Center of Excellence Center for Homogeneous DNA Analysis

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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

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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

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

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First year of COE -AchievementsApplications

• Mutation Scanning
• Software
• HLA Matching
• Unlabeled Probe Genotyping
• Amplicon melting - SNPs

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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 with A, C, G, or T at one position
• PCR primers on each side spaced 50 bp apart

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Mutation Scanning - Toolbox

• This data represents 1248 different calls in the
  Toolbox constructs

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Mutation Scanning - Toolbox
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Mutation Scanning - Toolbox
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Mutation Scanning - Toolbox
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Normalized and temperature shifted melting
profiles
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Dependence of area difference on product length
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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)

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Software Demonstrations

• Genotype clustering of high-resolution melting
  data
• Web SNPWizard
• Spiking animation for genotyping
• Genome-wide SNP nearest neighbor frequencies

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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

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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

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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

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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.

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HLA Inheritance
A1 A2
A3 A1-A3 A2-A3
A4 A1-A4 A2-A4
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CEPH Family UT1331
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Melting Curve of HLA-A Exon 2
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Sibling Genotypes
HLA Locus HLA Class Genotype 1 Genotype 2 Genotype 3 Genotype 4
A I 9, 10, 16 4, 7 8 3, 5, 6, 11, 17
B I 9, 10, 16 4, 7 8 3, 5, 6, 11, 17
C I 9, 10, 16 4, 7 8 3, 5, 6, 11, 17
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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

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Asymmetric PCR
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Amplicon and Probe Peaks with Asymmetric PCR
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Mismatch-detection of Homozygous Template
(LCGreen I)
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Mismatch-detection of Heterozygous Template
(LCGreen I)
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Mismatch-detection of Heterozygous Template (Sybr
Green I)
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Effect of Unlabeled Probe Length (LCGreen I)
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Genotyping of deltaF508 (LCGreen I)
deltaF508 hom
Wild type
deltaF508 het
-dF/dT
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SNP Genotyping with LCGreen I
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First year of COE - Achievements Amplicon
melting - SNPs

• Successful genotyping of all possible SNPs shown
  with plasmids.
• Demonstrated on clinically significant mutations.

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Homozygote Amplification
One Homoduplex
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Heterozygote Amplification
Two Homoduplexes
Two Heteroduplexes
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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

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Engineered SNP pBR322 Plasmids
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Clinical Samples
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Spiking Experiments
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Comparison of Methods for Real-Time SNP Typing
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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

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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)

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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)

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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)

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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 primers GC content be compared
  to the repeats GC content?

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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, 2071-75)
• 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, 31e121)
• Digestion with lambda exonuclease can eliminate
  one strand after PCR if one primer is
  5phosphorylated.

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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

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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

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DNAWizards commercialization

• Software purchase/upgrades
• Fee per use
• Contract design/analysis
• User support and education
• Oligonucleotide synthesis partnership
• Clinical lab partnership

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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

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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

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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

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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

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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

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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)

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Estimates

• Our methods will eliminate 95-99 of high-cost
  conventional DNA sequencing
• Global market for Centers 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

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Program Schedule
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Competitive Analysis-Homogeneous Repeat Typing
and Sequencing
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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

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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

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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

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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

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Economic Impact

• Create, attract and retain highly skilled
  technical workforce
• Attract possible out-of-state investment to fund
  COEs 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

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Organizational Structure
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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

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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
• CtWizard
• TypeWizard

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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

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Current and Pending Support
Title Agency Dates Amount
SNP Typing without Probes U of U Research Fund 7/3-6/05 70,000
Fluorescent Nucleic Acid Techniques Idaho Technology 1/03-12/07 1,652,000
Center for Homogeneous DNA Analysis State of Utah 7/03-6/04 150,000
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
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Financial Plan

• Projects initiated in 2nd year are expected to
  break even during 4th year
• Licensing of homogeneous repeat typing and
  sequencing possibly to Idaho Technology, Inc.
  (matching funds) or to Roche
• 4th and 5th year will focus more on market
  penetration
• Generic reagent and ASR revenue in 4th and 5th
  year will reach 2-3 Million/year
• Spin-off DNAWizard.com in 3rd 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