Quantitative detection of mutant alleles with minor groove binder-conjugated fluorogenic DNA probes

与肿瘤特异性mRNA表达相比，肿瘤特异性点突变是稳定的生物标志物，因此有助于检测隐匿性肿瘤细胞。这些突变从未用于实时定量聚合酶链式反应(RQ-PCR)分析，因为传统探针区分野生型和突变等位基因的能力很差。近年来，具有共轭小沟槽结合剂(MGB)的DNA探针已被开发出来。由于其较高的熔化温度，这些探针在检测单核苷酸错配方面取得了高性能。利用MGB技术，我们开发了一种新的针对K-ras点突变的RQ-PCR系统，用于检测结直肠癌(CRC)患者的隐匿性肿瘤细胞。针对所有先前报道的K-ras突变，设计了16个MGB结合的DNA探针。用已插入K-ras点突变的质粒DNA、来自15例大肠癌患者的32个癌细胞株和338个淋巴结来检测这些探针的性能。设计的16个MGB探针中有15个可用于准确的定量评估，并获得了高灵敏度(1/10^4~10^5个背景细胞)和高重复性(变异系数<10%)。与非MGB探针相比，MGB探针在区分单核苷酸错配方面的性能更好。我们在6例K-ras突变患者的110个淋巴结中检测到1个(0.9%)微转移(5.85/104个细胞当量)。9例K-ras基因突变阴性患者的209个淋巴结均未检出真假阳性。以K-ras基因突变为靶点的MGB RQ-PCR方法是一种准确定量检测结直肠癌隐匿性肿瘤细胞的方法。

Tumor-specific point mutations are stable biomarkers compared with tumor-specific mRNA expression, and are therfore useful to detect occult tumor cells. These mutations have never been used for real-time quantitative polymerase chain reaction (RQ-PCR) assays, because the ability of conventional probes to discriminate between wild-type and mutant alleles is poor. Recently, DNA probes with conjugated minor groove binder (MGB) have been developed. Because of their high melting temperature, these probes achieve high performance in detecting single nucleotide mismatches. Using the MGB technology, we developed a new RQ-PCR system for detecting occult tumor cells in patients with colorectal cancer (CRC), targeting K-ras point mutations. Sixteen MGB-conjugated DNA probes were designed for all previously reported K-ras mutations. The performance of these probes was examined with plasmid DNAs into which K-ras point mutations had been inserted, 32 cancer cell lines and 338 lymph nodes obtained from 15 CRC patients. Fifteen of the 16 MGB probes designed were useful for accurate quantitative assessment, and achieved high sensitivity (1/10^4-10^5 background cells) and high reproducibility (coefficients of variation < 10%). Performance in discriminating single nucleotide mismatches was superior for MGB probes compared with non-MGB probes. We detected a micrometastasis (5.85/10^4 cells equivalent) in one (0.9%) of 110 lymph nodes obtained from 6 patients with K-ras mutations. There was no true false-positive result in 209 lymph nodes obtained from 9 patients without K-ras mutations. The MGB RQ-PCR assay targeting K-ras mutations is an accurate quantitative method for detecting occult tumor cells in CRC.