'Broad-range' DGGE for single-step mutation scanning of entire genes: application to human phenylalanine hydroxylase gene.

Attempts to isolate human genes and characterize molecular defects underlying inherited diseases are in rapid progress. The evolving possibilities of improved diagnosis by analysis at the DNA level have prompted the need for efficient methods that enable rapid detection of mutations. For diseases caused predominantly by large gene deletions or expansion of a trinucleotide repeat, simple PCR-based assays are now available for diagnosis and evaluation of carrier status. However, for diseases that are caused primarily by point mutations, e.g. cystic fibrosis, /3-thalassaemia, and phenylketonuria (PKU), no mutation scanning method has yet proven satisfactory for rapid and comprehensive gene analysis in single individuals, and DNA sequencing still seems impracticable for determining whether a particular gene in an individual harbours a point mutation or not. For these reasons, diagnostic approaches have to a wide extent relied on methods for recognition of previously identified mutations (for a recent review see reference 1). Owing to a large number of different mutations and a marked divergence in mutational spectra between different populations, the sensitivity of these methods is often low. In this report we describe a modification of denaturing gradient gel electrophoresis (DGGE) (2), which we term 'broad-range' DGGE, that allows simultaneous analysis of multiple PCR-amplified DNA fragments, enabling rapid one-step scanning of entire genes for the presence or absence of any point mutation. The mutation-resolving power of DGGE relics on a physical separation between similar DNA fragments differing in melting properties due to differences in nucleotide composition (2). Two modifications greatly increase the sensitivity of the technique: Attachment of a thermostable GC-clamp to one of the ends of the DNA fragment (3), and analysis of heteroduplex molecules, i.e. hybrids formed between mutant and prototype strands (4). The clear advantages of DGGE for population studies have been substantiated by a number of recent studies, demonstrating a mutation detection efficiency near 100%. However, when there is a need to test a particular gene in only one individual, the conventional methodology is not rational due to the fact that different parts of the gene are generally analysed under different experimental conditions to optimize resolution of mutations. We have attempted to overcome this limitation by exploiting the flexibility of DGGE with respect to two critical parameters: gradient nange and running time. 8 9 10 11 12 13