center of excellence center for homogeneous dna analysis n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Center of Excellence Center for Homogeneous DNA Analysis PowerPoint Presentation
Download Presentation
Center of Excellence Center for Homogeneous DNA Analysis

Loading in 2 Seconds...

play fullscreen
1 / 69

Center of Excellence Center for Homogeneous DNA Analysis - PowerPoint PPT Presentation


  • 184 Views
  • Uploaded on

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

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Center of Excellence Center for Homogeneous DNA Analysis' - cleopatra


Download Now An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
center of excellence center for homogeneous dna analysis

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
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
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
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
First year of COE -AchievementsApplications
  • 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
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
Mutation Scanning - Toolbox
  • This data represents 1248 different calls in the Toolbox constructs
dependence of area difference on product length

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

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
First year of COE - Achievements Amplicon melting - SNPs
  • Successful genotyping of all possible SNPs shown with plasmids.
  • Demonstrated on clinically significant mutations.
slide35

Observed Combination

of 4 Duplexes

Heterozygote Amplification

Two

Homoduplexes

Two

Heteroduplexes

small amplicon primer design
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
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
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
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
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
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
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
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
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
DNAWizards commercialization
  • Software purchase/upgrades
  • Fee per use
  • Contract design/analysis
  • User support and education
  • Oligonucleotide synthesis partnership
  • Clinical lab partnership
software commercialization plan
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
financial plan
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