High sensitivity EndoV mutation scanning through real-time ligase proofreading
High sensitivity EndoV mutation scanning through real-time ligase proofreading
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DOI：
10.1093/nar/gnh150
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Barany, F
中科院分区：
文献类型：
--
作者：
Pincas, H;Pingle, MR;Barany, F
将癌症基因突变与疾病及其亚型相关联的能力对于了解肿瘤发生和识别临床诊断的生物标志物至关重要。我们实验室之前开发了一种两步突变扫描方法，依次使用核酸内切酶 V (EndoV) 在错配处进行切口，并使用 DNA 连接酶重新密封不正确或非特异性切口位点。在此，我们报告了一种优化的单步测定，使连接酶能够在缓冲液条件之间的折衷下实时校对 EndoV 切割。实时校对可大幅减少背景分裂。通用 PCR 策略同时采用未标记的基因特异性引物和标记的通用引物，允许多重基因扩增并排除引物二聚体的扩增。内部标记的 PCR 引物消除了 5' 末端的 EndoV 切割，从而实现高通量毛细管电泳读数。此外，通过生成仅包含两种可能的错配（例如 A/C 或 G/T）之一的异源双链体，可以增加信号强度并减少伪影。单步检测将 p53 和 K-ras 基因未知突变的灵敏度分别提高到 1:50 和 1:100（突变体：野生型），开辟了作为早期检测工具的前景。
The ability to associate mutations in cancer genes with the disease and its subtypes is critical for understanding oncogenesis and identifying biomarkers for clinical diagnosis. A two-step mutation scanning method that sequentially used endonuclease V (EndoV) to nick at mismatches and DNA ligase to reseal incorrectly or nonspecifically nicked sites was previously developed in our laboratory. Herein we report an optimized single-step assay that enables ligase to proofread EndoV cleavage in real-time under a compromise between buffer conditions. Real-time proofreading results in a dramatic reduction of background cleavage. A universal PCR strategy that employs both unlabeled gene-specific primers and labeled universal primers, allows for multiplexed gene amplification and precludes amplification of primer dimers. Internally labeled PCR primers eliminate EndoV cleavage at the 5' terminus, enabling high-throughput capillary electrophoresis readout. Furthermore, signal intensity is increased and artifacts are reduced by generating heteroduplexes containing only one of the two possible mismatches (e.g. either A/C or G/T). The single-step assay improves sensitivity to 1:50 and 1:100 (mutant:wild type) for unknown mutations in the p53 and K-ras genes, respectively, opening prospects as an early detection tool.