Simultaneous cycle sequencing assessment of (TG)m and Tn tract length in CFTR gene

The lengthy of the dinucleotide (TG), and mononucleotide T-n repeats, both located at the intron 8/exon 9 splice, acceptor site of the cystic fibrosis transmembrane conductance regulator (CFTR) gene whose mutations cause cystic fibrosis, (CF), have been shown to influence the skipping of exon 9 in CFTR mRNA_ This, exon 9-skipped mRNA encodes a nonfunctional protein and is associated with various clinical manifestations in CF As a result of growing interest in these repeats, several assessment methods have been developed, most of which are, however, cumbersome, multi-step, and time consuming. Here, we describe a rapid method for the simultaneous assessment Of the lengths of both (TG) and Tn repeats, based on a nonradioactive cycle sequencing procedure that can be performed even without DNA extraction. This method determines the lengths, of the (TG)(m) and T-n tracts of both alleles, which in our samples ranged from TG(8) to TG(12) in the presence of T-5, T-7, and T-9 alleles, and also fully assesses the aplotypes. In addition, the repeats in the majority of these samples can be assessed by single-strand sequencing, with no need to sequence the other strand, thereby saving a considerable amount of time and effort.