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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
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)Single Probes (Simple Probes)Unlabeled Probes (LCGreen)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 with A, C, G, or T at one position
PCR primers on each side spaced 50 bp apart:
X
Dependence of area difference on product length
0
5
10
15
20
25
30
70 100 150 200 250 300 350 400 450 550
Product Length (bp)
Are
a D
iffe
ren
ce
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.
Sibling GenotypesHLA
LocusHLA
ClassGenotype
1Genotype
2Genotype
3Genotype
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
Genotyping of [delta]F508 (LCGreen I)
5
3
2
60 70
Temperature (°C)
1
0
4Wild type
[delta]F508 hom
[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.
Melting Curves
Temperature
CG
TA
Heterozygote Amplification
TwoHomoduplexes
TwoHeteroduplexes
C
A
T
G
Observed Combinationof 4 Duplexes
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
AT
Forward Primer
Reverse Primer
5’5’3’
3’
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
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 high- resolution 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
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 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
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
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