Sensitive detection of low-abundance in-frame deletions in EGFR exon 19 using novel wild-type blockers in real-time PCR.
Sci Rep
; 9(1): 8276, 2019 06 04.
Article
em En
| MEDLINE
| ID: mdl-31164704
ABSTRACT
Epidermal growth factor receptor (EGFR) mutations are associated with response of tyrosine kinase inhibitors (TKIs) for patients with advanced non-small cell lung cancer (NSCLC). However, the existing methods for detection of samples having rare mutations(i.e. ~0.01%) have limits in terms of specificity, time consumption or cost. In the current study, novel wild-type blocking (WTB) oligonucleotides modified with phosphorothioate or inverted dT at the 5'-termini were designed to precisely detect 11 common deletion mutations in exon 19 of EGFR gene (E19del) using a WTB-PCR assay. And internal competitive leptin amplifications were further applied to enhance the specificity of the WTB-PCR system. Our results showed that WTB-PCR could completely block amplification of wild-type EGFR when 200 ng of DNA was used as template. Furthermore, the current WTB-PCR assay facilitated the detection of E19del mutations with a selectivity of 0.01% and sensitivity as low as a single copy. And, the results showed that the current WTB-PCR system exceeded detection limits afforded by the ARMS-PCR assay. In conclusion, the current WTB-PCR strategy represents a simple and cost-effective method to precisely detect various low-abundance deletion mutations.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oligonucleotídeos
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Carcinoma Pulmonar de Células não Pequenas
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Inibidores de Proteínas Quinases
Tipo de estudo:
Diagnostic_studies
Limite:
Adult
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Aged
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Aged80
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Female
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Humans
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Male
/
Middle aged
Idioma:
En
Revista:
Sci Rep
Ano de publicação:
2019
Tipo de documento:
Article
País de publicação:
ENGLAND
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ESCOCIA
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GB
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GREAT BRITAIN
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INGLATERRA
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REINO UNIDO
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SCOTLAND
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UK
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UNITED KINGDOM