title: Symmetric snapback primers for scanning and genotyping of the cystic fibrosis transmembrane conductance regulator gene.
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.
authors.affiliation: Department of Pathology, School of Medicine, University of Utah, Salt Lake City, UT 84132, USA. luming.zhou@path.utah.edu
authors.forename: Luming
authors.initials: L
authors.lastname: Zhou
authors.forename: Robert A
authors.initials: RA
authors.lastname: Palais
authors.forename: Felix
authors.initials: F
authors.lastname: Ye
authors.forename: Jimmy
authors.initials: J
authors.lastname: Chen
authors.forename: Jesse L
authors.initials: JL
authors.lastname: Montgomery
authors.forename: Carl T
authors.initials: CT
authors.lastname: Wittwer
mesh_terms: D045744:Cell Line, Tumor; D003550:Cystic Fibrosis / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D005838:Genotype; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic
chemical_list: C505032:CFTR protein, human; D017931:DNA Primers; D019005:Cystic Fibrosis Transmembrane Conductance Regulator