Setting up a PCR Facility Molecular Workshop

What is required?  Built environment of a Molecular facility is defined in

the NPAAC Molecular standards  Generally at least three functionally differentiated separate and controlled lab environments Clean/Mix Room DNA/RNA Extraction Room Amplification Room

 Lab design regulations primary focus is:

Preventing/minimising carry-back contamination from amplification

NPAAC Standard

What is the risk?  Amplified DNA represents the target region and will cause false

positive results in that assay if they make it back into front end processes  Is it really that bad?  40 cycles of PCR copies 1 molecule into 1 Trillion!

 How do amplicons move?  Hands, Shoes, Paper, Racks, Pens, Lab coats….

 Places found positive during contamination clean up:  Door handles, Fridge Freezer handles, centrifuges, buffers, pipettes,

Biohaz cabinet switches, racks, phones, mobile phones, lab surfaces, computer mice/keyboards, power and light switches

Contamination Prevention Contained lab spaces with directed airflow  Closed tube assays where possible  Rigorous adherence of lab procedure by operators 

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Lab coats – separate in each room Washing of hands prior to leaving rooms/touching door handles, Hand sensing taps Unidirectional workflow Unidirectional transition of racks with decontamination DNA destroying bench cleaning solutions (e.g. Bleach, DNAErase) Appropriate cleaning of amplicon spills if performing open tube manipulations Use of barrier pipette tips Use of IT solutions for data and procedures in preference to paperwork Appropriate discard of amplified PCR plates

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Appropriate lab cleaning regimen – clearly informing cleaners of requirements

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Use of Uracil bases in control materials and dNTPs of mastermixes

Example Lab Designs

Extraction Room

Clean Room/Mix

Amplification/Post Amplification

Red arrows indicate air flow

Example Lab Designs

Clean Room/Mix +30pa

Example Lab Designs

Clean Room/Mix +30Pa

Extraction Room +15Pa

Example Lab Designs

Clean Room/Mix +30Pa

Amplification/Post Amplification -30Pa

Extraction Room +15Pa

Example Lab Designs

Clean Room – PCR Reagents

Typical Clean Room  Highly positive pressure – air out  Primers, Probes, Mastermix reagents  -20C, -70C, refrigerator  Tube rollers for reconstituting primers/probes

 Centrifuges, vortexes  Stock and clean racks (can be moved to any other room)

 BSC/PCR Cabinets – to protect products  Dedicated cleaning equipment

DNA/RNA Extraction

Typical Extraction Room  Positive pressure – air out – not as high as clean room  BSC-2 Cabinets – product and operator  Automated extraction robotics

 Liquid handler robotics  Freezers -20,-70, Refrigeration  Centrifuges, Vortexes

 Spectrophotometer  Heating blocks/thermo devices

Amplification/Post-Amplification

Typical Amp/Post Amplification Room  High negative pressure – air IN and vented appropriately,    

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with redundancy if possible Cyclers, Real-time cyclers Containment area for open tube manipulation/nested PCR Electrophoresis and blotting apparatus, Electronic image capturing Pyrosequencers, Sanger sequencers, Next Gen sequencers Rack wash-up ‘system/process’ Dedicated cleaning equipment

Tricky question emerging  All-in-one extraction to result instruments  What room?  Standard:

 If you have the room – put integrated equipment in post-amp, and

cycle racks through decontamination process back to patient sample loading in extraction room

Future planning – new facilities  High throughput molecular equipment in core lab areas

and/or on track of core lab  Inert gas feeds (Helium) – low volume liquid handling (nanolitre/picolitre)  Acoustic droplet ejection picolitre dispensers

 Humidity control, dust filtration