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Large-scale screening for known polymorphisms will require techniques with few steps and the ability to automate each of these steps. In this regard, the 5' nuclease, or TaqMan, PCR assay is especially attractive. A fluorogenic probe, consisting of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye, is included in a typical PCR. Amplification of the probe-specific product causes cleavage of the probe, generating an increase in reporter fluorescence. By using different reporter dyes, cleavage of allele-specific probes can be detected in a single PCR. The 5' nuclease assay has been successfully used to discriminate alleles that differ by a single base substitution. Guidelines have been developed so that an assay for any single nucleotide polymorphism (SNP) can be quickly designed and implemented. All assays are performed using a single reaction buffer and single thermocycling protocol. Furthermore, a standard method of analysis has been developed that enables automated genotype determination. Applications of this assay have included typing a number of polymorphisms in human drug metabolism genes.

Sequencing of a human DNA ligase I cDNA clone derived from HeLa cells revealed two unreported differences with the published sequence: a single base change and a three-base deletion. Both differences are in exon 6, and were analyzed by amplifying a segment containing exon 5, intron 6, and exon 6. The first finding was that intron 6 is approximately 2.6 kb in size, not the 1 kb reported in the literature. By sequence analysis of amplified segments, the single-base difference in exon 6 was shown to be polymorphic, with HeLa cells heterozygous for the A/C difference. Analysis of 60 unrelated individuals found a frequency of 0.5 for each allele. Primer extension reactions across the exon 5/exon 6 boundary were performed on cDNA obtained from HeLa cells and human thymus. The results show that the three-base deletion is due to a variation in splicing. For both HeLa and thymus, two-thirds of the transcripts are like the published cDNA sequence and one-third have the three-base deletion. Finally, sequencing of part of intron 6 revealed the presence of a complex GT repeat consisting of a 48-50 nucleotide polypurine tract followed by a variable number of GT residues. This entire unit of polypurine tract plus GTs is repeated three times. Detection of the repeated sequences required the development of specialized cloning and PCR conditions. Analysis of a pedigree showed that this complex repeat is polymorphic.

The dopamine D4 receptor gene (DRD4) has an expressed polymorphism in the third exon that may have functional relevance. The polymorphism exists at two levels. At the higher level there is an imperfect tandem repeat of 48 base pairs (bp) coding for 16 amino acids; alleles have been identified with 2 (32 amino acids) to 10 (160 amino acids) repeats. The imperfect nature of the repeats is responsible for a more subtle level of variation since alleles with the same number of repeats can differ in the exact sequences or in the order of the variants of the 48-bp unit. We have undertaken a global survey of this expressed polymorphism as one approach to understanding the evolutionary significance and origins of the polymorphism as well as understanding what selective forces, if any, may be operating at this locus. As the first step, we have determined the repeat number genotype of the DRD4 repeat polymorphism in 1,327 individuals from 36 different populations. The allele frequencies differ considerably among the different populations. The 4-repeat allele was the most prevalent (global mean allele frequency = 64.3%) and appeared in every population with a frequency ranging from 0.16 to 0.96. The 7-repeat allele was the second most common (global mean = 20.6%), appearing quite frequently in the Americas (mean frequency = 48.3%) but only occasionally in East and South Asia (mean frequency = 1.9%). The 2-repeat allele was the third most common (global mean frequency = 8.2%) and was quite frequent in East and South Asia (mean frequency = 18.1%) while uncommon in the Americas (mean frequency = 2.9%) and Africa (mean frequency = 1.7%). The universality of the polymorphism with only three common repeat-number alleles (4, 7, and 2) indicates that the polymorphism is ancient and arose before the global dispersion of modern humans. The diversity of actual allele frequencies for this expressed polymorphism among different populations emphasizes the importance of population considerations in the design and interpretation of any association studies carried out with this polymorphism.

Genotype determination using the solid-phase minisequencing method of Syvänen et al. (1990, 1993) has been adapted for use with fluorescein-labeled dideoxynucleotides (F-ddNTPs). PCR is performed using one biotinylated primer and one unbiotinylated primer. The biotinylated products are captured in streptavidin-coated microtiter wells. Following removal of nonbiotinylated strands with NaOH, the bound strands are hybridized with a primer adjacent to the polymorphic site being tested. Using T7 DNA polymerase, the primer is extended using one F-ddNTP in the presence of the other three unlabeled ddNTPs. Incorporation of the F-ddNTP is detected by binding antifluorescein antibody conjugated with alkaline phosphatase followed by incubation with a chromogenic substrate. This assay was used to determine APOE genotypes for 75 subjects. The APOE genotypes were also determined using a method involving the incorporation of mobility-shifting nucleotide analogs (Livak et al., 1992). Investigation of the one discrepancy between the two methods revealed that one subject carries a rare APOE allele that has a 3 bp deletion.

The human dopamine D4 receptor contains a novel polymorphism within the putative third cytoplasmic loop of the protein. The polymorphism is characterized by a varying number of direct imperfect 48-bp repeats in the gene. Pharmacological characterization has suggested that this receptor is the site through which the atypical neuroleptic clozapine exerts its antipsychotic action and that some polymorphic variants display different pharmacological properties. Further analysis of the repeat region using innovative technologies indicates that the alleles vary not only in the number of repeats (2-8 or 10 repeat units) but also in the sequence of the repeats and the order in which they appear. In 178 unrelated chromosomes we have identified 19 different repeats in 25 different haplotypes coding for 18 different predicted amino acid sequences, making this one of the most variable functional proteins currently described.

A simple primer extension method for detecting nucleotide differences is based on the substitution of mobility-shifting analogs for natural nucleotides (1). This technique can detect any single-base difference that might occur including previously unknown mutations or polymorphisms. Two technical limitations of the original procedure have now been addressed. First, switching to Thermococcus litoralis DNA polymerase has eliminated variability believed to be due to the addition of an extra, non-templated base to the 3' end of DNA by Taq DNA polymerase. Second, with the analogs used in the original study, the mobility shift induced by a single base change can usually be resolved only in DNA segments 200 nt or smaller. This size limitation has been overcome by synthesizing biotinylated nucleotides with extraordinarily long linker arms (36 atom backbone). Using these new analogs and conventional sequencing gels (0.4 mm thick), mutations in the human beta-hexosaminidase alpha and CYP2D6 genes have been detected in DNA segments up to 300 nt in length. By using very thin (0.15 mm) gels, single-base polymorphisms in the human APOE gene have been detected in 500-nt segments.