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

A second potential limitation to screen directly for functional polymorphic vari- ants 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, in- volve relatively expensive technology that has not been completely validated (a sig- nificant 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 pos- sible sequence variants.28,29

TWO-DIMENSIONAL GENE SCANNING

The most reliable way of detecting DNA sequence alterations in genes is nucle- otide 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 rela- tive 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

gel electrophoresis (DGGE). DGGE allows for the separation of DNA differing by as little as one base pair, on the basis of differences in temperature.31 A given DNA fragment comprises one or more 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 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

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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.

introducing so-called GC-clamps, i.e., a stretch of 30 to 50 G and C bases, to the tar- get DNA fragments.32 A convenient way of attaching a GC-clamp to the target frag- ment is by making it part of one of the primers.33 Without GC-clamping, a DNA fragment consisting of one melting domain will become completely single stranded upon denaturation and runs off the gel. By adding a GC-clamp, a single high melting domain is artificially created at one end of the target fragment. This will prevent complete melting and ensures that the fragment halts at the melting temperature of the target sequence. However, when the target DNA fragment consists of multiple melting domains, only mutations in the lowest melting domain are readily detected. To facilitate detection of all possible mutations, it is imperative that the target frag- ment represents only one melting domain. Fortunately, since the addition of a GC- clamp allows for stacking interactions with neighboring bases, the entire target frag- ment will often behave as one melting domain. However, this is not always the case, and in practice, the target fragment needs to be “designed”, e.g., through the strategic positioning of PCR primers to achieve the ideal single melting domain.

duction of a heteroduplexing step using one round of denaturation/renaturation, usu- ally at the end of PCR amplification of the target fragment. In this manner, a heterozygous mutation is revealed as four different double-stranded fragments: two homoduplex molecules (one homoduplex wild type and one homoduplex mutant) and two heteroduplex molecules (each comprising one wild type and one mutant strand). The less stable mismatched heteroduplex molecules will melt earlier than the two homoduplex molecules.

% Although DGGE has the crucial advantage of having close to 100 sensitivity in detecting mutations,34,35 it has typically been applied in a serial fashion, e.g., on an exon by exon basis, for analyzing large genes. With the introduction of two-dimen- sional gene scanning (TDGS) we have transformed the method from the existing se- rial to a parallel system. The first step in TDGS is a large-scale multiplex PCR. Initial pre-amplification of multiple exons by long-distance PCR has been demon- strated to provide suitable template for a highly specific multiplex PCR. Indeed, the over-representation of the exon-containing genomic fragments, relative to the ge- nomic environment, permits multiplex groups of as many as 24 fragments.36