Michael T. Boyce-Jacino

Large-scale genotyping of complex DNA

Genetic studies aimed at understanding the molecular basis of complex human phenotypes require the genotyping of many thousands of single-nucleotide polymorphisms (SNPs) across large numbers of individuals. Public efforts have so far identified over two million common human SNPs; however, the scoring of these SNPs is labor-intensive and requires a substantial amount of automation. Here we describe a simple but effective approach, termed whole-genome sampling analysis (WGSA), for genotyping thousands of SNPs simultaneously in a complex DNA sample without locus-specific primers or automation. Our method amplifies highly reproducible fractions of the genome across multiple DNA samples and calls genotypes at >99% accuracy. We rapidly genotyped 14,548 SNPs in three different human populations and identified a subset of them with significant allele frequency differences between groups. We also determined the ancestral allele for 8,386 SNPs by genotyping chimpanzee and gorilla DNA. WGSA is highly scaleable and enables the creation of ultrahigh density SNP maps for use in genetic studies.

A SNPshot: pharmacogenetics and the future of drug therapy

Pharmacogenetics holds great promise for the optimization of new drug development and the individualization of clinical therapeutics in the 21st century. In this brief review, we trace the historical roots of pharmacogenetics, discuss its rapidly evolving processes and paradigms, and look towards future applications of pharmacogenetics in enhancing the efficiency of the drug development pipeline and in improving patient care.

Multicolor instrumentation for direct fluorescent detection of nucleic acids in a microchip format

Deposition of nucleic acids on solid support in the form of high density arrays (a DNA microarray) creates a powerful nonelectrophoretic technology for highly parallel genetic analysis. Microarrays have applications in the areas of DNA sequencing, genetic mutation detection and gene expression monitoring. We report here the design and utility of an experimental instrument for microchip parallel hyperspectral fluorescent imaging. The instrument integrates in-line laser excitation of microarray, parallel fluorescent spectrometry with cooled CCD and dye-base spectral classification software. Instrument has been applied for imaging detection, spectral analysis and base classification of Genetic Bit Analysis (GBA) reactions in a microchip format on a glass support. GBA is a solid phase DNA sequence analysis method that provides single nucleotide resolution by specific extension of dye-labeled dideoxynucleotidetriphosphates (ddNTPs). GBA testing yields one or two different ddNTPs on any given microarray spot, so analysis must resolve any pair wise combination of all possible ddNTPs labeled with distinct fluorescent dyes.

Fluorescence detection applied to nonelectrophoretic DNA diagnostics on oligonucleotide arrays

DNA analysis based on template hybridization (or hybridization plus enzymatic processing) to an array of surface-bound oligonucleotides is well suited for high density, parallel, low cost and automatable processing. Direct fluorescence detection of labelled DNA provides the benefits of linearity, large dynamic range, multianalyte detection, processing simplicity and safe handling at reasonable cost. Molecular Tool has applied a proprietary enzymatic method of solid phase genotyping (Genetic BitTM Analysis or GBATM) to DNA processing in 96-well plates and glass microscope slides. Glass slides are an inexpensive, convenient format with relatively low fluorescence and the capability for microfabrication of miniature arrays of oligonucleotides. Detecting the fluor-labelled GBATM dideoxynucleotides requires a detection limit of approximately 100 molecules/micrometer 2. Commercially available plate readers detect about 1000 molecules/micrometer 2, and an experimental setup with an Ar laser and thermoelectrically-cooled CCD can detect approximately 1 order of magnitude less signal. The current limit is due to glass fluorescence, and data is presented describing experimental modifications and analysis to improve the performance. Dideoxynucleotides labelled with fluorescein, eosin, tetramethylrhodamine, Lissamine and Texas Red are being characterized, and photobleaching, quenching and indirect detection with fluorogenic substrates have been investigated.