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
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
Hybridization Probe Formats Adjacent Hybridization Probes (HybProbes) Unlabeled Probes (LCGreen) Single Probes (Simple Probes) Amplicon as the Probe
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
First year of COE -AchievementsApplications • Mutation Scanning • Software • HLA Matching • Unlabeled Probe Genotyping • Amplicon melting - SNPs
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
Mutation Scanning - Toolbox • This data represents 1248 different calls in the Toolbox constructs
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
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)
Software – Demonstrations • Genotype clustering of high-resolution melting data • Web SNPWizard • Spiking animation for genotyping • Genome-wide SNP nearest neighbor frequencies
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
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
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
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.
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
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
First year of COE - Achievements Amplicon melting - SNPs • Successful genotyping of all possible SNPs shown with plasmids. • Demonstrated on clinically significant mutations.
Homozygote Amplification One Homoduplex
Observed Combination of 4 Duplexes Heterozygote Amplification Two Homoduplexes Two Heteroduplexes
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
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
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)
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)
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)
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?
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.
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
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
DNAWizards commercialization • Software purchase/upgrades • Fee per use • Contract design/analysis • User support and education • Oligonucleotide synthesis partnership • Clinical lab partnership
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