Allele-Specific PCR for KRAS Mutation Detection Using Phosphoryl Guanidine Modified Primers

Diagnostics

AlexanderA. Lomzov¹  AlexeyS. Chubarov¹  IgorP. Oscorbin¹  MaximL. Filipenko¹  DmitriiV. Pyshnyi¹ 

[1] Institute of Chemical Biology and Fundamental Medicine, SB RAS, 8 Lavrentiev Avenue, 630090 Novosibirsk, Russia;

关键词: mutation detection;    KRAS mutations;    allele-specific PCR;    blocker PCR;    modified oligonucleotides;    phosphoryl guanidine oligonucleotide (PGO);   

DOI  :  10.3390/diagnostics10110872

来源: DOAJ

【 摘 要 】

Establishing the Kirsten rat sarcoma (KRAS) 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 KRAS 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 KRAS 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.

【 授权许可】

Unknown