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Clin Chem. 2013 Jul;59(7):1052-61. doi: 10.1373/clinchem.2013.202689. Epub 2013 Mar 15.

Symmetric snapback primers for scanning and genotyping of the cystic fibrosis transmembrane conductance regulator gene.

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Department of Pathology, School of Medicine, University of Utah, Salt Lake City, UT 84132, USA.



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.


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.


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.


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.

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