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Mutation Scanning and Genotyping by High- Resolution DNA Melting Analysis Carl Wittwer, MD, PhD Professor of Pathology University of Utah Slide 2 High-Resolution DNA Melting Scanning PCR products Mutation Screening HLA Matching Genotyping Hybridization Probes Unlabeled Probes Small Amplicons Simultaneous scanning and genotyping Slide 3 Fluorescence Cycles Detection Quantification 0 30 2010 Detection Quantification Fluorescence Cycles Detection Quantification 0 30 2010 Amplification Temperature Time (min) 051015 Temperature (C) Product ID Allele ID 90 80 70 Fluorescence 20 Melting Analysis Temperature (C) Product ID Allele ID 90 80 70 Fluorescence Amplification & Analysis Slide 4 Temperature (C) Fluorescence -globin 53.2 536 Prostate 60.3 292 Specific Antigen Amplification %GC Length bp Hepatitis B 50.0 180 Melting Curves for Different Products (SYBR Green I) Slide 5 Heterozygote Amplification Two Homoduplexes Two Heteroduplexes Observed Combination of 4 Duplexes Slide 6 Heterozygote Amplification Two Homoduplexes Two Heteroduplexes Observed Combination of 4 Duplexes Slide 7 100 bp Product Homozygotes are easily distinguished from heterozygotes Different heterozygotes trace unique melting paths C/C Homozygote C/G Heterozygote C/T Heterozygote C/A Heterozygote Slide 8 0 20 40 100 80 60 Fluorescence (Normalized) Temperature (C) 82 85 84 83 CC TT TC SNP typing on a 544bp amplicon. (SNP is in the low temperature domain) SNP Typing in Long Amplicons Clin Chem. 2003 Jun;49(6 Pt 1):853-60. Slide 9 High-Resolution DNA Melting Scanning PCR products Finding heterozygotes among homozygotes Genotyping alleles Complete genotyping of wild type, heterozygous, and homozygous variants. Slide 10 1 2 (716) 3 (133) 4 (133) 5 (207) 6 (263) Mutation Scanning Typical Mutation Distribution Slide 11 Denaturing High Pressure Liquid Chromatography (DHPLC) Scanning by Heteroduplex Separation Temperature Gradient Capillary Electrophoresis (TGCE) Slide 12 Mutation Scanning Technologies All use PCR first Specimen Amplify DNA High-Resolution Melting Load on matrix Electrophoresis dHPLC Clean up Enzymatic reactions Clean up Mass Spec Arrays Sequencing Slide 13 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 Slide 14 High-Resolution Melting Platforms (Idaho Technology) LightScanner Slide 15 High-Resolution Melting Analysis Slide 16 Melting Analysis Slide 17 Slide 18 Slide 19 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 Use of a DNA toolbox as a model system for mutation scanning X Slide 20 HR-1 LightScanner Sensitivity and Specificity Dependence on Product Size N=1248, each instrument Three different targets All possible SNPs and WTs Clin Chem. 2004;50:1748-54. Slide 21 Sensitivity and Specificity (40% GC template) Slide 22 Sensitivity and Specificity (50% GC template) Slide 23 Sensitivity and Specificity (60% GC template) Slide 24 Missed Heterozygotes Homozygous Base A C G T Heterozygous Base cgt agt act acg bp 400 500 1000 400 800 40% GC 50% GC a = false negative a = correct calls Slide 25 Ability to detect allele fractions other than 50% (300 bp product) 100% 95% 90% 75% 50% Slide 26 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 Slide 27 GIST Mutation Detection by Melting Analysis (exon 11) Normal c-kit mutation Slide 28 HHT Exon Scanning Eng (exon 2 274 bp) Slide 29 Exon Scanning (Difference Plot) Eng (exon 2) 274 bp Fluorescence Difference Slide 30 Differences between Heterozygotes Clin Chem 51:1295-8 (2005) Slide 31 Transplantation Matching HLA: A1, A27, B17, B44, C2, C5, DR1, DR4 $1,200 per individual Slide 32 HLA-B Exon 3 Exon 2 Slide 33 HLA-C Exon 2Exon 3 Slide 34 HLA Matching, not Typing Tissue Antigens. 2004 Aug;64(2):156-64. Applications Living related donors Unrelated bone marrow transplantation ?? Phenotype/Genotype Correlation ?? ?? Identity ?? Slide 35 Genotyping Methods Open-tube (processing after amplification) Conventional methods (SNE) Arrays Mass spectrometry Closed-tube (real-time) Allele-specific Melting methods Slide 36 Closed-Tube Genotyping Popular Probe Designs Two AdditionsThree Additions ***One probe needed for each allele*** Slide 37 Genotyping by Melting Adjacent Hybridization Probes (HybProbes ) Am J Pathol. 1998;153:1055-61 ***One probe pair distinguishes many alleles*** Slide 38 Time (min) Temperature (C) Dynamic Dot Blot for Allele Analysis (Heterozygote) Anchor Probe Mutation Probe Match Mismatch Fluorescence Temperature (C) -dF/dT Slide 39 Temperature (C) -dF/dT Factor V Leiden Heterozygous Homozygous Mutant Homozygous WT Clin Chem 1997; 43: 2262 - 2267 Slide 40 Genotyping by Melting Adjacent Hybridization Probes (HybProbes ) Am J Pathol. 1998;153:1055-61 Single Probes (SimpleProbe ) Anal Biochem. 2001;290:89-97 Slide 41 Different Single Labeled Probes (CFTR) F508 F508C WT Slide 42 Genotyping by Melting HybProbes Am J Pathol. 1998;153:1055-61 Single Probes (SimpleProbe ) Anal Biochem. 2001;290:89-97 Unlabeled Probes (dsDNA dye) Clin Chem. 2004;50:1328-35 Slide 43 Unlabeled Probe Genotyping (Factor V Leiden) Clin Chem 2005; 51: 1770 - 1777 Slide 44 Automatic Clustering Wild Type Heterozygous Mutant Homozygous Mutant Slide 45 Genotyping by Melting HybProbes Am J Pathol. 1998;153:1055-61 SimpleProbe Anal Biochem. 2001;290:89-97 Unlabeled Probes (dsDNA dye) Clin Chem. 2004;50:1328-35 Amplicon Melting Anal. Biochem. 1997;245, 154-60 (SYBR Green I) Clin Chem. 2003;49:732-9 (LCGreen) ***One amplicon distinguishes many alleles*** Slide 46 Amplicon Melting (Factor V Leiden) Temperature (C) Slide 47 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 Slide 48 Most, but not all SNP homozygotes can be distinguished (Clin Chem. 2004;50:1156-64) Human SNP Percentage (C) Nearest Neighbor Symmetry Slide 49 H63D Genotype Melting Curves No Wild Type Addition Anal Biochem 2005, in press (Optimal Wild Type fraction = ?) With Wild Type Addition Slide 50 Genotyping by Melting with dsDNA dyes No covalent labels Multiplexing by temperature, not color Region of sequence interrogation Amplicon Melting Small amplicons Rapid cycling High PCR efficiency 4% of SNPs require Spiking Unlabeled Probe Asymmetric PCR 3-blocked oligos Region of sequence interrogation Slide 51 Comparison of Methods for Real-Time SNP Typing Amplicon Melting Unlabeled Probe Single Probe HybProbe TaqMan Beacons Scorpions MGB TaqMan Modifications 0011223300112233 Method # Probes 0112222201122222 Slide 52 HFE Mutations Small Amplicon Melting H63D (C187G) T189C S65C (A193T) C282Y (G845A) Unlabeled Probe Slide 53 HFE Genotyping Slide 54 Slide 55 Slide 56 Slide 57 Simultaneous Unlabeled Probe and Amplicon Melting (Factor V Leiden) Unlabeled Probe Melting Amplicon Melting Wild Type Heterozygous Mutant Homozygous Mutant Temperature (C) Slide 58 Scan & Genotype at once Temperature Time (sec) 0306090 120 Scan the full fragment Genotype by probe melting Slide 59 -dF/dT Temperature CFTR exon 10 scanning and genotyping Slide 60 Genotyping by Probe Melting 1 2 (716) 3 (133) 4 (133) 5 (207) 6 (263) High-resolution Melting to Scan PCR fragments Reported mutations Gene Analysis Slide 61 Eliminate 99% of sequencing? Scanning of PCR fragments for variants DNA sequencing ~1% Not identified Unlabeled probe genotyping of known variants Variant genotyped ~ 9% Identified ~ 90% Normal ~10% Abnormal Wild type Slide 62 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) Slide 63 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