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化学进展 2014, Vol. 26 Issue (11): 1840-1848 DOI: 10.7536/PC140808 前一篇   后一篇

所属专题： 酶化学

• 综述与评论 •

双链特异性核酸酶的生物学和医学应用

邱晓沛, 张洪, 蒋天伦, 罗阳*   

  1. 第三军医大学附属西南医院输血科 重庆 400038
  • 收稿日期:2014-08-01 修回日期:2014-09-01 出版日期:2014-11-15 发布日期:2014-09-12
  • 通讯作者: 罗阳 E-mail:luoyang@tmmu.edu.cn
  • 基金资助:

    国家自然科学基金项目(No. 81371899)、中国军队十二五课题(No. CWS13C046)、重庆市自然科学基金项目(No. CSTC2013JJB10012)和第三军医大学课题(No. SWH2013LC13)资助

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Biological and Medical Applications of Duplex-Specific Nuclease

Qiu Xiaopei, Zhang Hong, Jiang Tianlun, Luo Yang*   

  1. Department of Transfusion Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
  • Received:2014-08-01 Revised:2014-09-01 Online:2014-11-15 Published:2014-09-12
  • Supported by:

    The work was supported by the National Natural Science Foundation of China (No. 81371899), the Twelfth Five Year Plan of PLA, China (No. CWS13C046), the Natural Science Foundation of Chongqing (No. CSTC2013JJB10012), and the Third Military Medical University (No. SWH2013LC13)

双链特异性核酸酶DSN是一种能高选择性地识别并酶切完全匹配的DNA双链或者DNA-RNA杂交双链中的DNA,而对单链DNA和RNA几乎没有作用的核酸酶.DSN酶的上述特点使其在生物和医学等领域广泛应用,主要包括全长cDNA文库的均一化、单核苷酸多态性(SNP)检测和高通量测序等.近年来,DSN酶在microRNAs(miRNAs)的检测领域得以长足发展和应用.miRNAs是一组内源性、非编码短序列RNAs,在生理和病理过程中具有重要作用,但由于其序列短、丰度低等特点, miRNAs的检测一直是临床难题.新近研究主要基于DSN酶信号放大的特点,相继建立了一系列生物传感技术,通过采用不同检测原理如比色法、荧光法、电化学法等实现痕量miRNAs检测.本文就DSN酶在miRNAs检测、SNP检测、全长cDNA文库均一化及高通量测序等方面的生物学应用进行了综述.

关键词： 双链特异性核酸酶, microRNAs检测, SNP检测, cDNA文库均一化, 高通量测序

Duplex-specific nuclease (DSN) is a type of nuclease, that is isolated from the hepatopancreas of the Kamchatka crab. DSN displays a strong preference for cleaving double-stranded DNA or DNA in DNA-RNA heteroduplexes, and is practically inactive toward single-stranded DNA or RNA. Moreover, this enzyme shows excellent discrimination capability between perfectly and imperfectly matched (up to one mismatch) short duplexes. Owing to its unique feature of cleaving DNA, DSN enzyme is widely applied in the fields of biomedicine and molecular biology, including full-length cDNA library normalization,genomic single-nucleotide polymorphism (SNP) detection and high throughput sequencing. The recent research on DSN are mainly focused on the applications in microRNAs (miRNAs) detection using a DSN-mediated signal amplification strategy. miRNAs are a group of short, endogenous, noncoding RNAs that play vital regulatory roles in physiologic and pathologic processes, including hematopoietic differentiation, cell cycle, regulation, and metabolism. So miRNA is one of the most important biomarkers in individualized treatment, which has great value in terms of improving the diagnosis and treatment of diseases. However, detection of miRNAs is challenging owing to their unique characteristics, including a small size, sequence homology among family members, low abundance in total RNA samples, and susceptibility to degradation in solution. In recent years, isothermal signal amplification and detection of trace miRNA in fluids are reported by many researchers using DSN-mediated biosensors. According to different detection principle of biosensors, DSN-mediated biosensors can be classified as colorimetric, fluorescent, and electrochemical. In this review, we intensively summarize the advantages of DSN in miRNAs detection using DSN-based signal amplification strategy, expanded applications in SNP detection, high throughput sequencing and cDNA library normalization especially when being combined with SMART (Switching Mechanism At 5'end of RNA Transcript) technique.

Contents
1 Introduction
2 The application of DSN enzyme in microRNA detection
2.1 Colorimetric analyses
2.2 Fluorescent analyses
2.3 Electrochemical analyses
3 The application of DSN enzyme in SNP detection
4 Other biological applications of DSN enzyme
4.1 Construction of cDNA Library
4.2 High throughput sequencing
5 Conclusion and outlook

Key words: duplex-specific nuclease, microRNAs detection, SNP detection, cDNA library normalization, next-generation sequencing

中图分类号: 

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