Symmetric snapback primers for scanning and genotyping of the cystic fibrosis transmembrane conductance regulator gene.
Clin Chem
; 59(7): 1052-61, 2013 Jul.
Article
em En
| MEDLINE
| ID: mdl-23503723
ABSTRACT
BACKGROUND:
High-resolution melting of PCR products is an efficient and analytically sensitive method to scan for sequence variation, but detected variants must still be identified. Snapback primer genotyping uses a 5' primer tail complementary to its own extension product to genotype the resulting hairpin via melting. If the 2 methods were combined to analyze the same PCR product, the residual sequencing burden could be reduced or even eliminated.METHODS:
The 27 exons and neighboring splice sites of the CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] gene were amplified by the PCR in 39 fragments. Primers included snapback tails for genotyping 7 common variants and the 23 CFTR mutations recommended for screening by the American College of Medical Genetics. After symmetric PCR, the amplicons were analyzed by high-resolution melting to scan for variants. Then, a 5-fold excess of H2O was added to each reaction to produce intramolecular hairpins for snapback genotyping by melting. Each melting step required <10 min. Of the 133 DNA samples analyzed, 51 were from CFTR patient samples or cell lines.RESULTS:
As expected, the analytical sensitivity of heterozygote detection in blinded studies was 100%. Snapback genotyping reduced the need for sequencing from 7.9% to 0.5% of PCR products; only 1 amplicon every 5 patients required sequencing to identify nonanticipated rare variants. We identified 2 previously unreported variants c.3945A>G and c.4243-5C>T.CONCLUSIONS:
CFTR analysis by sequential scanning and genotyping with snapback primers is a good match for targeted clinical genetics, for which high analytical accuracy and rapid turnaround times are important.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Primers do DNA
/
Regulador de Condutância Transmembrana em Fibrose Cística
Limite:
Humans
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article