Exceptional healthcare, personally delivered
MLPA Automation Gemma Dennis
GT Training day – Newcastle23/11/11
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)
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.
Exceptional healthcare, personally delivered
MLPA Automation
• Centralised booking system
• All requests on one sheet – 96 well plate format
• All information needed for set up
Probe MastermixesWater Reservoir
DNA Rack 1 (4 x 6)
DNA Dilution Plate Hybridisation Plate1. 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 NoMLPA 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.11.05592 p300ARS 153 50 20 1.82 18.18 5 1.5 16 5 20
M.11.05708 p300ARS 153 50 20 4.35 15.65 5 1.5 16 5 20
PCR Plate
Reagent Rack
Hybridisation PlateThermocycler
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 NoMLPA 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.11.05592 p300ARS 153 50 20 1.82 18.18 5 1.5 16 5 20
M.11.05708 p300ARS 153 50 20 4.35 15.65 5 1.5 16 5 20
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
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
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
Exceptional healthcare, personally delivered
Any Questions?
AcknowledgmentsKenneth Smith
Mark Greenslade
Laura Yarram
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