Transcript Advanced Technology Group

Mutation Scanning and
Genotyping by High-
Resolution DNA Melting
Analysis
Carl Wittwer, MD, PhD
Professor of Pathology
University of Utah
High-Resolution DNA Melting
• Scanning PCR products
– Mutation Screening
– HLA Matching
• Genotyping
– Hybridization Probes
– Unlabeled Probes
– Small Amplicons
• Simultaneous scanning and genotyping
• Detection
••Quantification
Detection
• Quantification
• Detection
• Quantification
• Product ID
• Allele ID
Amplification & Analysis
10
0
20
30
Cycles
10
0
• Product ID
• Allele ID
20
70
70
30
Cycles
Amplification
0
5
10
Time (min)
80
Temperature (°C)
90
80
Temperature (°C)
15
90
Melting Analysis
20
Melting Curves for Different
Products (SYBR Green I)
Amplification
%GC
Fluorescence
Hepatitis B
50.0
 -globin
53.2
Prostate
60.3
Specific Antigen
Temperature (°C)
Length bp
180
536
292
Melting Curves
Heterozygote Amplification
Two
Heteroduplexes
Observed Combination
of 4 Duplexes
Two
Homoduplexes
T
A
C
A
C
G
T
G
Temperature
Derivative Plots
Heterozygote Amplification
Two
Heteroduplexes
Observed Combination
of 4 Duplexes
T
Two
Homoduplexes
T
A
G
C
G
C
A
Temperature
100 bp Product
C/C Homozygote
C/G Heterozygote
C/T Heterozygote
C/A Heterozygote
• Homozygotes are easily
distinguished from
heterozygotes
• Different heterozygotes
trace unique melting paths
Fluorescence (Normalized)
SNP Typing in Long Amplicons
CC
100
TC
SNP typing on a
544bp amplicon.
(SNP is in the low
temperature domain)
80
60
TT
40
20
0
82
83
84
Temperature (C)
Clin Chem. 2003 Jun;49(6 Pt 1):853-60.
85
High-Resolution DNA Melting
• Scanning PCR products
– Finding heterozygotes among homozygotes
• Genotyping alleles
– Complete genotyping of wild type,
heterozygous, and homozygous variants.
Mutation Scanning
1
2 (716)
3 (133)
4 (133)
5 (207)
Typical Mutation Distribution
6 (263)
Scanning by Heteroduplex Separation
Denaturing High Pressure
Liquid Chromatography
(DHPLC)
Temperature Gradient
Capillary Electrophoresis
(TGCE)
Mutation Scanning Technologies
• All use PCR first
High-Resolution Melting
Electrophoresis
Specimen
Amplify
DNA
Load on
matrix
dHPLC
Mass Spec
Arrays
Clean up
Enzymatic reactions
Clean up
Sequencing
Scanning by High-Resolution Melting
• Closed-tube
– dsDNA dye before PCR
– No processing, additions, or separations
– No exposure to the environment
• Rapid
– 1-2 min for single samples
– 5-10 min for 96/384 samples
• Non-destructive
– Downstream processing if necessary
High-Resolution Melting Platforms
(Idaho Technology)
LightScanner®
CCD Camera
Emmision Filter
LEDs
Excitation
Filter
Microtiter
Plate
High-Resolution Melting Analysis
Melting Analysis
Melting Analysis
Melting Analysis
Use of a DNA “toolbox” as a model
system for mutation scanning
• Highsmith et al., Electrophoresis (1999), 20: 188-1194
• Constructed plasmids of 40%, 50%, and 60% GC content
with A, C, G, or T at one position
• PCR primers on each side spaced 50 bp apart
     
X
     
Sensitivity and Specificity
Dependence on Product Size
HR-1
•N=1248, each instrument
•Three different targets
•All possible SNPs and WTs
LightScanner
Clin Chem. 2004;50:1748-54.
Sensitivity and Specificity
(40% GC template)
Sensitivity and Specificity
(50% GC template)
Sensitivity and Specificity
(60% GC template)
Missed Heterozygotes
Homozygous Base
A
C
G
T
bp
40% GC
400
500
1000
50% GC
400
800
Heterozygous Base
cgt
agt act acg
cgt agt act acg
cgt agt act acg
cgt
cgt
agt act
agt act
acg
acg
a = false negative
a = correct calls
Ability to detect allele fractions other than 50%
(300 bp product)
100%
95%
90%
75%
50%
Scanning by Melting
Applications
•
•
•
•
•
c-kit (GIST tumors) - Am J Clin Path, 122:206-16 (2004)
MCAD – Mol Genet Metab, 82:112-20 (2004)
SLC22A5 (Urea cycle) – Hum Mutat, 25:306-13 (2005)
BRAF (melanoma) – Hum Pathol, 36:486-93 (2005)
Cystic fibrosis – Am J Clin Path,124:330-8 (2005)
• Studies in progress
– PNH (Paroxysmal nocturnal hemoglobinurea)
– HHT (Hereditary hemorrhagic telangiectasia)
– Galactosemia
GIST Mutation Detection by Melting Analysis
(exon 11)
Normal
c-kit mutation
HHT Exon Scanning
Eng (exon 2 – 274 bp)
Exon Scanning (Difference Plot)
Fluorescence Difference
Eng (exon 2) – 274 bp
Differences between Heterozygotes
Clin Chem 51:1295-8 (2005)
Transplantation Matching
HLA: A1, A27, B17, B44, C2, C5, DR1, DR4
$1,200 per individual
HLA-B
Exon 2
Exon 3
HLA-C
Exon 2
Exon 3
HLA Matching, not Typing
• Tissue Antigens. 2004 Aug;64(2):156-64 .
• Applications
– Living related donors
– Unrelated bone marrow transplantation
• ?? Phenotype/Genotype Correlation ??
• ?? Identity ??
Genotyping Methods
• Open-tube (processing after amplification)
– Conventional methods (SNE)
– Arrays
– Mass spectrometry
• Closed-tube (“real-time”)
– Allele-specific
– Melting methods
Closed-Tube Genotyping
Popular Probe Designs
Two Additions
Three Additions
***One probe needed for each allele***
Genotyping by Melting
Adjacent Hybridization Probes
(HybProbes®)
***One probe
pair distinguishes
many alleles***
Am J Pathol.
1998;153:1055-61
Fluorescence
Time (min)
Dynamic Dot Blot for Allele
Analysis (Heterozygote)
Anchor Probe
Temperature (°C)
Mutation Probe
Match
-dF/dT
Temperature (°C)
Mismatch
Temperature (°C)
Factor V Leiden
Homozygous WT
Homozygous Mutant
-dF/dT
Heterozygous
Temperature (°C)
Clin Chem 1997; 43: 2262 - 2267
Genotyping by Melting
AdjacentSingle
Hybridization
Probes Probes
(SimpleProbe
(HybProbes®®))
Am JBiochem.
Anal
Pathol. 1998;153:1055-61
2001;290:89-97
Different Single Labeled Probes
F508C
WT
ΔF508
(CFTR)
Genotyping by Melting
Single
UnlabeledHybProbes
ProbesProbes
(dsDNA dye)
(SimpleProbe®)
Am
J Pathol.
Clin
Chem. 1998;153:1055-61
2004;50:1328-35
Anal Biochem. 2001;290:89-97
Unlabeled Probe Genotyping
(Factor V Leiden)
Clin Chem 2005; 51: 1770 - 1777
Automatic Clustering
Wild Type
Heterozygous Mutant
Homozygous Mutant
Genotyping by Melting
HybProbes
SimpleProbe
Amplicon
Melting
Unlabeled
Probes
(dsDNA dye)
Anal. Biochem. 1997;245, 154-60 (SYBR Green I)
Am
JChem.
Pathol.
Biochem.
1998;153:1055-61
2001;290:89-97
ClinAnal
2004;50:1328-35
Clin Chem.
2003;49:732-9
(LCGreen)
***One amplicon distinguishes many alleles***
Amplicon Melting
(Factor V Leiden)
Temperature (°C)
Distinguishing all 4 homoduplexes and all 6 heteroduplexes
A/A
C/C
G/G
T/T
A/C
A/G
A/T
C/G
C/T
G/T
Most, but not all SNP homozygotes
can be distinguished
(Clin Chem. 2004;50:1156-64)
Percentage
SNP Percentage
Human SNP
Human
10
8
Nearest Neighbor
Symmetry
6
4
2
0
0
0.5
1
|D Tm| (°C)
1.5
2
H63D Genotype Melting Curves
No Wild Type Addition
With Wild Type Addition
(Optimal Wild Type fraction = ?)
Anal Biochem 2005, in press
Genotyping by Melting
with dsDNA dyes
• No covalent labels
• Multiplexing by temperature, not color
Unlabeled
Amplicon Probe
Melting
Asymmetric
PCR
Small amplicons
3’-blocked
oligos
Rapid cycling
High PCR efficiency
4% of SNPs require “Spiking”
Region of sequence
interrogation
Comparison of Methods for
Real-Time SNP Typing
# Probes
Method
0
Amplicon Melting
1
Unlabeled Probe
1
Single Probe
2
HybProbe
2
TaqMan
2
Beacons
2
Scorpions
2
MGB TaqMan
Modifications
0
0
1
1
2
2
3
3
HFE Mutations
Small Amplicon Melting
Unlabeled Probe
T189C
H63D
(C187G)
S65C
(A193T)
C282Y
(G845A)
HFE Genotyping
HFE Genotyping
HFE Genotyping
HFE Genotyping
Simultaneous Unlabeled Probe
and Amplicon Melting
(Factor V Leiden)
Wild Type
Heterozygous Mutant
Homozygous Mutant
Amplicon
Melting
Unlabeled Probe
Melting
Temperature (°C)
Scan & Genotype at once
Scan the full fragment
Genotype by probe melting
0
30
60
Time (sec)
90
120
-dF/dT
CFTR exon 10 scanning and genotyping
Temperature
Gene Analysis
Reported mutations
1
2 (716)
3 (133)
4 (133)
5 (207)
High-resolution Melting to Scan PCR fragments
Genotyping by Probe Melting
6 (263)
Eliminate 99% of sequencing?
Scanning of PCR
fragments for variants
~ 90%
Normal
Wild type
~10% Abnormal
Unlabeled probe
genotyping of
known variants
~1% Not identified
DNA sequencing
~ 9%
Identified
Variant
genotyped
High-Resolution Melting Analysis
• Dyes
– SYBR™ Green I (1997)
– LCGreen high-resolution dyes
• LCGreen™ I (2003)
• LCGreen PLUS (2005)
• Instruments
– Single sample HR-1 (2003)
– 96/384 LightScanner (2005)
Thanks…
University of Utah
Mathematics
Bob Palais
Pathology
Luming Zhou
Gundi Reed
Rob Pryor
Josh Vanderstein
Joe Holden
Phil Bernard
ARUP
Mark Herrmann
Michael Liew
Mike Seipp
Becky Margraf
Bob Chou
Idaho Technology
Virginie Dujols
Derek David
Lyle Nay
Steve Dobrowolski
Jason McKinney
$$$$
NIH
Whitaker Foundation
State of Utah
University of Utah
ARUP
Idaho Technology
Roche