1. Mutation Scanning and Genotyping by High- Resolution DNA Melting Analysis Carl Wittwer, MD, PhD Professor of Pathology University of Utah

2. High-Resolution DNA Melting Scanning PCR products –Mutation Screening –HLA Matching Genotyping –Hybridization Probes –Unlabeled Probes –Small Amplicons Simultaneous scanning and genotyping

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

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)

5. Heterozygote Amplification Two Homoduplexes Two Heteroduplexes Observed Combination of 4 Duplexes

6. Heterozygote Amplification Two Homoduplexes Two Heteroduplexes Observed Combination of 4 Duplexes

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

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.

9. High-Resolution DNA Melting Scanning PCR products –Finding heterozygotes among homozygotes Genotyping alleles –Complete genotyping of wild type, heterozygous, and homozygous variants.

10. 1 2 (716) 3 (133) 4 (133) 5 (207) 6 (263) Mutation Scanning Typical Mutation Distribution

11. Denaturing High Pressure Liquid Chromatography (DHPLC) Scanning by Heteroduplex Separation Temperature Gradient Capillary Electrophoresis (TGCE)

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

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

14. High-Resolution Melting Platforms (Idaho Technology) LightScanner ®

15. High-Resolution Melting Analysis

16. Melting Analysis

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      

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.

21. Sensitivity and Specificity (40% GC template)

22. Sensitivity and Specificity (50% GC template)

23. Sensitivity and Specificity (60% GC template)

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

25. Ability to detect allele fractions other than 50% (300 bp product) 100% 95% 90% 75% 50%

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

27. GIST Mutation Detection by Melting Analysis (exon 11) Normal c-kit mutation

28. HHT Exon Scanning Eng (exon 2 – 274 bp)

29. Exon Scanning (Difference Plot) Eng (exon 2) – 274 bp Fluorescence Difference

30. Differences between Heterozygotes Clin Chem 51:1295-8 (2005)

31. Transplantation Matching HLA: A1, A27, B17, B44, C2, C5, DR1, DR4 $1,200 per individual

32. HLA-B Exon 3 Exon 2

33. HLA-C Exon 2Exon 3

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 ??

35. Genotyping Methods Open-tube (processing after amplification) –Conventional methods (SNE) –Arrays –Mass spectrometry Closed-tube (“real-time”) –Allele-specific –Melting methods

36. Closed-Tube Genotyping Popular Probe Designs Two AdditionsThree Additions ***One probe needed for each allele***

37. Genotyping by Melting Adjacent Hybridization Probes (HybProbes ® ) Am J Pathol. 1998;153:1055-61 ***One probe pair distinguishes many alleles***

38. Time (min) Temperature (°C) Dynamic Dot Blot for Allele Analysis (Heterozygote) Anchor Probe Mutation Probe Match Mismatch Fluorescence Temperature (°C) -dF/dT

39. Temperature (°C) -dF/dT Factor V Leiden Heterozygous Homozygous Mutant Homozygous WT Clin Chem 1997; 43: 2262 - 2267

40. Genotyping by Melting Adjacent Hybridization Probes (HybProbes ® ) Am J Pathol. 1998;153:1055-61 Single Probes (SimpleProbe ® ) Anal Biochem. 2001;290:89-97

41. Different Single Labeled Probes (CFTR) ΔF508 F508C WT

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

43. Unlabeled Probe Genotyping (Factor V Leiden) Clin Chem 2005; 51: 1770 - 1777

44. Automatic Clustering Wild Type Heterozygous Mutant Homozygous Mutant

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***

46. Amplicon Melting (Factor V Leiden) Temperature (°C)

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

48. Most, but not all SNP homozygotes can be distinguished (Clin Chem. 2004;50:1156-64) Human SNP Percentage (°C) Nearest Neighbor Symmetry

49. H63D Genotype Melting Curves No Wild Type Addition Anal Biochem 2005, in press (Optimal Wild Type fraction = ?) With Wild Type Addition

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

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

52. HFE Mutations Small Amplicon Melting H63D (C187G) T189C S65C (A193T) C282Y (G845A) Unlabeled Probe

53. HFE Genotyping

57. Simultaneous Unlabeled Probe and Amplicon Melting (Factor V Leiden) Unlabeled Probe Melting Amplicon Melting Wild Type Heterozygous Mutant Homozygous Mutant Temperature (°C)

58. Scan & Genotype at once Temperature Time (sec) 0306090 120 Scan the full fragment Genotype by probe melting

59. -dF/dT Temperature CFTR exon 10 scanning and genotyping

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

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

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)

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