KRAS genotyping by digital PCR combined with melting curve analysis.
Sci Rep
; 9(1): 2626, 2019 02 22.
Article
em En
| MEDLINE
| ID: mdl-30796246
ABSTRACT
Digital PCR (dPCR) has been developed as a method that can quantify nucleic acids more sensitively than real-time PCR. However, dPCR exhibits large fluctuations in the fluorescence intensity of the compartment, resulting in low accuracy. The main cause is most likely due to insufficient PCR. In this study, we proposed a new method that combines dPCR with melting curve analysis and applied that method to KRAS genotyping. Since the melting temperature (Tm) of the PCR product hardly depends on the amplification efficiency, genotyping accuracy is improved by using the Tm value. The results showed that the peaks of the distribution of the Tm values of DNA in the wells were 68.7, 66.3, and 62.6 °C for wild-type KRAS, the G12R mutant, and the G12D mutant, respectively, and the standard deviation of the Tm values was 0.2 °C for each genotype. This result indicates that the proposed method is capable of discriminating between the wild-type sequence and the two mutants. To the best of our knowledge, this is the first demonstration of the genotyping of single mutations by combining melting curve analysis and dPCR. The application of this approach could be useful for the quantification and genotyping of cancer-related genes in low-abundance samples.
Texto completo:
1
Coleções:
01-internacional
Temas:
Geral
Base de dados:
MEDLINE
Assunto principal:
Proteínas Proto-Oncogênicas p21(ras)
/
Técnicas de Genotipagem
/
Reação em Cadeia da Polimerase em Tempo Real
/
Desnaturação de Ácido Nucleico
Limite:
Humans
Idioma:
En
Revista:
Sci Rep
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
Japão