Single-tube method for determination of F508del genotype in the CFTR gene using bidirectional PCR amplification of specific alleles.

Cystic fibrosis is the most common severe autosomal recessive disorder in Caucasians, with a frequency of 1 in 2000 live births and a carrier frequency of approximately 5% (1). It is a multi-system disorder that leads to chronic pulmonary and exocrine pancreatic disease, associated with elevated sweat electrolytes and male infertility due to bilateral absence of the vas deferens (2–4). Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are the genetic cause of this disease (2–4), and their identification allows molecular diagnosis in patients, carrier and prenatal testing in cystic fibrosis families, and population screening (NIH consensus statement, 1997). The CFTR gene spans 189 kb, with a coding region of 6.2 kb, including 27 exons. More than 1000 mutations have been described in this gene (http://www. genet.sickkids.on.ca/cftr/rptTABLE2. html) (5), with a widespread distribution that complicates direct testing (1). The most common mutation in all populations is a deletion of three nucleotides that results in loss of a phenylalanine in the CFTR protein, at position 508 (F508del). Therefore, strategies for genetic testing in patients/families with cystic fibrosis are often initiated with direct detection of this particular mutation, such as the allele-specific PCR method described by Ballabio et al. (6). This method requires two PCRs for the determination of the genotype at this locus and does not allow the distinction between F508del (69% of cystic fibrosis alleles in the US) and the less common I507del mutation (0.31% in the US) (1), because the primers used for amplification of the mutant allele will match both of these sequences. Individuals with an I507del mutation will erroBenchmarks