Pascal Arp, André Uitterlinden
Genetic Laboratory, Dept Internal Medicine, Erasmus MC
Email: p.arp @ erasmusmc.nl, a.g.uitterlinden @ erasmusmc.nl
The main focus of our laboratory is genetic epidemiology of complex diseases, in particular the locomoter diseases osteoporosis and osteoarthritis. For this purpose we have many study populations available involving thousands of DNA samples of study participants in which DNA polymorphisms have to be determined. To quickly genotype these DNA samples for analysis of interesting SNPs we have generated a medium/high throughput (HT) genotyping set-up using a pipetting robot consisting of a Caliper/Zymark Sciclone AHL3000 and Twister II configuration. Sofar, we have been mostly focussed on candidate gene studies and optimizing systems to analyse of 1-40 SNPs in 400-30.000 DNA samples and on using as little DNA as possible per SNP at low cost.
To actually come to the step of HT genotyping, reformatting steps of the DNA samples are necessary (“DNA management”). We first transfer DNA samples from Eppendorf tubes to 96 deep well (DW) plates or 2D Matrix tubes in 96 well format, and normalizing the concentration to 50 ng/ul using an integrated Tecan Genios reader (96- and 384-well based). The samples of four 96 DW plates are transferred to a 384 DW plate and diluted to a concentration of 1 ng/ul. Subsequently, 384 well PCR plates with 1 or 2 ng of DNA are generated which are dried in a oven (500C) and stored untill further use. We include controls to check for sample swaps and plating errors.
For genotyping we most commonly use the Taqman allelic discrimination assay from Applied Biosystems. To the 384 well PCR plates 1,5 ul of Taqman mixture is added with a Deerac Equator NS808 liquid handler which can fill a 384 well plate in <3 seconds. The endpoint analysis of the Taqman results is done manually on the 7900 HT ABI Taqman machine.
We offer services to groups in- and outside ErasmusMC and have handled 50.000 DNA samples and produced 1,5 million genotypes over the last years. We will discuss the set-up and our experience with running this facility.