Enhancing radiation hybrid mapping through whole genome amplification.
Radiation hybrid (RH) mapping is limited by the inherent genomic instability of RH clones entailing both, limited DNA sample amounts and genomic heterogeneity of the clones. Here the instability of RH clones is quantified and the suitability of the multiple strand displacement whole genome amplification method (WGA) for radiation hybrid mapping is assessed. To quantify the instability of RH clones, eleven clones of a 10,000(Rad) rhesus macaque radiation hybrid panel were passaged ten times and analyzed by interspersed repeat sequence specific quantitative PCR and by genotyping of 46 macaque chromosome 5 STS markers. The quantitative PCR data indicate an average loss of 55% of the donor DNA over 10 passages. Over the same period, a dropout of 46.2% of the STS markers was observed. These data indicate a genome wide half-life of the donor DNA of 8.7 passages and of 10.6 passages for the chromosome 5 markers. The genotyping data of the genomic RH DNA were compared to three sets of WGA experiments: 1) single wgaDNA amplifications, 2) six WGA replicates, and 3) re-amplification of wga DNA. The assays demonstrated concordance rates of 97.6%, 98% and 99.3%, respectively, and indicated the marker specificity of some repeated WGA dropouts. The study confirms that WGA is suitable for RH mapping studies should enable the accurate analysis of almost an infinite numbers of markers. WGA will allow the analysis of earliest RH clone passages, thus limiting their heterogeneity and RH mapping artifacts.
Karere
Genesio M
GM
Department of Population Health and Reproduction, School of Veterinary Medicine, California National Primate Research Center, University of California - Davis, Davis, California, USA.
Lyons
Leslie A
LA
Froenicke
Lutz
L
D000818:Animals; D015894:Genome, Human; D006801:Humans; D006822:Hybrid Cells / Q000528:radiation effects*; D008253:Macaca mulatta; D016133:Polymerase Chain Reaction; D011839:Radiation, Ionizing