Allelic discrimination using fluorogenic probes and the 5′ nuclease assay
Allelic discrimination using fluorogenic probes and the 5′ nuclease assay
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DOI：
10.1016/s1050-3862(98)00019-9
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发表时间：
1999-02-01
期刊：
影响因子：
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通讯作者：
Livak, KJ
中科院分区：
文献类型：
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作者：
Livak, KJ
对已知多态性的大规模筛选将需要步骤少的技术和自动化这些步骤的能力。在这方面，5'核酸酶或TaqMan PCR测定特别有吸引力。在典型的PCR中包括由用荧光报告染料和猝灭染料两者标记的寡核苷酸组成的荧光探针。探针特异性产物的扩增引起探针的切割，产生报告荧光的增加。通过使用不同的报告染料，可以在单个PCR中检测等位基因特异性探针的切割。5'核酸酶测定已成功地用于区分通过单个碱基取代而不同的等位基因。已经制定了指南，以便可以快速设计和实施任何单核苷酸多态性（SNP）的测定。使用单一反应缓冲液和单一热循环方案进行所有测定。此外，已经开发了一种标准的分析方法，可以自动确定基因型。该测定的应用包括对人类药物代谢基因中的许多多态性进行分型。(C)1999年由Elsevier Science B.V.出版，版权所有。
Large-scare screening for known polymorphisms will require techniques with few steps and the ability to automate each of these steps. In this regard, the 5' nuclease, or TaqMan, PCR assay is especially attractive. A fluorogenic probe, consisting of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye, is included in a typical PCR. Amplification of the probe-specific product causes cleavage of the probe, generating an increase in reporter fluorescence. By using different reporter dyes, cleavage of allele-specific probes can be detected in a single PCR. The 5' nuclease assay has been successfully used to discriminate alleles that differ by a single base substitution. Guidelines have been developed so that an assay for any single nucleotide polymorphism (SNP) can be quickly designed and implemented. All assays are performed using a single reaction buffer and single thermocycling protocol. Furthermore, a standard method of analysis has been developed that enables automated genotype determination. Applications of this assay have included typing a number of polymorphisms in human drug metabolism genes. (C) 1999 Published by Elsevier Science B.V. All rights reserved.