Isothermal Nucleic Acid Amplification Strategy by Cyclic Enzymatic Repairing for Highly Sensitive MicroRNA Detection

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• 作者：Dian-Ming Zhou ; Wen-Fang Du ; Qiang Xi ; Jia Ge ; Jian-Hui Jiang
• 刊名：Analytical Chemistry
• 出版年：2014
• 出版时间：July 15, 2014
• 年：2014
• 卷：86
• 期：14
• 页码：6763-6767
• 全文大小：262K
• ISSN：1520-6882

文摘

Technologies enabling highly sensitive and selective detection of microRNAs (miRNAs) are critical for miRNA discovery and clinical theranostics. Here we develop a novel isothermal nucleic acid amplification technology based on cyclic enzymatic repairing and strand-displacement polymerase extension for highly sensitive miRNA detection. The enzymatic repairing amplification (ERA) reaction is performed via replicating DNA template using lesion bases by DNA polymerase and cleaving the DNA replicate at the lesions by repairing enzymes, uracil-DNA glycosylase, and endonuclease IV, to prime a next-round replication. By utilizing the miRNA target as the primer, the ERA reaction is capable of producing a large number of reporter sequences from the DNA template, which can then be coupled to a cyclic signal output reaction mediated by endonuclease IV. The ERA reaction can be configured as a single-step, close-tube, and real-time format, which enables highly sensitive and selective detection of miRNA with excellent resistance to contaminants. The developed technology is demonstrated to give a detection limit of 0.1 fM and show superb specificity in discriminating single-base mismatch. The results reveal that the ERA reaction may provide a new paradigm for efficient nucleic acid amplification and may hold the potential for miRNA expression profiling and related theranostic applications.