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Designing multiplex real-time PCR for disease diagnosis
Dr. Nurulhasanah OthmanProf Dr. Rahmah Noordin
Institute for Research in Molecular Medicine (INFORMM)
Universiti Sains Malaysia
28th National Science ConferenceBayview Hotel, Penang
16-17th August 2017
Outline
• PCR and history• Real-time PCR features• Designing multiplex real-time PCR• DNA extraction• Troubleshooting• Examples
PCR• Polymerase
chain reaction-Kary B. Mullis (1985)
• A method to copy and synthesize target sequences in a large amount.
https://www.ted.com/speakers/kary_mullis
• 2 most important technologies-a) Taq polymerase enzyme b) Thermal cycler machine.
• 1987, Perkin Elmer developed the first automated thermal cycler machine.
• Regulate the temperature of a reaction, heating or cooling the samples as needed
Real-time PCR• The word “real-time” indicates it can monitor the
amplification while its running.
• 1993- First real-time experiment conducted using EtBrfor quantization and detection by CCD camera.
• 1996- Introduction of TaqMan assay to replace EtBr.
• 1996-1997-The first real-time PCR machine was developed by Applied Biosystem (ABI)
Conventional PCR- Tell us “what”
Real-time PCRTell us “how much ”
Source: Bio-Rad
Real-time PCRConventional PCR
Ct value
Conventional VS Real-time
• It does not require post-PCR analysis ( No electrophoresis)
• Shorten time to obtain result
• Less contamination
• Less complexity for DNA quantitation analysis
Analysis
• Qualitative (presence or absence)• Quantitative (measure the DNA copies no)
Chemistry
• SYBR Green dye, Eva Green…• Hybridization probe: TaqMan Probe
Assay Category
• Real-time Assay (Gene Expression)a) 1 step RT-PCR -RNA b) 2 steps RT-PCR-RNAc) DNA quantification
• Endpoint Assay a) Allelic discrimination b) Plus/Minus
Designing multiplex real-time PCR
1) Model of instrumentHow many detection channels available?1, 2, 3 ??????
Example:1) Develop multiplex to detect 4 different targets2) Choose 4 different fluorophore colours (Depend on model of the instrument)3) ABI- FAM (Green)
VIC (Yellow)NED (Orange)CY5 (Red)
ProbeHydrolysis Probe
Source: ABI
• Taq Man MGB
2. How to design primer and probe?
• Primer 3 –free software• Oligos
IMPORTANT: All primers and probes have almost similar Tm
3. PCR Mastermix
• Choose conventional or ready-made mastermix???
• Total volume use??50, 25, 20, 15 or 10 µl?
• Optimize concentrations ofPrimer and probe
Source: Applied Biosystem
Thermal profile
• 2-steps PCR
• 3-steps PCR
Denaturing Denaturing Annealing/Extension
95°C for 10 min 95°C for 15 sec 60°Cfor 1 min
40-55 cycles
Denaturing Denaturing Annealing Extension
95°C for 10 min 95°C for 15 sec 58°C for 15 sec 72°C for 15 sec
40-55 cycles
• For all optimization steps, multiplexing, background test and testing panel:
1) Pure gDNA from target organisms
2) Plasmid containing target sequences(Cloning PCR product)
PositiveControl
4. Conventional PCR
• Primer working??
4. Primer concentration
100 80 60 40 20 nM
Primer concentration (nM) Mean Ct value
100 25
80 25
60 25
40 27
20 30
Example:
5. Multiplexing and background test
• Test with……
1.single template2. combination of 2 templates3. combination of 3 templates4. combination of 4/all templates….
Ct value showed a little changes in single and combination reactionsExample:Single Ct: 26.79
Combine (2) Ct: 27.1Combine (3) Ct: 27.4…….
6. LoD curve
Su et al., 2009
LoD Ct: 36
LoD Ct: 33
R square > 0.9E efficiency: 80-100%
7. Testing Panel
• Use gDNA from clinical specimens that are expected containing your target diseases
1) Body fluids: Blood, saliva, urine, CNS, tears,sweat..
2) Stools samples3) Tissues4) Buccal swab
8. DNA Extraction
• Conventional method??• Commercial kit???• Automated + commercial kit????
Things to ponder
• Need to remove inhibitor??Yes!!!! If stool samples
• Nature of target organismHave hard shell- egg of wormYes!!!! Must do pre-treatment before extraction (Exmp: Use of mechanical disruption)
Troubleshooting
Sigmaaldrich
Sigmaaldrich
Sigmaaldrich
Application for disease diagnosis
Acknowledgement• INFORMM,USM
Prof Rahmah Noordin Pn Maimunah AhmedPn Noorizan MiswanCik Madihah Basuni
• PPSP,USMProf Zeehaida Mohammed
• PPSK,USMAP. Dr. Lim Boon Huat
• Kementerian Kesihatan MalaysiaDr. Jamail Muhi
• Leiden University Medical CentreDr. Jaco J. Verweij
• National University of MexicoDr. Alfonso Olivos Garcia
Funding:MOHE: FRGS grantEU grant
Hands-on course on
Multiplex Real-Time PCRFrom primer and probe design to data analysis15 – 16 November 2017 | USM Main Campus, Penang
Thank you