FLAG assay as a novel method for real-time signal generation during PCR: application to detection and genotyping of KRAS codon 12 mutations.
FLAG assay as a novel method for real-time signal generation during PCR: application to detection and genotyping of KRAS codon 12 mutations.
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DOI：
10.1093/nar/gkm809
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发表时间：
2007
影响因子：
14.9
通讯作者：
Adlerstein D
中科院分区：
文献类型：
--
作者：
Amicarelli G;Shehi E;Makrigiorgos GM;Adlerstein D
用于检测和表征基因组 DNA 中低丰度突变的实时信号生成方法是癌症诊断和预后的强大工具。例如，癌基因 KRAS 密码子 12 的突变经常在几种类型的人类癌症中发现。我们开发了一种新型实时 PCR 技术 FLAG（荧光扩增子生成），并将其用于同时 (i) 扩增突变密码子 12 KRAS 序列，(ii) 实时监测扩增，以及 (iii) 对精确的核苷酸变化进行基因分型。 FLAG 利用极其耐热的核酸内切酶 PspGI，通过从 PCR 产物 5' 端裂解淬灭的荧光团来生成实时信号，同时用于选择 KRAS 突变（而非野生型）。通过在反应中加入肽-核酸探针，可以实现同步基因分型，从而避免了测序的要求。 FLAG 可实现高通量、闭管 KRAS 突变检测，检测突变率降至野生型的 ∼0.1%。该检测方法在模型系统上进行了验证，并与筛选 27 个癌症样本的等位基因特异性 PCR 测序进行了比较。预计 FLAG 在癌症、病毒学或传染病中的实时 PCR 或基因分型应用中有多种应用。
Real-time signal generation methods for detection and characterization of low-abundance mutations in genomic DNA are powerful tools for cancer diagnosis and prognosis. Mutations in codon 12 of the oncogene KRAS, for example, are frequently found in several types of human cancers. We have developed a novel real-time PCR technology, FLAG (FLuorescent Amplicon Generation) and adapted it for simultaneously (i) amplifying mutated codon 12 KRAS sequences, (ii) monitoring in real-time the amplification and (iii) genotyping the exact nucleotide alteration. FLAG utilizes the exceptionally thermostable endonuclease PspGI for real-time signal generation by cleavage of quenched fluorophores from the 5′-end of the PCR products and, concurrently, for selecting KRAS mutations over wild type. By including peptide-nucleic-acid probes in the reaction, simultaneous genotyping is achieved that circumvents the requirement for sequencing. FLAG enables high-throughput, closed-tube KRAS mutation detection down to ∼0.1% mutant-to-wild type. The assay was validated on model systems and compared with allele-specific PCR sequencing for screening 27 cancer specimens. Diverse applications of FLAG for real-time PCR or genotyping applications in cancer, virology or infectious diseases are envisioned.