Design of an automated multicapillary instrument with fraction collection for DNA mutation discovery by constant denaturant capillary electrophoresis (CDCE).

A fundamental goal ingenomics is the discovery of genetic variation that contributes to disease states or to differential drug responses. Single nucleotide polymorphism (SNP) detection has been the focus of much attention in the study of genetic variation over the last decade. These SNPs typically occur at a frequency greater than 1% in the human genome. Recently, low-frequency alleles are also being increasingly recognized as critical to obtain an improved understanding of the correlation between genetic variation and disease. Although many methods have been reported for the discovery and scoringof SNPs, sensitive, automated, and cost-effective methods and platforms for the discovery of low-frequency alleles are not yet readily available. We describe here an automated multicapillary instrument for high-throughput detection of low-frequency alleles from pooled samples using constant denaturant capillary electrophoresis. The instrument features high optical sensitivity (1 x 10(-12) M fluorescein detection limit), precise and stable temperature control (+/- 0.01degrees C), and automation for sample delivery, injection, matrix replacement, and fraction collection. The capillary array is divided into six groups of four capillaries, each of which can be independently set at any temperature ranging from room temperature to 90 degrees C. The key performance characteristics of the instrument are reported.

Detection and frequency estimation of rare variants in pools of genomic DNA from large populations using mutational spectrometry.

DNA variants underlying the inheritance of risk for common diseases are expected to have a wide range of population allele frequencies. The detection and scoring of the rare alleles (at frequencies of <0.01) presents significant practical problems, including the requirement for large sample sizes and the limitations inherent in current methodologies for allele discrimination. In the present report, we have applied mutational spectrometry based on constant denaturing capillary electrophoresis (CDCE) to DNA pools from large populations in order to improve the prospects of testing the role of rare variants in common diseases on a large scale. We conducted a pilot study of the cytotoxic T lymphocyte-associated antigen-4 gene (CTLA4) in type 1 diabetes (T1D). A total of 1228 bp, comprising 98% of the CTLA4 coding sequence, all adjacent intronic mRNA splice sites, and a 3' UTR sequence were scanned for unknown point mutations in pools of genomic DNA from a control population of 10,464 young American adults and two T1D populations, one American (1799 individuals) and one from the United Kingdom (2102 individuals). The data suggest that it is unlikely that rare variants in the scanned regions of CTLA4 represent a significant proportion of T1D risk and illustrate that CDCE-based mutational spectrometry of DNA pools offers a feasible and cost-effective means of testing the role of rare variants in susceptibility to common diseases.