title: Loopback rolling circle amplification for ultrasensitive detection of Kras gene.
abstract: Mutations in Kras gene may be used as a diagnostic marker and a target for treatment of the broad spectrum of human cancers. In this study, we developed a new class of amplification assay, double-hairpin molecular beacon (DHMB)-based cascade rolling circle amplification (RCA), for ultrasensitive and selective detection of Kras gene in a homogenous solution. Specifically, target DNA can hybridize with DHMB and activate cyclical target strand-displacement polymerization (CTDP) and nicking-mediated strand-displacement polymerization (NMDP). The resulting nicked/displaced fragments substantially outnumber target DNA and cause the cascade rolling circle amplification (C-RCA) and nicked fragment-induced strand-displacement polymerization (NFDP). Even if four amplification processes are designed, only DHMB, padlock probe and polymerization primer are involved. Under optimized conditions, this screening system exhibits a linear range of 5 orders of magnitude (from 100fM to 20nM), and the detection limit is down to 16fM. Moreover, the developed biosensing system offers a high assay specificity for perfectly matched target DNA, and the measured data from practical samples demonstrated the potential application in the cancer diagnoses. As a proof-of-concept genetic assay, the novel signaling strategy, as well as desirable analytical capability, would significantly benefit the development of versatile amplification gene profiling platforms, revealing great promise in biological studies and medical diagnostics.
authors.affiliation: Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350002, China.
authors.forename: Huo
authors.initials: H
authors.lastname: Xu
authors.affiliation: Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350002, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Wu
authors.affiliation: Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350002, China.
authors.forename: Yifan
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350002, China; Key laboratory of watershed science and health of Zhejiang Province, Institute of Functional Nucleic Acids and Personalized Cancer Theranostics, Key Laboratory of Laboratory Medicine, Ministry of Education of China, and Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, China.
authors.forename: Rongbo
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350002, China.
authors.forename: Qingzheng
authors.initials: Q
authors.lastname: Wu
authors.affiliation: Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350002, China.
authors.forename: Yiyun
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350002, China; Key laboratory of watershed science and health of Zhejiang Province, Institute of Functional Nucleic Acids and Personalized Cancer Theranostics, Key Laboratory of Laboratory Medicine, Ministry of Education of China, and Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Li
authors.affiliation: Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350002, China. Electronic address: wuzaisheng@163.com.
authors.forename: Zai-Sheng
authors.initials: ZS
authors.lastname: Wu
keywords: Cascade-rolling circle amplification; Double-hairpin molecular beacon (DHMB); Kras gene