Exploiting the enzymatic recognition of an unnatural base pair to develop a universal genetic analysis system
Exploiting the enzymatic recognition of an unnatural base pair to develop a universal genetic analysis system
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DOI：
10.1373/49.3.407
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发表时间：
2003-03-01
影响因子：
9.3
通讯作者：
Prudent, JR
中科院分区：
文献类型：
--
作者：
Moser, MJ;Marshall, DJ;Prudent, JR
背景：随着DNA芯片的发明，全基因组分析现已成为现实。不幸的是，固相检测系统，如DNA芯片的定量和灵敏度范围窄。如今，用于核酸的灵敏、宽动态范围定量和基因分型的最佳方法是实时PCR。然而，多重实时PCR技术需要复杂和昂贵的设计和制造单独的检测探针为每个新的target.Methods：我们开发了一种新的实时PCR技术，使用通用的能量转移探针构建从一个扩展的遗传信息系统（AEGIS）的定量和基因分型分析。逆转录-聚合酶链反应（RT-PCR）定量分析β-肌动蛋白信使RNA和18 S核糖体RNA的线性范围超过4个数量级。通过终点分析对176份既往基因分型的临床标本进行了单项试验验证研究，以检测凝血因子V Leiden和凝血酶原20210 A突变。对于173个样本，Invader（（R））试验和AEGIS通用能量转移探针系统对因子V Leiden和凝血酶原G20210 A的基因分型结果一致。两个凝血酶原和一个因子V样品给出了不确定的结果（无调用）。结论：AEGIS通用探针系统允许快速开发用于核酸定量和基因分型的PCR检测。(C)2003年美国临床化学协会。
Background: With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunately, solid-phase detection systems such as the DNA chip suffer from a narrow range in quantification and sensitivity. Today the best methodology for sensitive, wide dynamic range quantification and genotyping of nucleic acids is real-time PCR. However, multiplexed real-time PCR technologies require complicated and costly design and manufacturing of separate detection probes for each new target.Methods: We developed a novel real-time PCR technology that uses universal energy transfer probes constructed from An Expanded Genetic Information System (AEGIS) for both quantification and genotyping analyses.Results: RNA quantification by reverse transcription-PCR Was linear over four orders of magnitude for the Simultaneous analysis of beta-actin messenger RNA and 18S ribosomal RNA. A single trial validation study of 176 previously genotyped clinical specimens was performed by endpoint analysis for factor V Leiden and prothrombin 20210A mutation detection. There was concordance for 173 samples between the genotyping results from Invader((R)) tests and the AEGIS universal energy transfer probe system for both factor V Leiden and prothrombin G20210A. Two prothrombin and one factor V sample gave indeterminate results (no calls).Conclusion: The AEGIS universal probe system allows for rapid development of PCR assays for nucleic acid quantification and genotyping. (C) 2003 American Association for Clinical Chemistry.