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