An automated fluorescent single-strand conformation polymorphism technique for screening mutations in the hepatocyte nuclear factor-1alpha gene (maturity-onset diabetes of the young).

Maturity-onset diabetes of the young (MODY) is a form of NIDDM characterized by an early age of onset and autosomal-dominant inheritance (1). So far, three MODY-causing genes have been identified; namely, the hepatocyte nuclear factor4a (HNF-4a)/M0DYl on chromosome 20q, the glucokinase/M0DY2 on chromosome 7p, and the HNFla/M0DY3 on chromosome 12 q (2-4). The identification of the MODY genes has allowed diagnosis at the molecular level for a large portion of MODY patients. Mutations inM0DY3/HNF-la have been identified in several populations by means of direct sequencing (4-6). However, direct sequencing (expensive and time-consuming) or conventional single-strand conformation polymorphism (SSCP) are not routinely applicable. Radioactive SSCP involves radioactive labeling, and the silver-staining SSCP requires postlabeling. SSCP protocols using multiple fluorescent labeling and an automated DNA sequencer (7) may be an interesting alternative. We have developed a new SSCP protocol, based on fluorescent end-labeling with an automated sequencer, using primers extended by universal M13 sequences (8). This modification results in two advantages. First, the same couple of fluorescently labeled primers may be used to detect all exons and promoter regions of HNF-la, reducing the cost of the technique. Second, the polymerase chain reaction fragments extended at their 5' ends by the universal M13 primers can be readily sequenced using the M13 dye primers or the dideoxy terminator sequencing kits.