Analysis of whole genomes for mapping by admixture linkage disequilibium (MALD) and candidate genes requires an appropriate set of markers and the ability to accurately genotype hundreds of markers with hundreds of patients. 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 with sufficiently large differences between markers for use in analyses of African American patients. In FY 1997, we designed primers and multiplex analyses for an additional 220 markers to fill gaps in the 10-centiMorgan MALD microsatellite map of the human genome. Analyses of this second set of markers is ongoing with about 130 of them generating clean enough products in our first pass screening of approximately 45 individuals of Asian, Hispanic, African American and Caucasian descent. Determination of approximately 50,000 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, dried down and stored for later analyses. A panel of 37 plates of HIV-1-infected and -exposed individuals were developed for genotyping. 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, single strand conformation polymorphism and heteroduplex analysis) 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. Throughput will be further increased, particularly for microsatellites where only a small fraction of the PCR sample is analyzed, by an investment in 384-well technology that is currently coming to market. The adoption of 96-well matrix-based microtiter plate technology has allowed us address genetic questions that previously seemed virtually insurmountable.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010270-01
Application #
6161160
Study Section
Special Emphasis Panel (LGD)
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
1997
Total Cost
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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