genetics*; D005454:Fluorescence Polarization / Q000379:methods*; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results
keywords: 5'-nuclease reaction; Fluorescence polarization; Gene promoter; Homogeneous; Methylation