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ISSN 1027-2852 (Electronic)
ISSN 0864-4551 (Print)

2015, Number 4

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Biotecnol Apl 2015; 32 (4)

Development of a method to detect three frequent mutations in the CFTR gene using allele-specific real time PCR

Figueredo-Lago JE, Armas-Cayarga A, González-González YJ, Collazo-Mesa T, García RI, Perea-Hernández Y, Santos-González EN

Language: English
References: 22
Page: 4301-4306
PDF size: 528.41 Kb.


ABSTRACT

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A method was developed to detect three frequent CFTR mutations (F508del, 3120 + 1G › A and N1303K), using allele-specific real time PCR (ASPCR). Genomic DNA samples from nine patients carrying different CFTR mutations were evaluated in triplicate. The Ct variation (ΔCt) between wild type and mutant ASPCRs, and the mean value ± standard deviation (SD) were determined. The specificity of allele-specific primers was confirmed by High Resolution Melting (HRM) analysis. The results validated this test for genotype determination; the tested mutations were clearly identified in all the samples and in excellent agreement with the commercial panel StripAssay (Viennalab, Austria). The described method provides the necessary selectivity and specificity as required for clinical screening of CF population, mainly for F508del mutation due to its high prevalence among the Cuban population.


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