Chubarov Alexey S, Oscorbin Igor P, Filipenko Maxim L, Lomzov Alexander A, Pyshnyi Dmitrii V
Institute of Chemical Biology and Fundamental Medicine, SB RAS, 8 Lavrentiev Avenue, 630090 Novosibirsk, Russia.
Diagnostics (Basel). 2020 Oct 26;10(11):872. doi: 10.3390/diagnostics10110872.
Establishing the Kirsten rat sarcoma ) mutational status is essential in terms of managing patients with various types of cancer. Allele-specific real-time polymerase chain reaction (AS-PCR) is a widely used method for somatic mutations detection. To improve the limited sensitivity and specificity, several blocking methods have been introduced in AS-PCR to block the amplification of wild-type templates. Herein, we used a novel modified oligonucleotide with internucleotide phosphates reshaped 1,3-dimethyl-2-imino-imidazolidine moieties (phosphoryl guanidine (PG) groups) as primers and blockers in the AS-PCR method. Four common mutations were chosen as a model to demonstrate the advantages of the PG primers and blockers utilizing a customized PCR protocol. The methods were evaluated on plasmid model systems providing a mutation detection limit of 20 copies of mutant DNA in a proportion as low as 0.1% of the total DNA, with excellent specificity. PG-modification can serve as the universal additional mismatch-like disturbance to increase the discrimination between wild-type and mutated DNA. Moreover, PG can serve to increase primer specificity by a synergetic effect with additional mismatch and would greatly facilitate medical research.
确定 Kirsten 大鼠肉瘤病毒（KRAS）突变状态对于各类癌症患者的治疗至关重要。等位基因特异性实时聚合酶链反应（AS-PCR）是一种广泛用于检测体细胞突变的方法。为了提高有限的灵敏度和特异性，AS-PCR 中引入了几种封闭方法来阻断野生型模板的扩增。在此，我们使用一种新型修饰寡核苷酸作为引物和封闭剂，该寡核苷酸的核苷酸间磷酸被重塑为 1,3 - 二甲基 - 2 - 亚氨基咪唑烷部分（磷酰胍（PG）基团），用于 AS-PCR 方法。选择四个常见突变作为模型，利用定制的 PCR 方案来证明 PG 引物和封闭剂的优势。在质粒模型系统上对这些方法进行了评估，其突变检测限为在占总 DNA 低至 0.1%的比例下检测到 20 个拷贝的突变 DNA，具有出色的特异性。PG 修饰可作为通用的额外错配样干扰，以增加野生型和突变型 DNA 之间的区分度。此外，PG 可通过与额外错配的协同作用提高引物特异性，并将极大地促进医学研究。
