1. Exceptional healthcare, personally delivered MLPA Automation Gemma Dennis GT Training day – Newcastle 23/11/11

2. Exceptional healthcare, personally delivered MLPA @ Bristol CMT (Charcot-Marie Tooth) Telomeres FH (Familial Hypercholesterolaemia) SMA (Spinal Muscular Atrophy) ARS (Axenfeld-Rieger Syndrome) TAR (Thrombocytopenia-Absent Radius syndrome)

3. Exceptional healthcare, personally delivered Why Automate? Solid Tissue screening by MLPA was introduced in July 2010, resulting in a large increase in sample numbers Set-ups became complex and time consuming Result quality was reduced due to the large run size Automation = increased samples per set up, less staff time = cost saving.

4. Exceptional healthcare, personally delivered MLPA Automation Centralised booking system All requests on one sheet – 96 well plate format All information needed for set up

5. Probe Mastermixes Water Reservoir DNA Rack 1 (4 x 6) DNA Dilution PlateHybridisation Plate 1. Transfer Water (1-29ul) 2. Transfer DNA (1ul >) 3. Transfer normalised DNA (5ul) 5. Transfer Probemix (1.5-3ul) Thermocycler 4. Denature DNA on Thermocycler 6. Overnight hybridisation on thermocycler Sample No MLPA Name PCR No DNA Conc (reaction) DNA Dilution Volume DNA Volume (dilution) Water Volume (dilution) DNA Volume (reaction) Probe Mix Volume Ligase Mix Volume Ligation vol to PCR PCR Mix Volume M.11.05592p300ARS15350201.8218.1851.516520 M.11.05708p300ARS15350204.3515.6551.516520

6. PCR Plate Reagent Rack Hybridisation Plate Thermocycler 8. Ligation Rx on Thermocycler Thermocycler 10. PCR Rx on Thermocycler 11. Load on Analyser 7. Transfer Ligase Mix (16-32ul) 8. Transfer PCR Mastermix (20ul) 9. Transfer Ligated MLPA Product (5ul) Sample No MLPA Name PCR No DNA Conc (reaction) DNA Dilution Volume DNA Volume (dilution) Water Volume (dilution) DNA Volume (reaction) Probe Mix Volume Ligase Mix Volume Ligation vol to PCR PCR Mix Volume M.11.0559 2p300ARS15350201.8218.1851.516520 M.11.0570 8p300ARS15350204.3515.6551.516520

7. Exceptional healthcare, personally delivered MLPA Automation – Validation work Initial development work based on existing Biomek workflows Liquid handling was tested using coloured dyes (volume range 1.5-32µl) Single kit was run to asses if adding probe mix, ligase mix and/or PCR mix at room temperature had any adverse affects Large scale test was set up simultaneously with manual set up

8. Exceptional healthcare, personally delivered MLPA Automation – Observed results Robot was able to pipette all volume sizes accurately There were no adverse affects to all mixes being added at room temperature All results from automated set up were comparable to those from manual set up

9. Exceptional healthcare, personally delivered MLPA Automation - Conclusions Automation allows multiple MLPA tests to be set up simultaneously using a single plate Technical work is simplified and can be set up in less time System has ability for further tests to be added to the workflow without additional staff time being required

10. Exceptional healthcare, personally delivered Any Questions? Acknowledgments Kenneth Smith Mark Greenslade Laura Yarram