PDF(Pubmed)
Abstract：
Detection of the Kirsten rat sarcoma gene (KRAS) mutational status is an important factor for the treatment of various malignancies. The most common KRAS-activating mutations are caused by single-nucleotide mutations, which are usually determined by using PCR, using allele-specific DNA primers. Oligonucleotide primers with uncharged or partially charged internucleotide phosphate modification have proved their ability to increase the sensitivity and specificity of various single nucleotide mutation detection. To enhance the specificity of single nucleotide mutation detection, the novel oligonucleotides with four types of uncharged and partially charged internucleotide phosphates modification, phosphoramide benzoazole (PABA) oligonucleotides (PABAO), was used to prove the concept on the KRAS mutation model. The molecular effects of different types of site-specific PABA modification in a primer or a template on a synthesis of full-length elongation product and PCR efficiency were evaluated. The allele-specific PCR (AS-PCR) on plasmid templates showed a significant increase in analysis specificity without changes in Cq values compared with unmodified primer. PABA modification is a universal mismatch-like disturbance, which can be used for single nucleotide polymorphism discrimination for various applications. The molecular insights of the PABA site-specific modification in a primer and a template affect PCR, structural features of four types of PABAO in connection with AS-PCR results, and improvements of AS-PCR specificity support the further design of novel PCR platforms for various biological targets testing.
摘要：
检测Kirsten大鼠肉瘤基因（KRAS）突变状态是治疗各种恶性肿瘤的重要因素。最常见的KRAS激活突变是由单核苷酸突变引起的，通常通过使用PCR确定，使用等位基因特异性DNA引物。具有不带电荷或部分带电荷的核苷酸间磷酸修饰的寡核苷酸引物已证明其能够提高各种单核苷酸突变检测的灵敏度和特异性。提高单核苷酸突变检测的特异性,具有四种不带电和部分带电的核苷酸间磷酸修饰的新型寡核苷酸，磷酰胺苯并唑（PABA）寡核苷酸（PABAO），用于证明KRAS突变模型上的概念。评估了引物或模板中不同类型的位点特异性PABA修饰对全长延伸产物的合成和PCR效率的分子效应。与未修饰的引物相比，质粒模板上的等位基因特异性PCR（AS-PCR）显示分析特异性显着增加，而Cq值没有变化。PABA修饰是一种普遍的类失配干扰，可用于各种应用的单核苷酸多态性鉴别。引物和模板中PABA位点特异性修饰的分子见解影响PCR，四种类型的PABAO的结构特征与AS-PCR结果有关，和AS-PCR特异性的改进支持了用于各种生物靶标测试的新型PCR平台的进一步设计。
