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

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

0

5

10

15

20

25

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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.

HLA InheritanceHLA Inheritance

A1A1 A2A2

A3A3 A1A1--A3A3 A2A2--A3A3

A4A4 A1A1--A4A4 A2A2--A4A4

CEPH Family UT1331

Melting Curve of HLA-A Exon 2

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

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)

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3

2

60 70

Temperature (°C)

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0

4Wild type

[delta]F508 hom

[delta]F508 het

-dF

/dT

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

Temperature

Homozygote Amplification

OneHomoduplex

TA

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’

Engineered SNP pBR322 Plasmids

Clinical Samples

Spiking Experiments

Comparison of Methods for Real-Time 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:

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

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 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

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