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MOBILE CORONA DIAGNOSTIC LAB
THE PRINCIPLES OF
PCR, RT-QPCR AND RT-LAMP
ABOUT MYSELF
July 8th, 2021
MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management slide 2
M.Sc. in biochemistry (Technical University Munich)
PhD in Experimental Medicine (Helmholtz Zentrum
Munich)
Susanne Pettinger, M.Sc.
contact: susannepettinger@gmail.com
MY EXPERTISE
• Cell culture | microbiological cultures
• Molecular biology | biochemistry | different assays
• Protein expression and purification | protein engineering
• Electron microscopy | fluorescent imaging
Block 1:The PCR method
• How does PCR work?
• Reverse transcription
Block 2: RT-qPCR
• Why qPCR?
• Real-time detection methods
• RT-qPCR in the Mobile Lab
BREAK
Block 3: RT-LAMP
• Introduction
• How does LAMP work?
o LAMP primers
o The LAMP reaction cycle
• Detecting LAMP products
• RT-LAMP in the Mobile Lab
July 8th, 2021
MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management
SCHEDULE
BLOCK 1:THE PCR METHOD
PCR – A GAME CHANGER FOR SCIENCES
July 8th, 2021
MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management
• Polymerase Chain Reaction allows the amplification of DNA from a scarce sample
• The invention of PCR had a huge impact on
o Research: cloning, sequencing, measurement of gene expression, ….
o Medicine: identification of pathogens, typing tissue and blood, diagnosis of genetic defects
o Forensics: DNA comparison of blood or hair samples, parentage testing
o Paleontology: amplify and sequence DNA from trace amounts in fossils millions of years old
THE DISCOVERY OF PCR – A PARADIGM SHIFT IN THREE NOBEL PRIZES
July 8th, 2021
MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management
HOW DOES PCR WORK?
July 8th, 2021
MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management
• DNA template
• DNA polymerase
• complementary primers
• dNTPs
• Magnesium (Mg2+)
• buffer
REVERSE TRANSCRIPTION: EXPANDING PCR TO THE RNAWORLD
July 8th, 2021
MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management
• DNA polymerases can only bind double-stranded DNA → RNA templates have to be transcribed to cDNA
• Reverse transcriptase: enzyme originating in retroviruses

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2 - PCR.pdf

  • 1. MOBILE CORONA DIAGNOSTIC LAB THE PRINCIPLES OF PCR, RT-QPCR AND RT-LAMP
  • 2. ABOUT MYSELF July 8th, 2021 MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management slide 2 M.Sc. in biochemistry (Technical University Munich) PhD in Experimental Medicine (Helmholtz Zentrum Munich) Susanne Pettinger, M.Sc. contact: susannepettinger@gmail.com MY EXPERTISE • Cell culture | microbiological cultures • Molecular biology | biochemistry | different assays • Protein expression and purification | protein engineering • Electron microscopy | fluorescent imaging
  • 3. Block 1:The PCR method • How does PCR work? • Reverse transcription Block 2: RT-qPCR • Why qPCR? • Real-time detection methods • RT-qPCR in the Mobile Lab BREAK Block 3: RT-LAMP • Introduction • How does LAMP work? o LAMP primers o The LAMP reaction cycle • Detecting LAMP products • RT-LAMP in the Mobile Lab July 8th, 2021 MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management SCHEDULE
  • 5. PCR – A GAME CHANGER FOR SCIENCES July 8th, 2021 MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management • Polymerase Chain Reaction allows the amplification of DNA from a scarce sample • The invention of PCR had a huge impact on o Research: cloning, sequencing, measurement of gene expression, …. o Medicine: identification of pathogens, typing tissue and blood, diagnosis of genetic defects o Forensics: DNA comparison of blood or hair samples, parentage testing o Paleontology: amplify and sequence DNA from trace amounts in fossils millions of years old
  • 6. THE DISCOVERY OF PCR – A PARADIGM SHIFT IN THREE NOBEL PRIZES July 8th, 2021 MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management
  • 7. HOW DOES PCR WORK? July 8th, 2021 MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management • DNA template • DNA polymerase • complementary primers • dNTPs • Magnesium (Mg2+) • buffer
  • 8. REVERSE TRANSCRIPTION: EXPANDING PCR TO THE RNAWORLD July 8th, 2021 MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management • DNA polymerases can only bind double-stranded DNA → RNA templates have to be transcribed to cDNA • Reverse transcriptase: enzyme originating in retroviruses