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
Unlabeled
Single
Hybridization
Amplicon
Probes
Probes
as
(Simple
Probes
(LCGreen)
the Probe
Probes)
(HybProbes)
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 -Achievements
Applications



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
Mutation Scanning
Software
HLA Matching
Unlabeled Probe Genotyping
Amplicon melting - SNPs
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
     
Mutation Scanning - Toolbox
 This data represents 1248 different calls in the Toolbox
constructs
Mutation Scanning - Toolbox
Mutation Scanning - Toolbox
Mutation Scanning - Toolbox
Normalized and temperature
shifted melting profiles
Dependence of area difference on
product length
30
Area Difference
25
20
15
10
5
0
70
100
150
200
250
300
350
Product Length (bp)
400
450
550
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 highresolution 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.
HLA Inheritance
A3
A1
A1-A3
A2
A2-A3
A4
A1-A4
A2-A4
CEPH Family UT1331
Melting Curve of HLA-A Exon 2
Sibling Genotypes
HLA
Locus
HLA
Class
Genotype
1
Genotype
2
Genotype Genotype
3
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
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
Asymmetric PCR
Amplicon and Probe Peaks with
Asymmetric PCR
Mismatch-detection of Homozygous
Template (LCGreen I)
Mismatch-detection of Heterozygous
Template (LCGreen I)
Mismatch-detection of Heterozygous
Template (Sybr Green I)
Effect of Unlabeled Probe Length
(LCGreen I)
Genotyping of [delta]F508
(LCGreen I)
5
[delta]F508 hom
Wild type
-dF/dT
4
[delta]F508 het
3
2
1
0
60
Temperature (°C)
70
SNP Genotyping with LCGreen I
First year of COE - Achievements
Amplicon melting - SNPs
 Successful genotyping of all possible
SNPs shown with plasmids.
 Demonstrated on clinically significant
mutations.
Melting Curves
Homozygote Amplification
One
Homoduplex
T
A
Temperature
Melting Curves
Heterozygote Amplification
Two
Heteroduplexes
Observed Combination
of 4 Duplexes
Two
Homoduplexes
T
A
C
A
C
G
T
G
Temperature
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
Forward Primer
5’
3’
A
T
3’
5’
Reverse Primer
Engineered SNP pBR322 Plasmids
Clinical Samples
Spiking Experiments
Comparison of Methods for RealTime SNP Typing
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:

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Homogeneous sequencing and repeat typing (U-3601) [optioned to IT,
Inc through 7-2004]
Integrated primer synthesis and target amplification on arrays (U3570) [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, 20:7175)
 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
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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

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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
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
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
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 highresolution melting: $50,000 and $70,000
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
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)
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
Program Schedule
Competitive Analysis
-Homogeneous Repeat Typing and
Sequencing
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
Competitive Analysis
- Arrays for Real-Time PCR and HighResolution 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
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
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
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
Organizational Structure
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
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
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
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
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