Center of Excellence Center for Homogeneous DNA Analysis

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) Unlabeled Probes (LCGreen) Single Probes (Simple Probes) 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-1TM and LCGreenTMI available in US • Distributors in Japan, Italy, and Korea established

5. First year of COE -AchievementsApplications • Mutation Scanning • Software • HLA Matching • Unlabeled Probe Genotyping • Amplicon melting - SNPs

6. Mutation ScanningUse 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 withA, C, G, or Tat 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

9. Mutation Scanning - Toolbox

10. Mutation Scanning - Toolbox

11. Normalized and temperature shifted melting profiles

12. 30 25 20 15 Area Difference 10 5 0 70 100 150 200 250 300 350 400 450 550 Product Length (bp) Dependence of area difference on product length

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

21. CEPH Family UT1331

22. Melting Curve of HLA-A Exon 2

23. Sibling Genotypes

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. 5 4 3 2 1 0 60 70 Temperature (°C) Genotyping of [delta]F508 (LCGreen I) [delta]F508 hom Wild type [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. Observed Combination of 4 Duplexes Heterozygote Amplification Two Homoduplexes Two Heteroduplexes

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 • CtWizard: free web trial, $200 software • TypeWizard: free web trial, $100 software