1. Center of Excellence Center for Homogeneous DNA Analysis  new techniques  new instruments  new software  DNA analysis fast, simple and cost effective Genetics Infectious Disease Cancer  Commercialization

2. Background  1990s to present: Homogeneous DNA amplification and analyses  Probes or dyes are added prior to PCR  Focus on melting curve analysis 1997: Two “adjacent hybridization probes” 2000: Single hybridization probe 2003: Unlabeled probe 2003: Amplicon melting

3. Hybridization Probe Formats Adjacent Hybridization Probes (HybProbes)Single Probes (Simple Probes)Unlabeled Probes (LCGreen)Amplicon as the Probe

4. First year of COE - Achievements Instruments and Reagents  Development of method to scan PCR products for unknown mutations, licensed to Utah company  Reagents and instrument rights were licensed to IT, Inc  HR-1 TM and LCGreen TM I available in US  Distributors in Japan, Italy, and Korea established

5. First year of COE -Achievements Applications  Mutation Scanning  Software  HLA Matching  Unlabeled Probe Genotyping  Amplicon melting - SNPs

6. Mutation Scanning 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   

7. Mutation Scanning - Toolbox  This data represents 1248 different calls in the Toolbox constructs

8. Mutation Scanning - Toolbox

11. Normalized and temperature shifted melting profiles

12. Dependence of area difference on product length 0 5 10 15 20 25 30 70100150200250300350400450550 Product Length (bp) Area Difference

13. First year of COE - Achievements Software  Automatic melting curve classification (U-3703)  Primer design software for SNP analysis (SNPWizard U-3701)  Primer design software for exon analysis (ExonWizard U-3702)  Logistic quantification of real-time PCR (U-3704)

15. Software – Demonstrations  Genotype clustering of high- resolution melting data  Web SNPWizard  Spiking animation for genotyping  Genome-wide SNP nearest neighbor frequencies

16. Software  DNA duplex melting based on nearest-neighbor thermodynamic theory  Currently available estimates are based on non-PCR conditions  Determination of nearest-neighbor parameters via high resolution melting under PCR conditions  Development of a software suite of programs for primer and probe design to simplify SNP typing, exon analysis and clinical assay design to support novel techniques  Initial posting of programs on academic server: DNAWizards.path.utah.edu

17. Software –Methods  Increase the precision of Tm estimation to +/- 0.5C  Include parameters under PCR conditions, such as: Fluorescent labels DsDNA dyes Product concentration Mg ++, K + and Tris + effects

18. DNAWizards.path.utah.edu  DNAWizards site hosts Remotely controlled DNA analysis software  SNPWizard Downloadable data  Updated genomic SNP data Publications and supplementary materials  Optimal spiking visualization

19. First year of COE - Achievements HLA Matching  Determining HLA Genotypic Identity Among Siblings Siblings are the best first candidates for organ donation. They are most likely to share common HLA alleles.  Current HLA Typing Methods: Serotyping and DNA sequencing  Most widely used  Expensive  Requires several days for completion  High-resolution melting is a simple way to establish genotypic identity at polymorphic loci.

20. HLA Inheritance A1A2 A3 A1-A3 A2-A3 A4 A1-A4 A2-A4

21. CEPH Family UT1331

22. Melting Curve of HLA-A Exon 2

23. Sibling Genotypes HLA Locus HLA Class Genotype 1 Genotype 2 Genotype 3 Genotype 4 AI9, 10, 164, 783, 5, 6, 11, 17 BI9, 10, 164, 7 8 3, 5, 6, 11, 17 CI9, 10, 164, 7 8 3, 5, 6, 11, 17

24. First year of COE - Achievements Genotyping with Unlabeled Probes  No fluorescently-labeled probes required  Uses simple 3’-Blocked oligonucleotides  Asymmetric PCR  LCGreen I  Lower Cost  Greater probe stability  Greater flexibility

25. Asymmetric PCR

26. Amplicon and Probe Peaks with Asymmetric PCR

27. Mismatch-detection of Homozygous Template (LCGreen I)

28. Mismatch-detection of Heterozygous Template (LCGreen I)

29. Mismatch-detection of Heterozygous Template (Sybr Green I)

30. Effect of Unlabeled Probe Length (LCGreen I)

31. Genotyping of [delta]F508 (LCGreen I) 5 3 2 60 70 Temperature (°C) 1 0 4 Wild type [delta]F508 hom [delta]F508 het -dF/dT

32. SNP Genotyping with LCGreen I

33. First year of COE - Achievements Amplicon melting - SNPs  Successful genotyping of all possible SNPs shown with plasmids.  Demonstrated on clinically significant mutations.

34. Homozygote Amplification One Homoduplex

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

36. Small Amplicon Primer Design  Primers are designed to be as close as possible to the SNP site  The sequence of the primers must be checked for primer-primer dimer formation

37. Engineered SNP pBR322 Plasmids

38. Clinical Samples

39. Spiking Experiments

40. Comparison of Methods for Real- Time SNP Typing

41. First year of COE - Achievements Commercial  20 systems have been sold w/ gross revenue of $210,000  Six new jobs created, w/ average salary of $56,000

42. Technology Rights  U of U has 13 issued US patents in addition to foreign counterparts  About 13 further patents pending  Some technology rights have been licensed to Utah companies  Those NOT licensed as of yet: Homogeneous sequencing and repeat typing (U-3601) [optioned to IT, Inc through 7-2004] Integrated primer synthesis and target amplification on arrays (U- 3570) [optioned to IT, Inc through 5-2004] Homogeneous multiplex hybridization by color and Tm (US pat. #6,772,156) Simultaneous screening and identification of sequence alterations form amplified target (US pat. pending #2002-0142300) SNPWizard (U-3701) ExonWizard (U-3702) Automatic clustering and classification of homozygotes and heterozygotes by high-resolution melting curve similarity (U-3703) Logistic quantification of initial copy number from the plateau height, linear growth rate, and maximum second derivative of PCR amplification curves (U-3704)

43. Future Areas of Technology Development  Methods for homogeneous repeat typing and sequencing  Software for DNA analysis with the objective of spinning off “DNAWizards.com”  Developing a highly parallel hardware platform for real-time PCR an melting analysis in conjunction with proposed new COE by Dr. Bruce Gale (UU engineering)

44. Homogeneous Repeat Typing and Sequencing – Methods  Chain extension with dideoxynucleotide termination  High-resolution melting post PCR for direct Tm determination  Example: CA repeat determination: Amplification with dCTP, dATP and ddGTP. Amplification stops at first G after CA repeat. Melting peak will indicate length of repeat. Method works in an synthetic oligonucleotide system (see figure to right)

45. Homogeneous Repeat Typing and Sequencing – Experiments  What repeat lengths can be distinguished?  Can heterozygotes be easily identified?  What about small fractions of a repeat allele, as might be seen in cancer?  What should the primer’s GC content be compared to the repeat’s GC content?

46. Homogeneous Repeat Typing and Sequencing – Challenges  Asymmetric PCR needs to be coupled to cycle sequencing (closed tube!)  To separate the PCR reactions from the sequencing reagents, the sequencing reagents are added on top of an oil barrier. After amplification, a centrifugation step will mix reagents and sequencing can start. (described for nested PCR, J. Clin. Virol. 2001, 20:71- 75)  In a completely homogenous reaction, the use of two different polymerase can accomplish amplification and sequencing at the same time (described in Nucleic Acids Res. 2003, 31:e121)  Digestion with lambda exonuclease can eliminate one strand after PCR if one primer is 5’phosphorylated.

47. Homogeneous Repeat Typing and Sequencing – Commercialization Plan  Commercial partner or spin-off company will provide generic research reagents ($0.5/assay) 10 x dye optimized dye/buffer combination freeze dried PCR master mixes  Software for repeat typing ($1,000 per license)  Software for sequencing ($1,000 per license)  Analyte Specific Reagents (ASRs) sold to diagnostic laboratories ($20-40/assay). HCV genotyping bacterial identification by rDNA

48. Future DNAWizards.com  Software Goals User-friendly DNA manipulation/visualization Integrated platform from design to analysis  Projects Tm prediction under PCR conditions Primer design for SNP typing Primers/probes for exon mutation scanning Primers/probes for allele-differentiation by Tm Automatic normalization and genotype clustering Automatic genotyping by curve classification PCR target quantification

49. DNAWizards commercialization  Software purchase/upgrades  Fee per use  Contract design/analysis  User support and education  Oligonucleotide synthesis partnership  Clinical lab partnership

50. Software – Commercialization Plan  DNAWizards.com, a software and service enterprise will provide contract services and distribution of software and educational material. A bundled software package ($1,500) will include: TmWizard, free web trial, $200 software SNPWizard: free web trial, $25 custom design/assay, $200 software ExonWizard: free web trial, $100 custom design/gene, $300 software DxWizard: $100-$500 custom design/assay, $700 software C t Wizard: free web trial, $200 software TypeWizard: free web trial, $100 software

51. Arrays for Real-Time PCR – Objectives directing Methodology  Determine feasibility of amplifying and monitoring PCR and HR-melting in 1-10 nl volumes  There is no commercial array system for parallel real time PCR Closest competitor is ABI with their Prism 7900HT instrument

52. Arrays for Real-Time PCR – Anticipated Problems  Deposition of the primers in each compartment  Microfluidic introduction of the sample/PCR master mix to all cells  Sealing each compartment to prohibit intermixing

53. Arrays for Real-Time PCR – Commercialization Plan  Estimated price for the bare chips: $10  Estimated cost of analyte-specific chips will depend on the number of parallel reactions in the chip. i.e. 100 well chip (CF testing) costs $30 i.e. 300,000 well chip (human exon) costs $1,000  Instrument capable of PCR temperature cycling, real-time monitoring, and high- resolution melting: $50,000 and $70,000

54. How COE will Demonstrate Value of New Technology  Research publications  Providing access to analytical software through DNAWizards.path.utah.edu  Alpha-site testing at leading clinical diagnostic laboratories  As well as domestic and foreign academic centers

55. Further Considerations  Out-licensing of newer technologies  Formation of a new service/manufacturing company in Utah, which may or may not be independent of the new software company, DNAWizards.com  Product sales and distribution is best done through regional distributors or alliance partner(s)

56. Estimates  Our methods will eliminate 95-99% of high-cost conventional DNA sequencing  Global market for Center’s technology is ca $400 million (instruments plus reagents)  Annual growth of 9 – 10%  Annual revenue of $ 24 million (4% share) in 2008  Eventual financial independence from state  Development of newer technologies from years two through five will further strengthen competitive advantage of high resolution melting  Six additional new jobs created in year 2

57. Program Schedule

58. Competitive Analysis -Homogeneous Repeat Typing and Sequencing

59. Competitive Analysis -Software  There are over 30 oligonucleotide design web sites that offer free primer/probe design on-line  Several are linked to oligonucleotide synthesis services  Some are at least partly specific to a platform  Software for SNP typing, exon analysis, repeat typing and sequencing based on melting temperature are not available  Our techniques do not require probes and are less expensive  Tm predictions will be more accurate than prior methods by an order of magnitude

60. Competitive Analysis - Arrays for Real-Time PCR and High- Resolution Melting Analysis  There is presently no commercial array system for parallel real-time PCR  Closest competitor: ABI with Prism 7900HT instrument $200/card, $2/assay, 1-2ul/assay  Our system envisions 1-10nl/assay By flooding the system, highly parallel analysis on a genome-wide scale possible

61. Market Analysis - Sequencing and Repeat Typing  For clinical tests (HIV & HCV): 360,000 assays/year  HLA sequencing: 25,000 assays/year  Estimate for global market: 800,000 assays/year

62. Market Analysis - Microarray Market  Instrumentation estimated at $600 Million  Bioinformatics estimated at $110 Million  Affymetrix (50% of market) with 20% annual growth in sales 970 microarray analysis systems installed as of Jan 2004

63. Economic Impact  Create, attract and retain highly skilled technical workforce  Attract possible out-of-state investment to fund COE’s activities  Provide opportunity for infusion of federal funds through SBIR, STTR, and ATP programs  Attract visiting scholars for collaborative studies and international conferences  COE could interface with clinical diagnostic labs, such as ARUP and Myriad

64. Organizational Structure

65. Program Coordination -Method Group  Dr. Luming Zhou Rob Pryor (sr. lab. Technologist) Joshua Vandersteen (undergraduate) Matt Poulson (graduate Student) Dr. Gudrun Reed (sr. lab. Technologist)  Will also provide Market Intelligence  Measurable Milestones: Determine length and sequence dependence of melting analysis Obtain new parameters for Tm estimation under PCR and melting conditions

66. Program Coordination -Software Group  Dr. Bob Palais Ian Odell Allison Jarstad (undergrad)  Measurable Milestones: Development of Math of DNA course at U of U Posting web versions of  TmWizard  SNPWizard  ExonWizard  DxWizard  C t Wizard  TypeWizard

67. Program Coordination -Array Group  Dr. Bruce Gale Graduate Student (to be named)  Measurable Milestones: Demonstrate 1-10nl PCR reactions on a micro-machined chip substrate

68. Current and Pending Support TitleAgencyDatesAmount SNP Typing without Probes U of U Research Fund 7/3-6/05$70,000 Fluorescent Nucleic Acid Techniques Idaho Technology1/03-12/07$1,652,000 Center for Homogeneous DNA Analysis State of Utah7/03-6/04$150,000 Homogeneous Mutation Scanning NIH STTR (Phase I and II) 7/04-12/06$850,000 Integrated Amplification and Mutation Scanning NIH STTR (Phase I and II) 1/05-6/07$850,000

69. Financial Plan  Projects initiated in 2 nd year are expected to break even during 4 th year  Licensing of homogeneous repeat typing and sequencing possibly to Idaho Technology, Inc. (matching funds) –or to Roche  4 th and 5 th year will focus more on market penetration  Generic reagent and ASR revenue in 4 th and 5 th year will reach $2-3 Million/year  Spin-off DNAWizard.com in 3 rd year  Chip platform will be ready for the market in last year of center operation With a 5% market share this would equal $40Million/year