应用液滴数字PCR技术检测rs6983267三种多态性位点
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华北理工大学医学实验研究中心,华北理工大学医学实验研究中心,华北理工大学医学实验研究中心,华北理工大学医学实验研究中心,华北理工大学医学实验研究中心,华北理工大学医学实验研究中心,华北理工大学医学实验研究中心,华北理工大学医学实验研究中心

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国家自然科学基金(31050010),教育部归国留学基金([2015] No.311), 河北省归国留学人员择优资助项目(C201400358)和华北理工大学大学生创新创业计划(X2015052, X2016121)资助项目


Assessment of Quality of Tri-allelic Polymorphism Compassing rs6983267 Based on Droplet Digital PCR Taking Advantage of Probe Cross-reactivity
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Medical Research Center,North China University of Science and Technology,Medical Research Center,North China University of Science and Technology,Medical Research Center,North China University of Science and Technology,Medical Research Center,North China University of Science and Technology,Medical Research Center,North China University of Science and Technology,Medical Research Center,North China University of Science and Technology,Medical Research Center,North China University of Science and Technology,Medical Research Center,North China University of Science and Technology

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This work was supported by grants from National Natural Science Foundation of China (31050010),the Project Sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry([2015]No.311),Selected Foundation for the Returned Overseas Chinese Scholars by Hebei Government(C201400358) and NCST Undergraduate Training Programs for Innovation and Entrepreneurship (X2015052;X2016121)

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    摘要:

    核苷酸的多态性检测在临床以及基础生命科学研究中占据着重要地位.目前基于PCR的探针法应用最为广泛.由于在核苷酸上微小差异,探针的设计往往带有交叉活性反应,这阻碍了qPCR方法的推广.数字PCR(dPCR)是近年来已成功实现商业化的基于单分子分析的核酸检测技术.通过对条件的优化,dPCR可以消除探针的交叉活性,不过目前商业化的dPCR一般只有2个通道,对于同时检测3个多态性需要更加细致的优化.本研究以rs6983267位点的CCAT2基因3种多态性检测为例,利用探针的交叉活性反应检测其3个多态性位点.检测涉及到3个探针:2个针对多态性位点,另1个位于多态性位点外侧作为参照探针.结果表明,成功地区分了3个包含多态性位点基因片段的簇.在本研究中交叉活性反应可以为dPCR留出白空间,利于多样品的检测.

    Abstract:

    The detection of nuclear acid polymorphism has an important role in basic research and clinical application. The popular methods are PCR based on probe. Probe with cross-reactivity limits the application of traditional real-time quantitative PCR to quantitate allelic transcripts.Digital (d)PCR may overcome cross-reactivity defects, but available dPCR machines lack discrete optical channels, which limits the detection of more than two molecules. Colon cancer associated transcript 2 (CCAT2) is a non-coding transcript, encompassing rs6983267 SNP site. Here, we report a method based on two channels droplet digital PCR to quantitate three CCAT2 polymorphisms in one reaction. We designed a pair of primers and three hydrolysis probes including a reference and two competing probes. We successfully discriminated between the three CCAT2 transcript clusters. We took advantage of the cross-reactivity to provide a white space for more visible distinct clusters, although innate rain was detected. Labelling the reference probe with cross-reactivity probe resulted in more distinct clusters than the probe without cross-reactivity.

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沈阳丽,孙浩,蔡文臣,张品正,王旭莹,尚雨寒,石璐,辛倩倩,郭志义.应用液滴数字PCR技术检测rs6983267三种多态性位点[J].生物化学与生物物理进展,2018,45(11):1160-1165

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  • 收稿日期:2018-07-09
  • 最后修改日期:2018-08-16
  • 接受日期:2018-08-21
  • 在线发布日期: 2018-08-27
  • 出版日期: 2018-11-20
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