A brief History of PCR Dr. Richard Molenkamp Medical Molecular - - PowerPoint PPT Presentation

▶
a brief history of pcr
SMART_READER_LITE
LIVE PREVIEW

A brief History of PCR Dr. Richard Molenkamp Medical Molecular - - PowerPoint PPT Presentation

Oct 21, 2023 686 likes •1.2k views

A brief History of PCR Dr. Richard Molenkamp Medical Molecular Microbiologist Structure of DNA Visualisation of DNA I Staining Hybridisation Direct Visualisation of DNA II Small amounts of signal: Need amplification PCR: Polymerase Chain

slide-1
SLIDE 1

A brief History of PCR

  • Dr. Richard Molenkamp

Medical Molecular Microbiologist

slide-2
SLIDE 2

Structure of DNA

slide-3
SLIDE 3

Visualisation of DNA I

Direct Staining Hybridisation

slide-4
SLIDE 4

Visualisation of DNA II

Small amounts of signal: Need amplification PCR: Polymerase Chain Reaction

slide-5
SLIDE 5

Principle of PCR

Conventional thermocycle profile:

slide-6
SLIDE 6

Exponential amplification

Number of cycli on an agarose gel: 10 15 20 25 30 35 40

slide-7
SLIDE 7

Early PCR

Addition of polymerase in each cycle Breakthrough: Thermostable (Taq) polymerase Integrated Thermocyclers

slide-8
SLIDE 8

Conventional PCR = End point analysis no quantitative data Real-time PCR

slide-9
SLIDE 9

Sybrgreen reactions - Intercalating fluorescence

DNA Target Sequence Denaturation

Drawback: a-specifc product also fluoresent!

slide-10
SLIDE 10

Sample block

Lens Lens

Spectrograph

CCD camera LASER Dichrome Mirror

MUX

slide-11
SLIDE 11

Amplification curves

slide-12
SLIDE 12

Sybrgreen reactions – melting curve analysis

slide-13
SLIDE 13

Detection Formats (I) Probe-based

  • Taqman technology: specific double-dyed fluorescent hydrolysis probes
  • FRET (Fluorescence Resonance Energy Transfer): hybridisation probes
  • Partially double stranded, single-dyed, hybridisation probes

R Q R Q – Long target-specific probe w – – – – –

slide-14
SLIDE 14

Excitation / emission of fluorophores

Fluorescence

TAMRA quenching Deep Dark/Black hole quenchers

slide-15
SLIDE 15

5’ Nuclease Assay / Taqman assay

F = reporter  FAM, VIC, Hex… Q = quencher  Black hole quencher (BHQ)….

slide-16
SLIDE 16

Hybridization probes or FRET probes

slide-17
SLIDE 17

Emission Emission Excitation Excitation R Q R Q R – Long target-specific probe with fluor – Short quencher probe – Fluorescence quenched when probes are hybridized – Long probe preferentially binds target – Short quencher probe is dissociated – Fluorescence is detected

Partially double stranded linear DNA probes

slide-18
SLIDE 18

Probe free: MultiCode technology (I)

MultiCode Base Pair (isoC:isoG)

Scott C etal, Nucl. Ac. Res. 2004

Iso G Iso C

slide-19
SLIDE 19

MultiCode PCR (II)

slide-20
SLIDE 20

MultiCode technology

slide-21
SLIDE 21

Ct / Cp / Cq Calling

slide-22
SLIDE 22

PCR positivity measured at Cycle threshold (Ct)-level Number of cycles

10 20 30

Fluorescence

10x SD background Threshold Cycle Background fluorescence

Ct calling I

slide-23
SLIDE 23

PCR positivity measured at Cycle threshold (Ct)-level Number of cycles

10 20 30

Fluorescence

10x SD background Threshold Cycle Background fluorescence

Ct calling II

slide-24
SLIDE 24

PCR positivity measured 2nd derivative max. Number of cycles

10 20 30

Fluorescence

Crossing point Background fluorescence Calculates 2nd derivative and determines its maximum.  CP: Where the rate of increase of fluorescence is greatest

Cp calling I

slide-25
SLIDE 25

PCR positivity measured 2nd derivative max. Number of cycles

10 20 30

Fluorescence

Cp calling II

slide-26
SLIDE 26

Quantification

slide-27
SLIDE 27

Quantification with real-time PCR

slide-28
SLIDE 28

Principles of quantification – real time PCR

Calculation of efficiency

slide-29
SLIDE 29

Reverse transcription PCR

slide-30
SLIDE 30

Reverse transcription PCR

Mix of RT and PCR enzyme: M-MLV / Taqgold Enzyme with RT and DNA pol activity rTth

slide-31
SLIDE 31

Sequence variation Influences design of PCR PCR can be used to detect variation

slide-32
SLIDE 32

HIV-1 group M sequence variation in Gag and Pol genes

slide-33
SLIDE 33

Influence of mismatches on hybridization temperature

tgggaggttctctccagcactagcagg Length 27 nt GC content 60% Tm 69 ºC tgggaggttctctccagcactagcagg a t Tm 62.6 ºC tgggaggttctctccagcactagcagg a t a Tm 57.8 ºC

slide-34
SLIDE 34

How to deal with sequence variation

  • Degenerate oligos  GGTAYCCATGRTCAG
  • Dual target assay
  • Dual probe assay

IUB codes R = A or G Y = C or T

slide-35
SLIDE 35

Dual target real time PCR

LTR

Integrase

Genome HIV 5’-

  • 3’

If there is a mutation in either of the primer/probe sites  the other PCR will ‘take over‘

slide-36
SLIDE 36

Realtime PCR for detection of single mutations

  • Conventional Sanger sequencing: ~25%
  • Sensitive methods:
  • LIPA/DNA microarray (hybridisation) 5-10%
  • Allele-specific PCR ~5%
  • Next generation sequencing (0.5% ?)
  • Quantitative real-time techniques: 1-10%
  • LNA/MGB probes (short high affinity probes)
  • Digital PCR
slide-37
SLIDE 37

Probes used for detection of single mutations (I)

Minor groove binding probes Locked nucleic acid probes

  • Due to higher affinity binding shorter probes can be defined
  • Taqman probes are 22-30 nt long; LNA/MGB probes 8-20 nt long
slide-38
SLIDE 38

Hybridization temperature: effect in MGB and LNA probes

GGAGG(+T)T(+C)TCT(+C)CAG(+C)A

Length 17 nt Tm 69 ºC

GGAGG(+T)T(+C)TCT(+C)CAG(+C)A A

Tm 59 ºC

tgggaggttctctccagcactagcagg

Length 27 nt Tm 69 ºC

tgggaggttctctccagcactagcagg a t

Tm 62.6 ºC

tgggaggttctctccagcactagcagg a t a

Tm 57.8 ºC

slide-39
SLIDE 39

Detection of oseltamivir resistant influenza A/H1N1 H274Y by real-time discrimination PCR using LNA probes

NTC + control + control NTC + control + control

Wild-type cluster Mutant cluster

NA: 5’atcgaaaagggaaaggttactaaatcaatagagttaaatgcacccaattttCattatgaggaatgttcctgttacccagacactggc 3’ N1274Yfpr1 (30bp) N1274Yrpr1 (24bp) LNA:H274Y T (mut) LNA:H274H (16bp)

slide-40
SLIDE 40

Detection of lamivudine resistance in HBV

Pas et al., Journal of Clinical Virology 32 (2005) 166–172

slide-41
SLIDE 41

Effect of (enzyme) mastermix

  • n mismatch tolerance
slide-42
SLIDE 42

Influence of mastermix on primer bindingsite mismatch tolerance

> 50 different mutants

Stadhouders et al., J. Mol. Diag., 2010

slide-43
SLIDE 43

Primer bindingsite mismatch tolerance

slide-44
SLIDE 44

Influence of mastermix on primer bindingsite mismatch tolerance

MMLV / Taqgold combination (ABI): RT @ 48°C

slide-45
SLIDE 45

Influence of mastermix on primer bindingsite mismatch tolerance

rTth based mastermix: RT@60°C

slide-46
SLIDE 46

Digital PCR

Quant studio

Raindance technologies Biorad QX200 droplet PCR Fluidigm

slide-47
SLIDE 47

Principle of digital PCR (dPCR)

Limited dilution

0.368 1 0.368 2 0.184 3 0.061 4 0.015 5 0.003 >5 0.001

Poisson distribution

slide-48
SLIDE 48
  • 1. Manual dilution
  • 2. Droplet PCR (ddPCR, Biorad)
  • 3. Lab on a chip (Lifetechnologies)
  • 4. Microfluidics (Fluidigm)

1 2 3 4

slide-49
SLIDE 49

Applications of dPCR

  • Rare sequence / mutation detection (oncology, virology drug resistance)
  • Copy number quantification (standardisation controls)
  • Low level pathogen detection (in difficult samples)
  • Gene-expression (absolute quant. of (un)stimulated) gene expression)
slide-50
SLIDE 50

Questions ?