Analysis of whole genomes using mapping by admixture linkage disequilibrium (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 MALD in other admixed groups like Hispanics will also be explored by ourselves and other groups. To that end, we collected data for 3,669 high difference single nucleotide polymorphisms (SNPs) from Asians, Amerindians, Africans, African Americans and European Americans. These analyses identified 2,148 loci with sufficiently large differences between groups for use in analyses of African American patients. Determination of approximately hundreds of thousands of genotypes for these MALD marker and HIV-1/AIDS projects prompted us to search for more efficient and accurate genotyping strategies. Our research and development efforts now allow laboratory assays to be performed in 384-well microtiter plates for high-throughput genotyping. Currently DNA samples are pre-aliquoted into 384-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 14 plates of HIV-1-infected and -exposed individuals has been developed for genotyping, replacing the older 96-well format 42-plate """"""""Mega"""""""" panel. Our efforts using 384-well format has helped with the transition of most of the PIs in the Laboratory of Genomic Diversity (LGD) as they have been adopting 384-well technology. Genotyping with the 384-well panels has been largely automated. A Genetix automated pipettor can precisely add a few microliters of water to each of 384 wells in seconds. Then PCR cocktails and enzymatic genotyping reagents are added with 16-channel pipettors from Matrix. Total volumes have been reduced to 5 ul and DNA is used in quantities as low as 0.7 nanograms. These lower amounts are reducing the cost of genotyping reagents such that the increase in going from 96- to 384-well conserves DNA with little additional cost. Previously, setting up a few thousand PCR reactions took an hour or two. With these developments in automation and scale reduction it is possible to set up and analyze thousands of reactions at the same time. Always on the lookout for bottlenecks, the current one is now analysis of polymorphisms as they are determined, and subsequent epidemiological analyses. 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. In the MALD map project, our error rate was low with 61 discrepancies out of 12,447 (0.49%) replicate and independent determinations. Development and implementation of new 1532-well technology which holds tremendous promise will be the future of these efforts, but that equipment and plastic ware is still in its infancy. The implementation of 384-well matrix-based microtiter plate technology is allowing us to address genetic questions that previously required heroic handling of many plates and detailed sample tracking.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010270-07
Application #
6950962
Study Section
(LGD)
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2003
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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