1. FilmArray: Automated PCR

2. Genetics In the Real World
How are genetics used in real world applications?
What can an undergrad student do with knowledge gained from Biology?

3. PCR: A Quick Review
Polymerase Chain reaction:
Quiz: what do you know?

4. Components of PCR 5’ 3’ TCCACAGGCGCTATCTGCT AT C
Free nucleotides C C G A T
Taq polymerase T
Primer 5’ 3’
TCCACAGGCGCTATCTGCT AT C G
AGGTGTCCGCGATAGACGATAGCGAGTAGCAGAGGTTTAGA 3’ 5’
Template DNA
Buffers

5. PCR Process -Heat denatures template strand
-Forward and reverse primers are annealed to single stranded DNA
-Taq polymerizes dNTP’s to elongate the replicated strand.

6. PCR Amplification Original DNA Copy
5 cycles of PCR amplify 1 copy into 32………and so on

7. Instruments for Viewing PCR Results
Gel Electrophoresis and Camera image of agarose gel

8. PCR in the Classroom
How long does PCR take?

9. Improving the Process: PCR today
New Concepts
Biotechnology industry utilizes many new improvements in conducting and analyzing the PCR process

10. Fluorescent DNA: DNA Binding Molecules
GCAATCGTGTCATGTCTG =
CGTTAGCACAGTACAGAC +
GCAATCGTGTCATGTCTG
CGTTAGCACAGTACAGAC
Fluorescent molecules that bind to double stranded DNA help make it visible. Works like ethidium bromide in gel electrophoresis

11. Camera Records a Fluorescent Image Every Cycle
Visible Realm
Greater Than 10 Billion Copies
Number of DNA Copies
PCR Cycle

12. Uses a camera and Software to plot fluorescence during PCR
Real-Time PCR
Uses a camera and Software to plot fluorescence during PCR

13. Nested PCR Nested reaction includes: 1. outer PCR (PCR1) 2. dilution
3. inner PCR (PCR2)
*allows for more specific amplification of selected organisms

14. Nested RT-PCR Primer Designs
Virus Genome
Outer RT-PCR
200bp
Inner PCR
90bp

15. Multiplex Assays Uses multiple primers in one reaction to
amplify several different DNA templates
present in a sample
i.e.: clinical sample run on a panel of 20
organisms to determine presence of
infection

16. Schematic of Nested Multiplex PCR
Primary RT-PCR
Secondary PCR 1F 1R 2F 2R 3F 3R 4F 4R 5F 5R 6F 6R
Dilute 100 fold

17. Primer Design
Primer of 17 base pairs has 1 in 10 billion chance of laying down on human genome. Design never goes below 17 bp (usually 18-20bp)
AGGCGCTATCTGCT ATC 5’ 3’
AGGTGTCCGCGATAGACGATAGCGAGTAGCAGAGGTTTAGA 3’ 5

18. Our Project: FilmArray

19. Collaboration Chemists: PCR reactions occurring in the pouch
Engineers: Pouch development and Instrument development
Software: Communication from instrument to computer, analysis of data
Film Array instrument allows for automated PCR with results in approximately 1 hour

20. FilmArray Pouch Pouch substitutes pipettes and tubes for mixing
Cell
Lysis
PCR1
PCR2
Mag
Bead
Capture
Dilute
Wash
FilmArray Pouch
Pouch substitutes pipettes and tubes for mixing
substitutes chemist on a bench-top
FLASH ANIMATION

21. FilmArray Beta Prototype
Substitutes mechanical actions of chemist and bench-top instruments (bead whacker=vortex, mag. beads and blisters=filter tubes and centrifuge, peltier=thermo block, array=DNA detection

22. Run Protocol DNA Melt Green indicates camera acquisitions:
2nd PCR
1st PCR
Green indicates camera acquisitions:
Once per PCR2 cycle
2500 in the 5 min melt
There are two Peltier Thermocyclers
The software displays the temperature during each PCR and the melt

23. Example of Results Software makes call, positive or negative result
PCR1 Control PCR2 Control Sc DNA assay Sc RNA assay
Sp DNA Assay
Sp RNA Assay
2nd PCR
Post PCR Melt
Software makes call, positive or negative result

24. Film Array Utilizes Faster Process
Sample utilization: 120 1uL reactions = 1/10 price
Pre amplification (PCR 1= enrichment) amplifies enough to cover entire array every well
Some micro-array processes have difficulty having enough sample for every well

25. Risks of PCR Contamination is a HUGE risk factor
Nesting not to popular because of how easily you can contaminate your assays
False positives look the same as true positives
The pouch is an all enclosed environment that eliminates the risk of contamination

26. Reagent Lyophilization
All reagents in the pouch or lyophilized (freeze dried) so all that is needed is water
Freeze Dried reagents can have a shelf life of approximately 1 year
Wet Bench Top reagents have short shelf life
Proteins are protected with “cake” so they don’t die

27. Film Array Applications: Projects Under Development
Respiratory Panel: Clinical Samples
Febrile Infant Risk Stratification Tool (FIRST): bacterial identification for infant fever
Biothreat Pouch:Department of Defense
Methicillin Resistant Staph. aureus detection:characterizing outbreaks of Staph infection in the hospital and community
Tuberculosis Screening

28. Respiratory Panel
Resp. Panel screens samples for 16 viruses or bacteria in 1 hour

29. Micro-array: Automated Pipetting

30. Spots Primers in specific layout

31. Kody (BioChemistry Intern)
Pouch Production
Freeze Dry Reagents
Reagent QC
Primer Validation and Tracking
Assay Optimization
Pouch/Protocol Optimization
Build and Update Database

32. Meghan (Research Associate I)
NanoPlotter validation and QC
Film Array instrument optimization
Assist with pouch production

33. Idaho Technology Broad range of projects
Great experience, resume builder
Offers career opportunities, internships, and benefits
Visit:
Join the ITI Team
                                                    

34. Questions?