releasing the promise of hrmliterature is sparse and controversial no standardisation of the method...
TRANSCRIPT
![Page 1: Releasing the promise of HRMLiterature is sparse and controversial No standardisation of the method Complex studies are hard to replicate. Custom designed HRM assays HRM reagents](https://reader030.vdocuments.site/reader030/viewer/2022011921/6033a75496494e4ac01c2e0c/html5/thumbnails/1.jpg)
Releasing the promise of HRM
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Custom designed real-time PCR assays
Real-time PCR reagents
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Custom designed HRM assays
HRM reagents
![Page 4: Releasing the promise of HRMLiterature is sparse and controversial No standardisation of the method Complex studies are hard to replicate. Custom designed HRM assays HRM reagents](https://reader030.vdocuments.site/reader030/viewer/2022011921/6033a75496494e4ac01c2e0c/html5/thumbnails/4.jpg)
High Resolution Melting analysis (HRM)
ssDNA
Primer
Step 1. Real-time PCR in presence of intercalating dye
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High Resolution Melting analysis (HRM)Step 1. Real-time PCR in presence of intercalating dye
![Page 6: Releasing the promise of HRMLiterature is sparse and controversial No standardisation of the method Complex studies are hard to replicate. Custom designed HRM assays HRM reagents](https://reader030.vdocuments.site/reader030/viewer/2022011921/6033a75496494e4ac01c2e0c/html5/thumbnails/6.jpg)
High Resolution Melting analysis (HRM)Step 1. Real-time PCR in presence of intercalating dye
![Page 7: Releasing the promise of HRMLiterature is sparse and controversial No standardisation of the method Complex studies are hard to replicate. Custom designed HRM assays HRM reagents](https://reader030.vdocuments.site/reader030/viewer/2022011921/6033a75496494e4ac01c2e0c/html5/thumbnails/7.jpg)
55˚C
High Resolution Melting analysis (HRM)
95˚C
Step 2. Melting analysis
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High Resolution Melting analysis (HRM)
![Page 9: Releasing the promise of HRMLiterature is sparse and controversial No standardisation of the method Complex studies are hard to replicate. Custom designed HRM assays HRM reagents](https://reader030.vdocuments.site/reader030/viewer/2022011921/6033a75496494e4ac01c2e0c/html5/thumbnails/9.jpg)
Genotyping by amplicon melting analysis
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The Promise of HRM
SimpleSimple Post PCR melting analysis
Automated software of genoptying
Explosive range of applicationsClinical diagnostics
Clinical researchGene Scanning
Reliable All types of mutation can be
determined
Low costNo expensive probes required
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The reality of HRM
Literature is sparse and controversial
No standardisation of the method
Complex studies are hard to replicate
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Custom designed HRM assays
HRM reagents
Any variationAny species
Any machine
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A MasterMix for HRM
Real-time PCR MasterMix HRM Mastermix
Priority:Melting Curve
Priority:Amplification plot CT
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Leading commercial real-time PCR MasterMix with HRM dye
PrimerDesign Precision HRM MasterMix with HRM dye
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Chromofy®
A novel asymmetric Cyanine dye
A dye for HRM
Minor groove binder with saturating chemistry
Improved fluorescent properties over BeBo
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Comparison with other Saturating dyes
SYBR green ® EVA green ® SYTO9 ® LCgreen ®Chromofy ®
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Custom designed HRM assays
HRM reagents
Any variationAny species
Any machine
![Page 18: Releasing the promise of HRMLiterature is sparse and controversial No standardisation of the method Complex studies are hard to replicate. Custom designed HRM assays HRM reagents](https://reader030.vdocuments.site/reader030/viewer/2022011921/6033a75496494e4ac01c2e0c/html5/thumbnails/18.jpg)
HRM assays - Simple HRM analysis of the amplicon
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Problem : Some variations are difficult to type
SNP Class 1 2 3 4
Base Change C/T & G/A C/A & G/T C/G A/T
Frequency 65% 19% 9% 7%
Tm Shift Large > 0.5˚C → Small < 0.2˚C
SNP Class 1 2 3 4
Base Change C/T & G/A C/A & G/T C/G A/T
Frequency 65% 19% 9% 7%
Tm Shift Large > 0.5˚C → Small < 0.2˚C
→
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HRM with a raZor probeTM
ssDNA
Primer
Step 1. Asymmetric real-time PCR in presence of intercalating dye and raZor probeTM
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HRM with a raZor probeTM
Step 1. Asymmetric real-time PCR in presence of intercalating dye and raZor probeTM
![Page 22: Releasing the promise of HRMLiterature is sparse and controversial No standardisation of the method Complex studies are hard to replicate. Custom designed HRM assays HRM reagents](https://reader030.vdocuments.site/reader030/viewer/2022011921/6033a75496494e4ac01c2e0c/html5/thumbnails/22.jpg)
HRM with a raZor probeTM
Step 1. Asymmetric real-time PCR in presence of intercalating dye and raZor probeTM
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55˚C
95˚C
Step 2. Melting analysis
HRM with a raZor probeTM
![Page 24: Releasing the promise of HRMLiterature is sparse and controversial No standardisation of the method Complex studies are hard to replicate. Custom designed HRM assays HRM reagents](https://reader030.vdocuments.site/reader030/viewer/2022011921/6033a75496494e4ac01c2e0c/html5/thumbnails/24.jpg)
Problem : Some variations are difficult to type
SNP Class 1 2 3 4
Base Change C/T & G/A C/A & G/T C/G A/T
Frequency 65% 19% 9% 7%
Tm Shift Large > 0.5˚C → Small < 0.2˚C
![Page 25: Releasing the promise of HRMLiterature is sparse and controversial No standardisation of the method Complex studies are hard to replicate. Custom designed HRM assays HRM reagents](https://reader030.vdocuments.site/reader030/viewer/2022011921/6033a75496494e4ac01c2e0c/html5/thumbnails/25.jpg)
Problem : Some variations are difficult to typeProblem solved : raZor probeTM
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Normalised data on the Rotorgene 6000
Amplicon
raZor probeTM
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Optimisation of raZor probeTM
ampliconraZorTM probe
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Custom designed HRM assays
HRM reagents
Any variationAny species
Any machine
![Page 29: Releasing the promise of HRMLiterature is sparse and controversial No standardisation of the method Complex studies are hard to replicate. Custom designed HRM assays HRM reagents](https://reader030.vdocuments.site/reader030/viewer/2022011921/6033a75496494e4ac01c2e0c/html5/thumbnails/29.jpg)
Roche480 normalisation across the amplicon TNFα252A→G
Only Heterozygotes identified
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Roche480 normalisation across the probe TNFα252A→G
Gene Scanning analysis module
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Roche480 probe based normalisation TNFα252A→G
Melt curve genotyping module
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Amplicon gene scanning is strong (heterozygote detection)Amplicon genotyping is weakerraZor probeTM based genotyping very strong
Roche480 (and others)
raZor probeTM genotyping data on the Roche 480
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Roche LightCycler 1.5TNFα252A→G
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Bio-Rad iCycler iQ5 TNFα252A→G
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Sample no. % confidence RG60001 98.71 HET
2 98.65 HET
3 97.675 A
4 97.49 HET
5 99.69 HET
6 99.425 G
7 99.565 G
8 99.285 HET
9 96.965 HET
10 97.73 A
11 99.365 HET
12 97.495 HET
13 98.63 HET
14 98.375 G
15 98.49 HET
16 96.505 HET
17 93.57 HET
18 96.03 HET
19 99.03 HET
20 96.425 HET
21 99.375 A
22 94.12 A
23 99.255 G
24 98.63 A
25 99.52 A
Sample no. % confidence RG6000 Bio-Rad iQ51 98.71 HET HET
2 98.65 HET HET
3 97.675 A A
4 97.49 HET HET
5 99.69 HET HET
6 99.425 G G
7 99.565 G G
8 99.285 HET HET
9 96.965 HET HET
10 97.73 A A
11 99.365 HET HET
12 97.495 HET HET
13 98.63 HET HET
14 98.375 G G
15 98.49 HET HET
16 96.505 HET HET
17 93.57 HET HET
18 96.03 HET HET
19 99.03 HET HET
20 96.425 HET HET
21 99.375 A A
22 94.12 A A
23 99.255 G G
24 98.63 A A
25 99.52 A A
Sample no. % confidence RG6000 Bio-Rad iQ5 Roche LC 1.51 98.71 HET HET HET
2 98.65 HET HET HET
3 97.675 A A A
4 97.49 HET HET HET
5 99.69 HET HET HET
6 99.425 G G G
7 99.565 G G G
8 99.285 HET HET HET
9 96.965 HET HET HET
10 97.73 A A A
11 99.365 HET HET HET
12 97.495 HET HET HET
13 98.63 HET HET HET
14 98.375 G G G
15 98.49 HET HET HET
16 96.505 HET HET HET
17 93.57 HET HET HET
18 96.03 HET HET HET
19 99.03 HET HET HET
20 96.425 HET HET HET
21 99.375 A A A
22 94.12 A A A
23 99.255 G G G
24 98.63 A A A
25 99.52 A A A
Sample no. % confidence RG6000 Bio-Rad iQ5 Roche LC 1.5 Roche LC4801 98.71 HET HET HET HET
2 98.65 HET HET HET HET
3 97.675 A A A A
4 97.49 HET HET HET HET
5 99.69 HET HET HET HET
6 99.425 G G G G
7 99.565 G G G G
8 99.285 HET HET HET HET
9 96.965 HET HET HET HET
10 97.73 A A A A
11 99.365 HET HET HET HET
12 97.495 HET HET HET HET
13 98.63 HET HET HET HET
14 98.375 G G G G
15 98.49 HET HET HET HET
16 96.505 HET HET HET HET
17 93.57 HET HET HET HET
18 96.03 HET HET HET HET
19 99.03 HET HET HET HET
20 96.425 HET HET HET HET
21 99.375 A A A A
22 94.12 A A A A
23 99.255 G G G G
24 98.63 A A A A
25 99.52 A A A A
raZor probeTM genotyping on different platformsTNFα252A→G
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Machine limitations for HRM
Gene scanning by amplicon
AmpliconGenotyping
raZor probeTM
Genotyping
Rotorgene 6000
Idaho LightScanner
Roche 480
ABI7900
Roche LightCycler 1.5
Bio-Rad iQ5
Gene scanning by amplicon
AmpliconGenotyping
Rotorgene 6000
Idaho LightScanner
Roche 480
ABI7900
Roche LightCycler 1.5
Bio-Rad iQ5
Gene scanning by amplicon
Rotor-Gene 6000
Idaho LightScanner
Roche 480
ABI7900
Roche LightCycler 1.5
Bio-Rad iQ5
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Factor V Leiden
SNP mutation in the factor V gene in exon 10
Type 1 SNP : A to G
5% of Europeans carry the variant form
Overproduction of thrombin leading to excess fibrin generation and excess clotting
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Factor V Leiden Factor V Leiden raZor probeTM kit- raw melting curve data
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Factor V Leiden Factor V Leiden raZor probeTM kit- normalised data (rotorgene 6000)
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Factor V Leiden raZor probeTM kit - difference plots
amplicon raZorTM probe
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Factor V Leiden raZor probeTM kit- clinical validation
Acknowledgement
World Reference ReagentFactor V Leiden Human gDNA
Reference PanelNIBSC code: 04/224
National Institute for Biological Standards and Control
Blanche LaneSouth Mimms
Potters BarHertfordshire
EN6 3QG
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Custom designed HRM assays
HRM reagents
Any variationAny species
Any machine
![Page 43: Releasing the promise of HRMLiterature is sparse and controversial No standardisation of the method Complex studies are hard to replicate. Custom designed HRM assays HRM reagents](https://reader030.vdocuments.site/reader030/viewer/2022011921/6033a75496494e4ac01c2e0c/html5/thumbnails/43.jpg)
razor sharp genotyping
HRM
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High Resolution Melting Analysis
Quality Reagents
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razor sharp genotyping
HRM