Digital quantification of gene methylation in stool DNA by emulsion-PCR coupled with hydrogel immobilized bead-array.
Aberrations of gene methylation in stool DNA (sDNA) is an effective biomarker for non-invasive colorectal cancer diagnosis. However, it is challenging to accurately quantitate the gene methylation levels in sDNA due to the low abundance and degradation of sDNA. In this study, a digital quantification strategy was proposed by combining emulsion PCR (emPCR) with hydrogel immobilized bead-array. The assay includes following steps: bisulfite conversion of sDNA, pre-amplification by PCR with specific primers containing 5' universal sequences, emPCR of pre-amplicons with beaded primers to achieve single-molecular amplification and identification of hydrogel embedding beads coated with amplicons. The sensitivity and the specificity of the method are high enough to pick up 0.05% methylated targets from unmethylated DNA background. The successful detection of hypermethylated vimentin gene in clinical stool samples suggests that the proposed method should be a potential tool for non-invasive colorectal cancer screening.
Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China.
Yunlong
Y
Liu
Huadong Research Institute for Medicine and Biotechnics, Nanjing 210002, China.
Haiping
H
Wu
Key Laboratory of Drug Quality Control and Pharmacovigilance of Ministry of Education, China Pharmaceutical University, Nanjing 210009, China.
Qiang
Q
Zhou
Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China; Key Laboratory of Drug Quality Control and Pharmacovigilance of Ministry of Education, China Pharmaceutical University, Nanjing 210009, China.
Qinxin
Q
Song
Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China.
Jianzhong
J
Rui
Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China. Electronic address: zbj523@163.com.
Bingjie
B
Zou
Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China; State Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing 210093, China. Electronic address: ghzhou@nju.edu.cn.
Guohua
G
Zhou
D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000032:analysis; D015374:Biosensing Techniques / Q000295:instrumentation*; D015179:Colorectal Neoplasms / Q000175:diagnosis; D004247:DNA / Q000032:analysis; D019175:DNA Methylation; D017931:DNA Primers / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D004655:Emulsions / Q000737:chemistry; D005243:Feces / Q000737:chemistry*; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry*; D061925:Immobilized Nucleic Acids / Q000737:chemistry*; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation; D016133:Polymerase Chain Reaction / Q000295:instrumentation*
D014408:Biomarkers, Tumor; D017931:DNA Primers; D004277:DNA, Single-Stranded; D004655:Emulsions; D061925:Immobilized Nucleic Acids; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D004247:DNA
Emulsion PCR; Gene methylation; Hydrogel immobilized bead-array; Stool DNA