Analysis of whole genomes using mapping by admixture linkage disequilibium (MALD) and candidate genes requires an appropriate set of markers and the ability to accurately genotype hundreds to thousands of patients with hundreds to thousands of markers. Markers appropriate for MALD must have large differences between racial groups. While our first applications of MALD are in African Americans, we expect that other admixed groups like Hispanics will also be explored by ourselves and other groups. To that end, we initially genotyped 254 microsatellite markers in Asians, Hispanics, African Americans and Caucasians with sample sizes of approximately 22 for each group. These analyses identified 154 loci with sufficiently large differences between groups for use in analyses of African American patients. We designed primers and multiplex analyses for an additional 220 markers which have now been typed to fill gaps in the 10-centiMorgan MALD microsatellite map of the human genome. We have about 450 markers to choose from in MALD analyses depending on the racial/ethnic group being examined. Determination of approximately 75,000+ microsatellite genotypes for these MALD marker projects alone prompted us to search for more efficient and accurate genotyping strategies. The first efforts screening microsatellite markers were handled essentially eight or twelve tubes at a time. Our research and development efforts now allow laboratory assays to be performed in 96-well microtiter plates for high-throughput genotyping. Currently DNA samples are pre-aliquotted into 96-well plates with a pre-polymerase chain reaction (PCR) Hydra pipettor that pipettes all of the samples at once which are then dried down and stored for later analyses. A panel of 38 plates of HIV-1- infected and -exposed individuals has been developed for genotyping. This ?Mega? panel has been used by the most of the PIs in the LGD. PCR cocktails for a locus are later added with 12-channel pipettors. Subsequent microsatellite pooling for multiple analyses is performed with a post-PCR Hydra pipettor, and other genotyping assays (e.g., PCR- restriction fragment length polymorphisms, denaturing high performance liquid chromatography and 5?exonuclease) are handled with multichannel pipettors. Previously, setting up 500 PCR reactions took an hour or two. With these developments it is possible to set up 2000-3000 reactions in the same time. The reduced sample handling has resulted in fewer missing data points that were the result of pipetting errors made in the complex set-up of reactions. Development and implementation of new 384-well technology has been successful with proof in principle now in place so that throughput can be further increased, particularly for microsatellites where only a small fraction of the PCR sample is analyzed. The adoption of 96-well matrix-based microtiter plate technology has allowed us to address genetic questions that previously seemed virtually insurmountable and the 384-well technology holds tremendous promise. High Throughput Genotyping of HIV-1-infected and - exposed Individuals - admixture, Genetic epidemiology, African Americans, genetic map, genotyping technology, Hispanics, - Human Tissues, Fluids, Cells, etc.
Showing the most recent 10 out of 13 publications
