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 or obtained data for 742 short tandem repeats (STRs) in Asians, Hispanics, African Americans and Caucasians. These analyses identified 315 loci with sufficiently large differences between groups for use in analyses of African American patients and 213 for Hispanics. 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. In the first efforts of screening STRs, samples were handled essentially 96 at a time. 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-aliquotted 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 13 plates of HIV-1-infected and -exposed individuals has been developed for genotyping replacing the older 96-well format 42-plate """"""""Mega"""""""" panel. The 384-well format """"""""Dense"""""""" panel will likely be used by the most of the PIs in the LGD once they adopt 384-well technology. Genotyping with the Dense panel has been largely automated. A Genetix automated pipettor can add a few microliters of water to each of 384 wells in seconds. Then PCR cocktails for a locus are added with a Packard Multiprobe II robot. Total volumes have been reduced to 7.5 ul and DNA can be used in quantities as low as a few nanograms. These lower volumes are reducing the cost of genotyping reagents such that the increase in going from 96- to 384-well is essentially in increased analysis time, which conserves DNA. Subsequent pooling for multiple analyses is performed with a post-PCR Hydra pipettor, and other genotyping assays (e.g., fluorescent single base extension and denaturing high performance liquid chromatography) are handled with multichannel pipettors and creative use of the pre-PCR multiprobe II and the post-PCR 384-well hydra. 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 thousands of reactions at the same time. Always on the lookout for bottlenecks, the current one is now analysis of STRs and single nucleotide polymorphisms on an automated sequencer. 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 1532-well technology which holds tremendous promise will be the future of these efforts, but that equipment and plasticware are in their infancy. The implementation of 384-well matrix-based microtiter plate technology is allowing us to address genetic questions that previously required heroic handling of lots of plates and detailed sample tracking.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010270-05
Application #
6559144
Study Section
(LGD)
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2001
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Zhang, Lin; Kao, W H Linda; Berthier-Schaad, Yvette et al. (2006) Haplotype of signal transducer and activator of transcription 3 gene predicts cardiovascular disease in dialysis patients. J Am Soc Nephrol 17:2285-92
Chretien, J-P; Coresh, J; Berthier-Schaad, Y et al. (2006) Three single-nucleotide polymorphisms in LPA account for most of the increase in lipoprotein(a) level elevation in African Americans compared with European Americans. J Med Genet 43:917-23
Liu, Yongmei; Berthier-Schaad, Yvette; Fallin, Margaret D et al. (2006) IL-6 haplotypes, inflammation, and risk for cardiovascular disease in a multiethnic dialysis cohort. J Am Soc Nephrol 17:863-70
Laud, K; Marian, C; Avril, M F et al. (2006) Comprehensive analysis of CDKN2A (p16INK4A/p14ARF) and CDKN2B genes in 53 melanoma index cases considered to be at heightened risk of melanoma. J Med Genet 43:39-47
Shrestha, Sadeep; Strathdee, Steffanie A; Galai, Noya et al. (2006) Behavioral risk exposure and host genetics of susceptibility to HIV-1 infection. J Infect Dis 193:16-26
Liu, Yongmei; Berthier-Schaad, Yvette; Fink, Nancy E et al. (2005) Beta-fibrinogen haplotypes and the risk for cardiovascular disease in a dialysis cohort. Am J Kidney Dis 46:78-85
Oleksyk, T K; Thio, C L; Truelove, A L et al. (2005) Single nucleotide polymorphisms and haplotypes in the IL10 region associated with HCV clearance. Genes Immun 6:347-57
Shrestha, Sadeep; Smith, Michael W; Beaty, Terri H et al. (2005) Theory and methodology for utilizing genes as biomarkers to determine potential biological mixtures. Ann Epidemiol 15:29-38
Smith, Michael W; O'Brien, Stephen J (2005) Mapping by admixture linkage disequilibrium: advances, limitations and guidelines. Nat Rev Genet 6:623-32
Oleksyk, T K; Goldfarb, L G; Sivtseva, T et al. (2004) Evaluating association and transmission of eight inflammatory genes with Viliuisk encephalomyelitis susceptibility. Eur J Immunogenet 31:121-8

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