With the rapid emergence of complete sequence information for all human genes, it is theoretically possible to scrutinize all genes or families of genes suspected to be involved in a complex phenotype for all possible polymorphic variation in a group of subjects comparedeasier as a result of this technology. In a study by Cargill et al.26, the coding regions of 106 genes relevant to cardiovascular disease, endocrinology, and neuropsychiatry were systematically surveyed for all possible variants in a sample of mixed ethnic groups. Oligonucleotide chip array hybridization and DHPLC were used for this purpose. A total of 560 single nucleotide polymorphisms were discovered in these 106 genes. Halushka et al.27 used the same technology to assay 75 candidate genes to identify potentially functional SNPs involved in blood pressure homeostasis and hypertension. They screened a sample of 74 ethnically heterogeneous individuals with a range of hypertension phenotype diversity and discovered 387 SNPs in gene coding sequences, 54 of which were predicted to lead to altered protein.

There are two potential limitations to this approach. The first involves the relatively small number of genes that have been completely identified in terms of structure and function. Although almost all human genes are now available as ESTs, only a limited number have been defined by a complete cDNA sequence and functional pathway. Indeed, for only a few genes is a complete genomic sequence including 3 UTR, promoter, and flanking regions readily available. With the genome program drawing to a close in the next few years, it is reasonable to expect that most of these gaps will be filled soon and that complete sequence information and pathway assignments will be available.

A second potential limitation to screen directly for functional polymorphic variants involved in a complex trait involves the technology to screen multiple genes in large numbers of individuals for all possible sequence variation, which is still in its infancy. Most technology in this respect is limited by the need to PCR amplify each individual target sequence for mutational scanning. Moreover, the most advanced methods, such as the DNA chip method used in the two studies described above, involve relatively expensive technology that has not been completely validated (a significant number of variants are missed). A cost-effective method developed in our laboratory is based on extensive multiplex PCR to obtain the target fragments, which are subsequently analyzed on two-dimensional denaturing gradient gels for all possible sequence variants.28,29

The most reliable way of detecting DNA sequence alterations in genes is nucleotide sequencing, the gold standard in mutation detection. Sequencing is PCR-based with preparation of template on an exon-by-exon basis. This is expensive and labor-intensive, even in the semi-automated setting. Other mutation scanning techniques, e.g., single strand conformation polymorphism analysis, trade off accuracy for relative throughput. Many novel methods with the potential of both a high throughput and a high accuracy remain immature and unproven in practice. Moreover, virtually all methods rely on PCR amplification to obtain the target sequences.30

DGGE is a gel electrophoresis technique that allows for the separation of DNA differing by as little as one base pair, based on differences in temperature.31 A given DNA fragment comprises one or more melting domains representing a stretch of between about 50 and 500 base pairs with equal temperature (the temperature at which each base pair has a 50% probability in either the helical or the denatured state). Therefore, when a fragment migrates parallel to the gradient of denaturants (urea/formamide), the lowest melting domain will denature first and, because of the double-strandedness of the DNA strands, the fragment will be retarded in the gel. The melting of a domain is dependent on the base composition of the fragment; G-C base pairs have a higher melting temperature than A-T base pairs and the stacking interactions of the bases. Therefore, DNA sequence variations will be reflected by the positions in the gel where the fragments are halted.