SBIR Phase II Proposal “A DNA Taggant Watermarking System“
0 Project Overview
DNA Taggants have moved to the forefront of technologies for supporting covert,
highly multiplexed, track-and-trace applications. While brand security is one
market niche that has been exploited by a number of companies, there is an
unmet need to develop DNA taggant technology, which can be used robustly for
covert operations by the military and police. The components lacking heretofore,
was a simple, compact, low-cost means to amplify and detect DNA-tagged items.
We propose in this Phase II application a complete solution to meet this covert,
taggant market niche. Such a complete solution includes a reliable amplification
procedure, which is both enzyme-free and isothermal; a lateral-flow assay
format, which is low-cost and used in simple pregnancy test kits; and, a simple
colorimetric readout, which can be measured both by eye, and, with a suitable
reader, quantitated for highly sensitive readout.
In Phase I of this grant, we generated two novel protocols (one of which is
enzyme-free) for amplifying or detecting a DNA Taggant isothermally – methods
which have generated significant attention from external companies.
Nonetheless, these protocols fulfill our Phase I goals such as i) the number of
distinct DNA Taggants are scalable to the millions and more, ii) the DNA
Taggants are not easily detectable without certain secret information, and iii) with
this information, only a few DNA Taggants molecules are required for detection.
Furthermore, by creating isothermal amplification and detection protocols, we
have not only gone beyond the goals of Phase I, but also superseded them with
a method that allows simple low-cost readout in the field.
Current DNA Taggant technologies are termed “forensic”. In the brand-security
market, ”forensic” is defined as meaning that the sample must be sent back to a
lab for PCR amplification and detection. In military and policing applications,
specifically espionage-based, the removal of the article of interest and delivery to
a testing facility is unacceptable. Indeed, even after lab delivery, the amount of
time necessary to verify a DNA signature is, at best, on the order of hours. Our
DNA Taggant system, however, allows the user to covertly test an article in the
field that has had a DNA taggant either directly applied, or indirectly transferred
to its surface using a simple colorimetric assay based on gold-nanoparticles, all
performed on a “paper” strip.
The “paper” strip, actually a cellulose-based
membrane embedded with the necessary reagents, will both amplify the taggant
and provide a visual cue for its presence.
As mentioned, our isothermal DNA amplification and detection protocols are
central components developed in Phase I that are now key to our current
capability to develop a complete, field-deployable system in Phase II. These
isothermal reactions offer significant portability by omitting the need for the
presence of a bulky thermocycler. Furthermore, not only is the presence of a
thermocycler obviated, but also the isothermal amplification reaction can occur
on a membrane strip using colloidal gold particles, with a visual readout. One of
our detection protocols can amplify a DNA taggant without the use of enzymes,
which not only lowers cost, but also more importantly, improves portability by
discarding of the most fragile reagent in DNA amplification, the enzyme (not only
do enzymes need ultra-cold refrigeration for reasonable lifetimes, but they also
are thermally and chemically sensitive in situ, the amplification reaction). For
increased quantization and sensitivity of readout, we will also test (optional to the
system) a commercial portable optical reader of the membrane strips.
Our Phase II tasks will be to assemble a full-scale prototype of our DNA Taggant
system and field test the aforementioned system. The project has numerous
facets, and we envision the following breakdown in tasks:
Task I will be software development for design of DNA Taggants and its use for
expansion of our DNA taggant library to 20 distinct DNA strands (allowing
representation of 380 virtual Taggants as pairs of distinct DNA strands).
Task II will be optimization of the two isothermal amplification and detection
protocols for our expanded DNA taggant library. This will involve “wet-lab” work
and computer software simulations to optimize our protocol over a range of
environmental conditions (e.g. temperature, buffer solutions, contaminants, etc.).
Task III will be to combine a commercially available colorimetric DNA detection
system based on colloidal gold, from Nanosphere Inc., with our proprietary
isothermal DNA amplification protocols. We will also in this task extend this
colorimetric DNA detection method to detect the AND of a pair of distinct DNA
strands chosen from the library.
Task IV will involve transferring the detection protocols of Task II to a lateral flow
(dipstick) format on membrane strips. (We don’t envision this to be a significant
hurdle as Nanosphere’s detection format is already membrane-based.) The
demonstration of our detection apparatus includes (see detailed work schedule)
demonstration of manufacturing via automation (the spotting machine used),
which will provide a key demonstration of our ability to manufacture the product
using automation. The extreme portability of our resulting DNA taggant detection
apparatus will demonstrate it has a unique and critical commercial advantage
over any other DNA detection method with similar sensitivity.
Task V will be extensive tests of the DNA Taggant system, and optimization of
the detection protocols and colorimetric readout on membrane strips. (Here we
will employ a commercial portable optical reader for increased quantization and
sensitivity of colorimetric readout of the membrane strips. The optical reader will
mostly be used to optimize performance; it will be optional for use in the actual
product.)
Finally Task VI will be demonstrations of use of full-scale prototype for various
applications of commercial interest. Our planned application demonstrations will
provide dramatic demonstrations in key application market areas that will be of
critical use for marketing (and they will also substantially aid and foster partnering
with companies that produce products in these markets).
(We also include an optional Task VII to demonstrate DNA nanostructure
Taggants that are hard to detect and that degrade in the environment even
slower.)
1 Identification and Significance of the Problem and Opportunity
1A Identification of the Problem
Challenge 1: We address a key challenge in many military and commercial
systems: the tracking of materials and/or documents, and the certification of their
origin (watermarking). Many material distribution and administration systems, in
both the military and commercial sectors, are plagued with a vast number of
materials and documents without adequate methods for certification of their
origin (watermarking). The materials of concern include weapon parts and
ammunition as well as a wide variety of commercial products that are vulnerable
to counterfeiting. The interjection of counterfeit parts and/or documents is
extremely disruptive to these military and commercial systems.
Challenge 2: We also address another key challenge in intelligence: the tracing
of personnel so that their movements can be monitored, and networks of
interactions mapped. Counter-terrorist efforts can require targeting an
organization consisting of a network of many individuals. (Similar efforts are
required for targeting an organized crime organization.) This is a central need for
intelligence gathering and surveillance. Monitoring by conventional intelligence
methods would be extremely costly (or impractically asset-intensive).
A common solution to the tracking of materials is to make use of Taggants, which
are small objects, placed on materials and/or documents. More precisely,
Taggants can be defined to be any substance that can be added to an object or a
surface so that the origin of such objects can be traced, or the movement
monitored. Taggants have many uses. They can be used for watermarking
objects and/or documents. Taggants can also added to or placed on a material or
individual to trace movement. For example, unique plastic taggants are added to
explosive compounds to later aid identification of explosive origins. But to date
commercial taggants have limited diversity (compared to the number of part
types) and are easily detectable.
DNA Taggants are an application of DNA biotechnology that provides potentially
revolutionary improvements in intelligence-gathering and surveillance
capabilities. Briefly, a DNA taggant is a molecular taggant system using DNA and
employing amplicon and oligonucleotide taggants. More precisely, DNA
Taggants are unique combinations of uniquely synthesized DNA that can be
added to compounds or the surfaces or objects and/or personnel so that the
origin of such objects can be traced, the movement monitored, or networks of
interactions mapped.
The use of DNA Taggants potentially provides high impact and revolutionary
improvements to intelligence-gathering and surveillance capabilities. Since the
DNA Taggants operate on a molecular level they are extremely discreet, it is
difficult to determine their use from all the other DNA in the environment.
A set of distinct DNA Taggants used together can represent a unique molecular
barcode, increasing the encoding capacity of the taggant system (as
demonstrated in prior work of Reif). Example uses of DNA Taggants that have
been demonstrated by Reif include:
-Opening detection: detection of use of a laptop using two taggants:
one outside and one inside,
-Authentication of a person via a DNA Taggant,
-Detection of Taggant residual after sequential chaining of multiple
handshakes.
The drawback to the above use of DNA Taggants is that, up to now, the
detection apparatus is a PCR machine, and the equipment required for running
and monitoring the reaction is rather bulky (additionally, the enzymes required
refrigeration). This severely limits the application of DNA Taggants to these
applications.
1B Current state of art: prior research and development
First we will provide a brief historical overview of DNA Taggant technology. Then
this is followed with a discussion of a prior approaches to DNA detection.
Prior DNA Taggant Systems:
Note of Prior Pioneering AFSOR Support of DNA Taggant Research:
Interesting, the AFSOR has been the contract support for all of the major
research projects in DNA Taggants (the co-funding partners have included the
CIA and DARPA) that we have been able to learn about.
Competing Methodologies
Several varieties of alternative molecular and nanoscale taggant systems have
been developed. These include fluorescent chemical tags, plastic microbeads,
barcoded nanoparticles, etc. There are direct-read and amplification-based
strategies where, respectively, the signaling components are detected at the
labeling concentrations or the signal molecules are replicated prior to detection.
The majority of these techniques require instrumentation or equipment that may
inhibit certain field applications.
Barcoded nanoparticles labeled with a mixed SAM of organic chemicals sealed
onto the NP surface by deposition of silica can be sensitively detected using
surface-enhanced RAMAN scattering. Although the cost and size of RAMAN
spectrometers have dropped markedly in recent years, the laptop-sized or
toaster-sized instrument may still be an undesired burden during rapid field
analysis compared to a simple dipstick or drip-stick assay.
Many competing methods have interesting applications but none offers the
combination of ease of use, low cost, wide applicability, and rapid analysis
provided by Eagle Eye DNA taggant systems, as descried below.
We will characterize prior DNA Taggant Systems as follows (using the Gen
terminology used for night vision systems):
Gen1 DNA Taggants: These use no amplification. They have widespread use
in commercial sector. DNA molecules are used to embed fluorescent dyes for
tags of authenticity.
GEN2 DNA Taggants: [Ellington, contract by AFSOR/CIA in middle 1990s]
Ellington used PCR Amplification for single Taggant detection. These are now
termed “forensic taggants” in commercial sector and are employed when
increase detection sensitivity is required.
GEN3 DNA Taggants: [Reif, supported by AFSOR/DARPA in 2002-2003] Reif
developed and experimentally demonstrated advance protocols for multiplexed
PCR amplification to allow one to simultaneously identify many distinct DNA
Taggants at the same time.
GEN4 DNA Taggants: [current AFSOR SBIR] Vastly improved portability of the
DNA detection apparatus by use of isothermal detection protocols and
colorimetric output visible to the eye.
Now we discuss these various generations of DNA Taggant Systems in more
detail.
GEN1 DNA Taggant Systems
The potential for artificial DNA or other nucleic acid sequences to be used as
taggants has long been recognized. There are two major advantages to using
DNA sequences as taggants: first, DNA sequences can be amplified and
sensitively detected using the polymerase chain reaction. Theoretically, even a
single molecule of a particular DNA sequence can be identified using off-theshelf technologies. In practice, at least a few dozen molecules are generally
required for PCR amplification. Second, the chemical composition of DNA can be
readily and easily varied by varying the sequence of the DNA. Indeed, short (<
150 residue) strands of DNA can easily be synthesized using automated
methods. Thus, many different DNA molecular taggants can be created at will,
without resorting to complex synthetic methods. For these reasons, there is a
substantially growing market for using DNA molecular taggants to mark sports
memorabilia and other valuable objects, as a way of guarding against forgery.
However, it should be noted that most so-called DNA Taggants that are
advertised by companies are not amplicons. Instead, DNA molecules are used
as platforms to embed fluorescent dyes, and it is in fact the dyes that are the real
tags of authenticity. These methods are primarily used for guarding against
forgery, for example, to mark sports memorabilia and other valuable objects.
Current Drawbacks: since these DNA Taggants are just platforms to embed
fluorescent dyes used as tags of authenticity, and are not amplicons, their use
requires large amounts of Taggant that is not easy to disguise, and their
fluorescent detection method is not at all sensitive.
GEN2 DNA Taggants:
Pioneering prior research on molecular taggants was executed by Andrew
Ellington, under ONR contract. Ellington designed and synthesized a series of
random sequence libraries of nucleic acid taggants. By generating and cloning
oligonucleotides in which a large number (ca. 10 15) different random sequences
are flanked by constant sequences, he proved it possible to immediately
generate very large numbers of these taggants. He assessed the amplification
properties of individual taggants from these libraries using real-time PCR on the
Cepheid SmartCycler, and detected down to 4 individual molecules (in the
context of a plasmid, in order to stabilize taggants against degradation by
environmental nucleases). In addition, he identified how well nucleic acid
taggants survive on common surfaces (up to 23 days in the context of a plasmid)
and in soil (up to 14 days in the context of a plasmid), and how readily taggants
can be detected in soil (down to 100 individual molecules). He looked at the
ability of taggants to be transferred between individuals (1 microgram of plasmid
DNA, coresponding to ca. 1011 molecules), and observed detection following six
serial dilutions via handshake. He has further examining taggant recovery and
detection under a wide variety of conditions relevant to field operations.
In addition to developing appropriate methods and assays for amplicon taggants,
the Ellington lab has begun to work towards methods for utilizing oligonucleotide
taggants. In particular, he developed deoxyribozyme ligases that can capture
oligonucleotide taggants.
Eric Kool and his co-workers have previously
developed a novel ligation chemistry in which a 3' phosphorothioate residue
displaces a 5' iodine to form an unnatural internucleotide linkage with a bridging
sulfur atom (Xu and Kool, 1997, 1999). Ellington used this chemistry as the basis
for selecting novel deoxyribozyme ligases that accelerate the basal rate of the
template-directed iodine displacement reaction (Levy and Ellington, 2001). This
prior research will guide us in examining DNA taggant recovery and detection
under a wide variety of conditions relevant to field operations.
GEN3 DNA Taggants:
This prior GEN2 DNA Taggant system developed by Reif was funded by
AFSOR/DARPA 2002-2003 via a special increment for developing antiterrorist
technology in response to 9/11. (To keep costs down, Reif volunteered his time
free of charge and also financed from other funds a smart cycler real time PCR
system). Reif developed and experimentally demonstrated advanced protocols
for multiplexed PCR amplification to allow one to simultaneously identify many
distinct DNA Taggants at the same time. The protocols made combinatorial use
of pairs of k=16 DNA strands to represent k(k-1)/2 = 120 taggants.
The project demonstrated example uses of the DNA Taggant system:
-Opening detection: detection of use of a laptop using two taggants: one
outside and one inside
-Authentication of a person via a DNA taggant
-Detection of Taggant residual after sequential chaining of multiple
handshakes
-Detection of DNA Taggants on various surfaces: painted(oil and latex) metal,
wood, plastic, skin, clothes, money.
This GEN3 DNA Taggant project used a stand-alone, off-the-shelf commercial
real-time PCR machine known as the Cepheid SmartCycler™ system, which had
a thermal cycler and multiple wells with built-in optical excitation and observation.
But this equipment was rather bulky and the enzymes required refrigeration.
GEN4 DNA Taggants:
Eagle Eye's AFSOR SBIR Phase I has demonstrated the basic capabilities of a
DNA amplification and detection apparatus with vastly improved portability. The
DNA detection apparatus make use of isothermal amplification and detection
protocols and colorimetric output visible to the eye. This makes the application of
DNA Taggants much more feasible, and creates a very attractive commercial
opportunity, as described in detail in the following sections.
Prior Approaches to DNA detection:
Conventional PCR Methods using Thermal Cycling:
- Conventional PCR provides an exponential DNA amplification, and hence is
exquisitely sensitive. However, most of the conventional detection methods
based on PCR require thermal cycling, which makes the DNA amplification
apparatus bulky. Also, it uses energy to do the thermal cycling, and its enzymes
generally require refrigeration. After amplification, there are many known
detection methods including fluorescence, chemiluminescence, as well as
molecular beacons. There are many commercial real-time PCR systems which
have thermal cyclers with multiple wells with built-in optical excitation and
observation, but this the equipment required for running and monitoring the
reactions are rather bulky (and generally, the enzymes require refrigeration).
- TMA Detection (GEN-PROBE): Uses Thermal Cycling with Polymerase &
Reverse Transcriptase for Amplification. It is very sensitive, but is not isothermal.
- LCR Detection (Abbott Labs): Uses Thermal Cycling with antibody-antigen
reaction to detect ligated probes using fluorescent labels. However, this method
is does not provide highly sensitive detection of DNA and is not isothermal.
Isothermal DNA Amplification Systems:
- Rolling Circle Amplification.
Rolling circle amplification (see [Dolinnaya et al, 1993], as [Nallur, et al, 2001]
and [Dean, et al 2001]) is an isothermal DNA amplification technique, which uses
circularization and primer extension, to provide amplification of specified DNA
strands. It has the advantage over PCR of not requiring thermal cycling, and
thus making the amplification apparatus somewhat more transportable. However,
this method results in only a linear rate of amplification (as opposed to the usual
exponential rate of amplification of the detected signal observed by PCR), so is
not exquisitely sensitive.
References for DNA Rolling circle amplification:
Dolinnaya et al, Oligonucleotide circularization by template-directed chemical
ligation, (1993) N.A. Res 21, 5403-5407
Nallur, G. et al. Signal amplification by rolling circle amplification on DNA
microarrays. Nucleic Acids Res. 29, e118 (2001).
Dean, F.B., Nelson, J.R., Giesler, T.L. & Lasken, R.S. Rapid amplification of
plasmid and phage DNA using phi29 DNA polymerase and multiply primed rolling
circle amplification. Genome Res. 11, 1095-1099 (2001).
- Strand Displacement Amplification.
Bectondickinson, Inc (BD Technologies) have designed an isothermal DNA
detection system known as “strand displacement amplification” with exponential
amplification. Their strand displacement amplification method uses a polymerase
and a protein restriction enzyme that require refrigeration to avoid degradation.
Their detection methods include fluorescence polarization, chemiluminescence,
or colorimetric signals. However, this method is quite difficult to use in practice,
and requires many months to optimize for a single amplification experiment for a
novel DNA strand.
References for Strand Displacement Amplification:
Walker GT, Linn CP, Nadeau JG, Nucleic Acid detection by strand displacement
amplification and fluorescence polarization with signal enhancement using a
DNA binding protein, NUCLEIC ACIDS RES 24: (2) 348-353 JAN 15 1996.
Walker GT, Nadeau JG, Linn CP, et al. Strand displacement amplification (SDA)
and transient state fluorescence polarization detection of Mycobacterium
tuberculosis DNA, CLIN CHEM 42: (1) 9-13 JAN 1996.
- NanoGold Colorimetric Detection (Mirkin, NanoSphere, Inc): Uses aggregation
of DNA labeled Gold to provide a color change visible to the eye to detect DNA.
This method is isothermal. However, this method is does not provide highly
sensitive detection of DNA. (We use similar techniques for output, but only after
the DNA is amplified.)
References:
Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A.
Selective Colorimetric Detection of Polynucleotides Based on the DistanceDependent Optical Properties of Gold Nanoparticles, Science, 1997, 277, 10781080.
Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. One-Pot
Colorimetric Differentiation of Polynucleotides with Single Base Imperfections
Using Gold Nanoparticle Probes, J. Am. Chem. Soc., 1998, 120, 1959-1964.
Figure: Comparison of Major Current methods for DNA detection and
amplification.(Gen-Probe)
For a survey of current methods for DNA detection and amplification, see also:
DNA amplification: Current Technologies and Applications, ed by Vadim V
Demidov and Natalia E. Broude, Horizon Bioscience, Norfork, UK, 2004
1C Identification of the Opportunity for a Commercial Product
DNA computing needs commercial applications
DNA computing is defined as the use of DNA to execute computations. To date,
there has been one successful commercialization of DNA computing: a Japanese
device for biomedical applications. Do date, there are no established markets for
DNA computing capabilities or the software for design of DNA libraries used in
DNA computing, outside of biomedical applications.
Why DNA Taggants Present great commercial opportunities
However, there are considerable existing commercial markets for Taggants
(using known data, we determine this market to be at least 1 billion dollars per
year). A DNA Taggant system needs to detect minute amounts of specific DNA
in environments with many other distinct DNA. The well-known PCR assay for
DNA amplification can be used for DNA detection, but suffers from a number of
disadvantages: the apparatus for PCR is not very portable, requires high energy,
and moreover the enzymes used degrade rapidly without refrigeration. This nonportability characteristic severely limits the application of DNA Taggants to the
intended military and commercial applications.
Current DNA Taggant technologies are termed “forensic”. In the brand-security
market, ”forensic” is defined as meaning that the sample must be sent back to a
lab for PCR amplification and detection. In military and policing applications,
specifically espionage-based, the removal of the article of interest and delivery to
a testing facility is unacceptable. Indeed, even after lab delivery, the amount of
time necessary to verify a DNA signature is, at best, on the order of hours.
The Specific Opportunity: A Highly Portable DNA Taggant Detection
System
To ensure a major and significant commercial advantage over other Taggant
systems (both on the market and/or proposed), the most significant requirement
our DNA Taggant system is that the detection device is highly portable; also it
requires the following additional properties: the software for design of these DNA
Taggants allows for scaling to multiple distinct Taggants, the DNA detection
assay is exquisitely sensitive and swift, and be able to discriminate between
distinct Taggants and not generate false positives from environmental DNA
and/or other sample containments, and if possible that the assay use reagents
that degrade slowly without refrigeration.
Why Development of a Portable Isothermal DNA Taggant Detection Apparatus
are key to Commercial Viability:
The recent AFSOR SBIR Phase I have developed a very portable DNA detection
apparatus, which is far more transportable, and provide us with a powerful
commercial advantage over other more conventional PCR amplification methods.
This advantage would hold both for DNA taggant detection applications for
government customers including military intelligence, CIA, FBI, homeland
defense and police. Our DNA taggant system allows the user to covertly test an
article in the field that has had a DNA taggant either directly applied, or indirectly
transferred to its surface using a simple colorimetric assay based on goldnanoparticles, all performed on a “paper” strip. The “paper” strip, actually a
cellulose-based membrane embedded with the necessary reagents, will both
amplify the taggant and provide a visual cue for its presence. This portability
makes the application of DNA Taggants much more feasible, and creates a very
attractive commercial opportunity, and described in detail in the following
sections.
1D Conforming to SBIR Solicitation Objectives
Why we are meeting the SBIR Solicitation Objectives:
The SBIR SBIR Phase I Solicitation Topic “High Performance Biomolecular
Computing Architectures for Information Technology” description includes the
text “Areas of interest with near term potential include, but are not limited to
application of DNA as a taggant or authentication/watermark material where
information is stored in the DNA and retrieved as a function of detecting the tag.”
We meet this solicitated objective since our major goal is to develop a DNA
Taggant system.
2 Phase II Technical Objectives
2A Our Prior Phase I Results
Eagle Eye recently demonstrated the basic capabilities of a portable DNA
Taggant system while funded by an AFSOR Phase I SBIR contract FA8750-04C-0147 titled “A DNA Taggant Watermarking System“.
Overview of Phase I SBIR Results:
(i) Software for design of DNA Taggants. In our Phase I SBIR we have
developed software for design of DNA Taggants. For details, see Section 3.
(ii) Isothermal DNA amplification and detection protocols. Our key technical
challenge was to develop isothermal (that is, they operate without thermal
cycling) biochemical protocols to detect minute amounts of specific DNA in
environments with many other distinct DNA. Unlike conventional PCR, our DNA
amplification and detection protocols are isothermal and so our apparatus needs
no external energy source. The isothermal property is important, for it allows the
DNA detection device to be easily transportable and to not require significant
energy. Nevertheless, our DNA amplification and detection protocols are
exquisitely sensitive: given only few molecules of single stranded DNA, the
methods increase its concentration at an exponential rate with time. We
experimentally demonstrated two novel biochemical protocols for isothermal DNA
amplification and detection. (Details of these protocols are given in Section 3.)
One of our protocols uses no enzymes, and so requires no refrigeration of
reagents. In contrast, all known prior “exponential, isothermal” DNA amplification
and detection methods (such as the strand displacement developed by DB, Inc.)
require restriction enzymes.
(iii) Readout Methods. In our Phase I SBIR we also developed two distinct
methods for readout that are error-resilient: they both use self-assembled DNA
nanostructures that provides visible reporting of the logical AND of multiple
independent DNA strand detection tests, and considerably drops the falsepositive rate of detection errors. The methods are:
o
A fluorescent (FRET-based) readout method, and
o
A colorimetric readout method using color changes due to the clustering of
gold spheres with affixed DNA.
Our colorimetric readout method, derived from methods developed by Mirkin,
uses nanostructures to provide the indication of DNA detection via a color
change detectable by the eye. This read-out method can be made error-resilient
since it uses a self-assembled DNA nanostructure that provides visible reporting
of the logical AND of multiple independent DNA strand detection tests, and
considerably drops the false-positive rate of detection errors.
(iv) Design for the detection apparatus. In our Phase I SBIR we made a design
for the detection apparatus using one of our isothermal DNA detection assays
with colorimetric output. The envisioned detection device is very compact: it is on
a dip stick of just a few centimeters long where one puts a drop of liquid sample
at one end and the other end produces, in less than 30 minutes, a colorimetric
change to indicate detection of a specific strand of DNA.
(v) Protecting DNA Taggants from environmental degradation and discovery.
Furthermore, in our Phase I SBIR we developed methods for protecting DNA
Taggants from environmental degradation and discovery by the use of DNA
nanostructures; these protect the DNA from action by restriction enzymes and
make the DNA difficult to detect by conventional methods (e.g., PCR).
The above results have considerable potential intellectual property right value
and we have already made provisional patent applications.
2B List of Phase II Technical Objectives
Our full scale DNA Taggant system to be constructed will include:
(i) A detection device and associated biochemical assay for detection of the
DNA Taggants, and
(ii) Software to design the DNA strands comprising the DNA Taggants.
(Means for synthesis, dispersal and capture of the DNA Taggants are well
known.)
Objective 1:
Software for design of DNA Taggants.
The software for design of DNA Taggants will allows for scaling to multiple
distinct Taggants.
Objective 2:
Our major Objective is development of a full-scale prototype portable DNA
detection apparatus:
- The DNA detection device will be a dip stick of just a few centimeters long
where one puts a drop of liquid sample at one end and the other end produces,
in less than an hour, a colorimetric change to indicate detection of a specific
strand of DNA.
- The colorimetric readout method will provide the indication of DNA detection via
a color change detectable by the eye. This readout method is error-resilient since
it uses a self-assembled DNA nanostructure that provides visible reporting of the
logical AND of multiple independent DNA strand detection tests, and
considerably drops the false-positive rate of detection errors.
It will have the following properties:
- The detection of specified DNA strands will be exquisitely sensitive: detecting
as few as 10-20 DNA strands/ sample. This entails the use of an assay providing
exponential DNA detection.
- The DNA detection apparatus will be very compact (Approx. 2 cm x 3 mm x 1
cm) and transportable (with a weight of approx. 3 to 5 grams), and with no
external energy cost.
- Ideally, we require that the DNA detection chemicals not require refrigeration.
- The DNA Taggant assay cost will be less than 25 cents per detection.
- The Taggant Assay will have a rate of false positives = approx. 1% (without
AND protocol), and approx. 0.01% (with AND protocol for two independent DNA
Taggants).
2C Phase II demonstration of commercial product
Planned Application Demonstrations of full-scale prototype DNA Taggant system:
(A) Demonstrations of counterfeiting protection:
- For a machine part in a material distribution system,
- For a copyrighted high cost product (e.g., a watch), and
- For an administration system (e.g., a document).
(B) Demonstrations of tamper detection to determine if a particular container or
object has been opened or tampered, for:
- For a shipping container,
- For a laptop, and
- For a restricted room.
(C) Demonstrations of intelligence applications:
- Demonstrations of surveillance of suspects,
- Demonstrations of mapping contact networks, and
- Demonstrations of use of DNA Taggants for agent authentication.
2D Measurable characteristics of planned product that indicate commercial
value:
acceptable ranges to be demonstrated
Taggant Assay Sensitivity:
- 10-20 DNA strands/ sample.
Taggant Assay Precision:
- Rate of False Positives = approx. 1% (without AND protocol)
- Rate of False Positives = approx. 0.01% (with AND protocol for two
independent DNA Taggants)
Weight of DNA detection Apparatus:
- Approx. 3 to 5 grams
(In comparison, sensitive DNA assays using PCR amplification require many
pounds of weight.)
Size of DNA detection Apparatus:
- Approx. 2 cm x 3 mm x 1 cm
Operating Temperature Range for DNA detection Apparatus:
- Isothermal (no change in temperature). Can be optimized for any given
ambient temperature T between 10 to 35 Centigrade; will perform within specs
within 5 degrees variation of chosen T.
Energy Consumption of DNA detection Apparatus:
- No external energy consumption required, since assays are isothermal
Longevity of Unrefrigerated Reagents of DNA detection Apparatus:
- Approx. 10 to 20 days for first assay using enzymes
- Over 120 days for second assay that makes no use of enzymes.
Longevity of DNA Taggant:
- Approximately 15 to 30 days under direct sunlight.
- At least 120 days away from direct sunlight.
Taggant Assay Cost:
10 and 25 cents per detection.
Cost Analysis: Current gel-based detection for the RCA protocol requires less
than 3x10-4 cents worth of DNA (for example 50 pmoles @ $85.00 for 1400
micromoles of a 50 nucleotide sequence). Cost for buffer components, dNTPs,
and nanoparticles are also quite negligible.
Preparation of custom
oligonucleotide derivatized nanoparticles requires portions of 3 days of technician
time but will decrease in per assay cost due to economy of scale when
manufactured in large production-scale batches. The major reagent expense for
the first detection assay using enzymes is the enzymes themselves (polymerase
and restriction endonuclease), which cost between 10 and 25 cents per reaction,
in total. The second assay, which is enzyme-free, avoids this expense and will
therefore cost less than a penny per assay for reagents. A reduction in the cost
for these procedures is expected when we complete conversion of the test to a
lateral flow strip method. (The exact amount of savings will not be known until
prototype test strips can be fabricated and the enzyme requirements are better
known.)
2E Reduction of risk in Phase II of new technology and evidence of reduced
risk
We take risk mitigation very seriously as a method to ensure the Phase II project
succeeds in its technical objectives.
(i) Risk mitigation for the amplification and detection protocols:
To mitigate risks inherent in the development of the amplification and detection
protocols for DNA Taggant, we will continue development of our two distinct
isothermal amplification and detection protocols for DNA detection. We expect
both to be viable, but that each will have specific advantages (e.g., speed of
detection vs. sensitivity) that may make them more attractive to use for specific
applications. Nevertheless, the detection apparatus is expected to be same for
both assays; the distinct assays just will entail the use of distinct reagents.
Furthermore, the optimization of the two distinct protocols may require somewhat
distinct buffer concentrations, sample preparation, etc.
Note: We will stress the development of the second (enzyme-free) protocol, since
it requires no enzymes and hence no refrigeration of reagents.
(ii) Other Risk: Safety Issues in use of DNA Taggants
One basic issue is the non-toxicity of DNA Taggants. To date, after numerous
studies, no toxic properties of DNA have been observed in studies in the
literature. Literature on environmental DNA indicates that approximately 15
microgram of DNA is found per gram of soil, mostly derived from living or dead
bacteria. A comprehensive survey is given in reference:
ANKE HENNE, ROLF DANIEL, RUTH A. SCHMITZ, AND GERHARD
GOTTSCHALK, Construction of Environmental DNA Libraries in Escherichia coli
and Screening for the Presence of Genes Conferring Utilization of 4Hydroxybutyrate, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept.
1999, p. 3901–3907 Vol. 65, No. 9.
One possible explanation for the non-toxicity of DNA is that the large and very
diverse environmental DNA indicates that most DNA is nontoxic. A side benefit of
this is that diffuse quantities of environmental DNA will help disguise covert use
of DNA Taggants.
2F Why our innovative approach is the best avenue to reaching goals
Here we give discriminators between our proposal and other approaches and a
list of advantages of our approach, describing why our approach is more feasible.
Advantages to using Synthetic DNA Sequences as Taggants
(i) The relative high stability of DNA
(ii) The chemical composition of DNA can be readily and easily varied by altering
the sequence of the DNA.
(ii) Established related biotechnology:
many different short (< 100 base) strands of DNA can easily be synthesized
using automated methods.
(iii) DNA sequences can be amplified and sensitively detected using the
polymerase chain reaction. Theoretically, a single molecule of a particular DNA
sequence might be identified. In practice, onecan detect as few as a dozen per
sample using off-the-shelf technologies.
(iv) DNA Taggants themselves can exist undetected on surfaces. Taggant
dispersion can be designed so that taggants can be transferred from surfaces
upon contact
(v) The taggant is itself the amplicon and therefore those who know the exact
sequence can amplify the amount of the taggant allowing for secured detection
Advantages over competing technologies for DNA Taggants
Most existing sensitive detection assays for DNA require some sort of very
sensitive detection assay such as PCR or its variants, requiring both thermal
cycling and optical detection apparatus. Also, PCR and its variants use the
enzymes such as polymerase that need to be refrigerated.
Our new amplification and detection protocols appear to have a major
"competitive advantage" in the major markets for DNA Taggants. This is primarily
due to the fact that our protocols are both ultra sensitive and also follow the
"homogenous assay" format- you only add reagents, and need to make no use of
any working devices (e.g., for thermal cycling and optical detection apparatus).
Our DNA amplification and detection protocols allow for very portable kits for
detection of DNA Taggants normally requiring a PCR assay in a lab. Our
detection assays could be done anywhere by a user, rather than at a lab, using
just a very small dipstick and using their own eyes for optical detection.
Therefore, the cost for the test would be reduced to the cost for the kit, and the
costs associated with the lab would not exist. Also, since our second DNA
detection protocols use no enzymes, it does not need to be refrigerated. So it
could be easily stored.
To our knowledge, there are no other known assays for ultra sensitive DNA
detection are even close to being feasible to develop as portable kits, and so our
assays may have the required "completive advantage" in the DNA Taggants
markets. That is, by avoiding a lab test, it may have a dramatic cost advantage.
Summary of Our Advantages over competing technologies:
There are many facets of our new technology that exceed those technologies
currently in the marketplace. The most notable features include
•
Sensitivity,
•
Ease-of-use,
•
Multiplicity,
•
Track-and-trace ability,
•
Transferable,
•
Secureness (need to know what you’re amplifying),
•
and Covertness.
In terms of sensitivity, our technique can amplify DNA Taggants at the most
sensitive level of only a dozen or so DNA strands per sample. Only DNA-based
technologies using assays for exponential amplification are capable of this
amplification process before detection.
Companies in the Taggant field include:
i) Sure Trace Security Corporation (produces many types of taggants including
DNA Taggants, generally not amplified)
For a description of their S-DNA marking technology, see:
http://www.suretrace.com/home.html
and http://www.smallcap.ca/Sure%20Trace%20Profile%2009%2029%2004.doc .
ii) InkSure Technologies (encoded by mixing chemical markers into inks for
document and product packaging tracking)
See http://www.inksure.com/html/Products/SmartInk.html .
iii) Authentix, Inc. (DNA taggant produc, generally not amplified)
See http://www.authentix.com/ .
iv) November, AG (markets DNA Taggants in ink)
http://www.november.de/english/portal.php?pageid=204&cat=product .
v) SmartWater Technology Limited (DNA taggant product, generally not
amplified)
See http://www.smartwater.com/about/about_smartwater.html
and
http://www.smartwater.com/news/archive/news33.html .
vi) Trace Tag International (DNA taggant product, generally not amplified)
See http://www.tracetag.com/ .
vii) Applied DNA Sciences (DNA taggant product, amplified)
See http://www.adnas.com/ .
viii) DNA Technologies, Inc (DNA taggant product, amplified)
See http://www.dnatechnologies.com/ .
Many companies are supplying upconverting phosphors as a means of covert
detection in brand security. While this technology does advertise field-based
simple readout methods, this technology lacks a number of other attributes, such
as multiplicity – only a handful of “flavors” of taggants exist; secureness – dozens
of companies offer this technology with its prevalence being its largest detractor,
due to the ease of counterfeiting; and sensitivity – a large amount of taggant
must be present on the article of interest to be visible to the eye (no inherent
amplification mechanism).
Other DNA taggant technologies, exist, for example, DNA Matrix™ developed by
DNA Technologies, Inc. and Applied DNA Sciences, Inc. which both market DNA
tags with track-and-trace ability. While verification of the presence of DNA is
available at the field level, actual authentication of a specific sequence must be
performed in a forensic mode at a fully equipped laboratory. Hence their
detection apparatus is not at all portable. With our complete taggant system, we
believe it is possible to verify the sequence, itself, at the field level. This belief is
made possible due to the inherent field-based amplification process of our
system, which allows a known probe matched to the specific item’s tag to
produce a colorimetric readout. Indeed, while hardening of our DNA taggant
molecules remains a goal throughout our investigations, we make the claim that,
again, as a result of amplification, we can tolerate significant degradation before
an item’s tag is undetectable.
Other track and trace technologies include Microtrace, Inc.’s polymeric striped
taggant, originally developed at 3M, which has most notably been used to tag all
explosives manufactured in Switzerland. While high multiplicity is possible, the
technology lacks other significant qualities such as sensitivity, transferability,
secureness, and most importantly covertness. Indeed, it is possible to make out
these relatively large particles by eye whose size ranges exceed 100um.
Other companies are focusing on fluorescent dyes as a means of security
tagging. While readout is relatively easy, these taggants have a host of other
problems. Probably the most significant shortcoming is the relatively small
codespace these taggants occupy.
They are more suited for simple
authentication, rather than any serialization application and even under these
conditions they are subject to counterfeiting, due to their ubiquitous nature.
3 Phase II work plan
3A Detailed Description of full-scale prototype Product to be developed
3A1 Design of Libraries of DNA Taggants and of DNA Nanostructures used
by our 2nd Detection Assay
Existing Software for Design of DNA Sequences
A key computational challenge in DNA self-assembly is to design DNA
sequences that form or do not form prescribed secondary structures. Abundant
efforts have been made in the DNA computing community to develop algorithms
and software that produce a pool of DNA sequences such that each sequence is
of maximal difference from others. These sequences can either be used as DNA
libraries with unique sequences or associate into linear DNA molecules. In
contrast, research efforts attacking the problem of designing DNA sequences
that uniquely assemble into desired secondary structures are comparatively
scarce. The current software tool widely used is a semi-automatic software
package is called Sequin, which was developed by Seeman in 1990. Though
being a very useful tool, Sequin requires a significant amount of manual
operation by the molecular architect in the sequence optimization process. In
addition, Sequin only provides a text interface that is not user-friendly and
intuitive for inputting and displaying the graphical nature topological information
of the desired secondary structures. As such, desirable improvements of Sequin
include relieving the molecular architect of the laborious process of manually
carrying out the designing tasks and providing a graphical user interface that can
take in the input as well as display of the final designed sequences.
In addition, there is a wide variety of other software developed for design of DNA
libraries which is too numerous to list here; in fact, virtually every group engaged
in DNA computing has developed such software. Generally, such software has
been released (by NYU, MIT, Caltech, Duke Univ., etc.) as shareware for no
cost, since the design software for DNA sequences is not generally considered to
have commercial value. (In contrast, the DNA sequence design software used for
the design of DNA chips and other biotechnology uses quite distinct criteria for
sequence design dependent on the properties of the DNA array or biotechnology
detection assay.)
The Software we will use for Design of DNA Sequences
We will use a known evolutionary optimization procedure to find a set of DNA
strands of specified length such that at a specified temperature and buffer
conditions, they do not hybridize with each other, or with strands used in the
assays, and they do not form unintended secondary structure. We will employ
standard software models for DNA hybridization and secondary structure
determination. We will use as our basis, software known as TileSoft developed
by our group. TileSoft has two components, a graphical user interface (GUI)
developed in Java and an optimization module written in C. The user friendly GUI
allows the molecular architect to specify the tile templates directly on the
interface as well as to display the optimized sequence information pictorially. The
optimization module is fully automated and hence frees the user completely from
potentially time-consuming and tedious manual selection of the DNA sequences.
It implements an evolutionary algorithm that produces satisfactory results
efficiently. We also note that though the GUI and the optimization module are
smoothly integrated from user's perspective, they are actually well separated in
terms of software architecture. Hence the two components can be improved
independently. Currently, TileSoft runs on a FreeBSD Unix platform.
Reif’s software for design of DNA sequences useful in tiles and nanostructures:
http://www.cs.duke.edu/~tinab/dnawork/docs/index.html
(ii)
Software
for
simulation
of
queries
in
a
DNA-encoded
image
databases:
http://cgi.cs.duke.edu/~clk/dnasrch.
Our design of Libraries of DNA Taggants
We will use this software for design of our DNA taggant library of 20 distinct DNA
strands. This will allow representation of 380 virtual Taggants as pairs of distinct
DNA strands. Our DNA taggant library may need to be subsequently slightly
revised due to needs to optimize our detection assays.
Our design DNA Nanostructures used by our 2nd Detection Protocol
We will also use this software for design of DNA nanostructures used for the 2nd
(enzyme-free) detection protocol. The DNA nanostructures used for that 2nd
protocol may need to be subsequently slightly revised due to needs to optimize
the protocol.
3A2 Our DNA Detection and Amplification Protocols and Their Optimization
We have developed in our Phase I SBIR two distinct amplification and detection
protocols for “exponential, isothermal” detection of a specific segment of DNA. In
Phase II these protocols will need to be further optimized to a larger DNA
Taggant library and for use with the intended dipstick detection apparatus.
Like PCR, our amplification and detection protocols are exquisitely sensitive,
requiring only a dozen or so DNA strands in the sample for the detection to
occur. Also, like PCR, both methods increase the input DNA strand’s
concentration at a rate increasing exponentially with time.
Unlike conventional PCR, a key property of our improved protocols for DNA
amplification and detection is that they are isothermal, that is not requiring
thermal cycling. This will allow our DNA detection apparatus to be demonstrated
in Phase II to be far more transportable and provide us with a commercial
advantage over other more conventional detection methods based on PCR.
Our amplification and detection protocol to be discussed are as follows:
 Our first isothermal DNA amplification and detection protocol combines
rolling circle PCR with restriction enzyme operations, and gives
exponential increase of the detected DNA strand with time.
 Our second isothermal DNA detection protocol uses DNA nanostructures to
induce a hybridization cascade signal amplification and exposure of
previously sequestered sequences. It has the advantage of not using
restriction enzymes (which can require refrigeration to avoid degradation).
It also gives exponential increase of the detected DNA strand with time. In
contrast, all known prior “exponential, isothermal” DNA detection methods
(such as the strand displacement developed by DB, Inc.) required
enzymes.
Our Isothermal DNA Amplification Protocol Using Enzymes:
Here we describe our first isothermal DNA amplification and detection protocol
that combines a prior protocol known as rolling circle PCR with restriction
enzyme operations. It produces a number of products growing exponentially with
time but requires no thermal cycling. Hence it is isothermal and autonomous. The
Figure below illustrates the basic design of the assay.
Inputs:
- A very large number of circular single strand DNA templates (C)
containing a restriction enzyme recognition site (indicated by purple
rectangles).
- The restriction enzyme recognition sites are methylated and hence the
restriction is inhibited.
- A very large number of short linear single strand DNA containing the
restriction site (A). An initial primer (P).
Output. A number of linear products (P) growing exponentially with time.
Step 1. The primer P hybridizes with the circular template C and
a polymerase starts extending the primer P.
Step 2. After finishing one full circle, the linear strand product is displaced by
polymerase, in a similar fashion as in the standard circular PCR. The extended
linear strand hybridizes with the assisting strand (short strands in the above
figure).
Step 3. The duplex is cut into short linear primers P. Now each primer can serve
as a new starting point for a new round of synthesis. As such the reaction goes
on in an exponential way.
Step 4. Detection of the exponentially increased product X by colorimetric
signals (or various other possible methods, including fluorescence polarization,
chemiluminescence).
Discussion: We would like to emphasize the following points.
(a) The hybridization product between the circular DNA strand C and the
linear product E will not be restricted due to the methylation of the recognition
site on the circular DNA C.
(b) Though we require an exponential number of C and A, we only require a
tiny amount of P that is synthesized in numbers growing exponentially in
time by the above assay.
Optimization to be done in Phase II
In Phase II this assays will need to be further optimized to a larger DNA Taggant
library. We will put considerable effort in this optimization work. Optimization of
this modified rolling circle reaction is dependant on factors that relate to enzyme
kinetics. Since a polymerase is necessary to generate the amplified strand and a
restriction enzyme is necessary to cut the strand, the buffer formulation is critical
for the system to work robustly for field applications. Buffer components such as
pH, salt, and magnesium concentration are classical parameters for optimization,
but with new chain reaction technologies currently available picogreen dye
concentration, non-ionic detergents, and Bovine Serum Albumin could also be
added to enhance the rolling circle amplification process.
The strategy used for optimization is to first optimize the reaction to be used with
picogreen dye so that the operator can track the reaction in real time. This will
yield data related to efficiency, and reaction profile; is the reaction linear or
exponential? Next we will optimize choice of polymerase. Different polymerases
have different features, and stability factors. Once the enzymatic components
are formulated, primer and template titrations will help to determine the sensitivity
of the assay.
Our DNA Detection Assay Using DNA Nanostructures and No Enzymes
Our other isothermal DNA detection has the advantage of not using enzymes,
which require refrigeration to avoid degradation. In contrast, all known prior
“exponential, isothermal” DNA detection methods (such as the strand
displacement developed by DB, Inc.) required restriction enzymes.
Note: The recent PNAS paper of Dirks & Pierce (Robert M. Dirks and Niles A.
Pierce, Triggered amplification by hybridization chain reaction, PNAS, Oct. 26,
2004, Vol 101, No. 43, pp 15275-15278) provided an amplification of DNA by use
of a chain reaction of hybridization. However, the amplification resulted in linear
growth of product over time, rather than exponential growth of product as
desired. Hence, it can’t be used to give exquisitely sensitive detection of small
amount of DNA within a short time.
A brief overview for this 2nd detection assay is given below. The main idea is to
make use of DNA nanostructures that hide within their structure the DNA strand
to be detected. If it is detected, these DNA nanostructures open up (to a new
confirmation that decreases their potential energy) to expose further copies of the
detected strand, providing for a chain reaction with the concentration of the
detected DNA strand exponentially increasing with time.
Overview of the 2nd Detection Protocol using DNA Nanostructures and no
enzymes.
A and B are DNA nanostructures that are separately formed and added
together. Without the input strand I, they are stable. I is the input DNA strand to
be detected. If the input strand I is added to a solution with nanostructures A and
B, then a series of hybridizations occur which will open up a single stranded
section of B that contains a copy of the input strand (portion a-b). Repetitions of
this reaction involving the newly exposed copies of input strand I induces a chain
reaction of amplification of copies of the input strand I. These copies of the input
stand grow exponentially with time, as illustrated below. In the figure, the portion
corresponding to I is indicated with a red circle. (The protocol however is
completely autonomous and requires no thermal cycling.)
Figure: Exponential (over time) of Growth of Exposed Target DNA
In summary, our second isothermal DNA detection protocol uses DNA
nanostructures to induce a hybridization cascade signal amplification and
exposure of previously sequestered sequences.
The Phase II of this DNA detection protocol will also need to be further optimized
to a larger DNA Taggant library and for use with the intended dipstick detection
apparatus. We intend to use our in-house software TileSoft for this, which
essentially uses an evolutionary optimization procedure to find the required set of
DNA strands forming A and B with the intended secondary structure. We may
also use some kinetic simulations of the reactions to predict the efficiency of the
cascading reactions.
3A3 Our Output Methods: Colorimetric Assays
We will combine a commercially available colorimetric DNA detection system
based on colloidal gold, from Nanosphere Inc., with our proprietary isothermal
DNA amplification protocols. We will also in this task extend this colorimetric
DNA detection method to detect the AND of a pair of distinct DNA strands
chosen from the library.
We assume here we have first applied one of our two proprietary isothermal DNA
amplification protocols (our first assay gives a enzymatic amplification of target
strand sequences, or the 2nd assay provides a hybridization cascade signal
amplification and exposure of previously sequestered sequences). We can now
assume the resulting solution now has large amounts of the exposed single
stranded DNA to be detected.
Summary of Nanosphere’s Nanoparticle Colorimetric Detection
We now describe how we can apply a commercially available colorimetric DNA
detection system based on colloidal gold, from Nanosphere Inc. Detection of
product can be visualize by the naked-eye using nanoparticle-based color
change. Briefly, two types of gold nanoparticles (X and Y) carrying multiple
copies of particular DNA strands (x and y) placed together do not associate and
remain red. When crosslinking strand (x’y’) is add to the mix the nanoparticles
are brought together into aggregates and the sample turns blue. The process is
summarized in the figure below.
Figure NPD: Two types of unassociated DNA functionalized nanoparticles (left)
are brought together by target strands (middle) containing sequence
complements for both immobilized strands thus forming aggregates of dsDNA
linking nanoparticles. The macroscopic sample changes color from red separated
particles (left) to blue aggregates (right).
References
Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A.
Selective Colorimetric Detection of Polynucleotides Based on the DistanceDependent Optical Properties of Gold Nanoparticles, Science, 1997, 277, 10781080.
Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. One-Pot
Colorimetric Differentiation of Polynucleotides with Single Base Imperfections
Using Gold Nanoparticle Probes, J. Am. Chem. Soc., 1998, 120, 1959-1964.
Optical detection of the AND of Two Amplified Products
We will also develop a readout method that is error-resilient: it uses a simple
duplex self-assembled DNA nanostructure that provides visible reporting of the
logical AND of multiple independent DNA strand detection tests, and
considerably drops the false-positive rate of detection errors.
The idea in the case of the detection of two distinct DNA strands, S and T is
simply to add an auxiliary DNA strand A that has end segments that are
complementary to end segments of S and T. The resulting duplex nanostructure
contains A bound to both s and t, with single stranded DNA x’ and y’ at the end of
S and T, respectively. Then we simply apply the same colorimetric DNA
detection system described above, with this nanostructure in place of the
crosslinking strand (x’y’) described above that links the gold spheres.
3A4 The Detection Apparatus
The lateral flow assay format has rapidly become the premiere low cost choice
for point-of-care diagnostic testing of analytes. Not only is the method simple to
use, it requires no external instrumentation for readout other than the human eye.
Nonetheless, if accurate quantization is desired, simple low cost OEM
instrumentation is available for readout.
The lateral flow assay was developed in the late 80’s apparently through the
combination of a 1987 patent by RL Campbell, et. al. (US4,703,017) and a 1989
patent by Rosenstein, et. al. (US4,855,240). Today they are used routinely for
home pregnancy tests, human fecal occult blood detection, HIV-1 diagnostics,
mycobacterium tuberculosis diagnostics, and detection of drugs of abuse, to
name a few.
Introduction to Lateral flow assay formats
Rapid DNA detection assays can be configured in single-use disposable lateral
flow strips for field use. The lateral flow format consists of a plastic-backed strip
with sample application area on one end and an absorbent pad on the other end,
which causes the sample to be drawn through the reaction area and detection
pads by the absorbent action of the terminal pad. DNA molecules in the sample
are exposed to reagents in the proper order by patterning them in sequence on
the various pads during the manufacturing process. The figure below illustrates
a typical DNA detection assay in lateral flow strip format.
Figure LFA. Overview of a DNA detection assay on lateral flow strip. The top
cartoon shows the liquid sample being applied on the left and flowing through the
reaction pad then the detection pad on the way to the absorbent pad on the right.
The lower cartoon shows the appearance of both a control line and a capture line
indicating that the assay has executed properly and that DNA was present in the
sample.
Application of Lateral flow assay format for our Detection Apparatus
The premise of this assay is as follows. A lateral strip of nitrocellulose will
incorporate the reagents needed to amplify and collect the target DNA in the test
region of the strip. Amplification will involve the use the reagents used by the
assay (we will call these the A and B reagents) discussed in Section 3A2 and
3A2. In addition, a positive control region of the strip wills the DNA taggant itself
immobilized, which will be used as a reference when comparing to the test area.
Test DNA will be applied to the sample pad area. Capillary flow will draw the test
DNA solution into the Test Region whereby amplification will occur if the test
DNA matches the DNA to be detected. Subsequently, gold nanoparticles with
complementary oligo probes to the taggant DNA will be introduced into both the
test region and the positive control region. If the correct target is present, the
gold will collect and aggregate in this region resulting in a color change. If
however, no target is present, the gold nanoparticles will simply pass through the
membrane. (See ref Mirkin, J. Am. Chem. Soc. 1998, 120, 1959-1964,
specifically bottom right of page 1962, the “Northwestern spot test”.)
Test
DNA
taggant
Sample
Pad
Flow
A and B
reagents
+DNA
taggant
control
Absorbant
pad
Probe-coated
gold colloid
Test Region
Test Spot
Positive
Control Region
PositiveControl Spot
Assays configured in lateral flow format should yield results within 5-10 minutes.
Tests strips should be stable for at least several months and perhaps up to a
year.
As noted in Section 3A2 and 3A2, use of our isothermal and colorimetric
detection assays obviates the necessity for any incubation or detection
equipment. Furthermore, for enzyme-free detection assay, we do not need
refrigeration of the reactants.
Transfer the detection protocols and assays of Task II to a lateral flow (dipstick)
format on membrane strips. We don’t envision this to be a significant hurdle as
Nanosphere’s detection format is already membrane-based. However it will
involve a key manufacturing step, and subsequent automation step, involving the
use of a computer controlled mechanical spotter.
We will need to develop manufacturing capability to reliably assemble the
detection reagents on the lateral flow (dipstick) format on membrane strips. This
involves, as a key-manufacturing step, the use of a computer controlled
mechanical spotter to deposit the various reagents along the length of the
membrane strips of the dipstick. We will then make improved use of
programming of the mechanical spotter to provide partial automation of the
manufacture of the lateral flow (dipstick) format on membrane strips.
We will need to test the liquid flow rate and re-hydration rates and
determine reagent concentrations to ensure optimized performance.
A variety of available chromophore systems will also be investigated to optimize
the colorimetric assay for maximum detection using naked-eye observation.
Cost could be as low as pennies per assay.
Several potential partner companies offer assistance with optimization and
manufacturing of lateral flow assays including detection strategies employing
semiconducting nanoparticles (http://www.qdots.com/), antibody-based immunoassays
(http://www.strategicbiosolutions.com/serv_assay.html).
and
dye
containing liposomes (http://www.ibi.cc/advantages_of_liposomes.htm).
3B Tasks for Development & Testing of a full-scale prototype Product
(3B.1) List and Schedule for Tasks, Subtasks and Milestones
Our proposed Phase II tasks will be to assemble a full-scale prototype of our
DNA Taggant system and field test the aforementioned system. The project has
numerous facets, and we envision the following breakdown in tasks:
Task I (Months 1-4): Software development for design of DNA Taggants and its
use for expansion of our DNA taggant library to 20 distinct DNA strands (allowing
representation of 380 virtual Taggants as pairs of distinct DNA strands). Also
design of DNA nanostructures use for the 2nd Assay.
Subtask Ia (Month 1-2): Software development for design of DNA
Taggants. We will use a known evolutionary optimization procedure to find a set
of DNA strands of specified length such that, at a specified temperature and
buffer conditions, do not hybridize with each other, nor with strands used in the
assays, and do not form unintended secondary structure. We will employ
standard software models for DNA hybridization and secondary structure
determination.
Subtask Ib (Month 3): Use of this software for design of a DNA taggant
library for 20 distinct DNA strands (allowing representation of 380 virtual
Taggants as pairs of distinct DNA strands). (DNA taggant library may need to be
subsequently slightly revised due to needs to optimize detection assays in Task
II.)
Milestone (end of Month 3): Determination of initial choice of expanded
DNA taggant library to 20 distinct DNA strands.
Subtask Ic (Month 4): Design of DNA nanostructures used for the 2nd
(enzyme-free) detection assay. (DNA nanostructures used for the 2nd Assay
may need to be subsequently slightly revised due to needs to optimize 2nd
detection assay in Task IIb.)
Task II (Months 3-24): Optimization of our two isothermal detection assays for
our expanded DNA taggant library. This will involve “wet-lab” work and computer
software simulations to optimize our protocol over a range of environmental
conditions (e.g. temperature, buffer solution, contaminants, etc.).
SubTask IIa (Months 3-24): Optimization, for our expanded DNA taggant
library, of our first (modified rolling circle) exponential isothermal amplification
and detection assays using enzymes.
SubTask IIb (Months 4-24): Optimization, for our expanded DNA taggant
library, of our 2nd exponential isothermal detection assay which uses no
enzymes.
Milestone (end of Month 6): Determination of initial determination of
optimized designs of both detection assays for expanded DNA taggant library.
Task III (Months 6-24): Combine a commercially available colorimetric DNA
detection system based on colloidal gold, from Nanosphere Inc., with our
proprietary isothermal DNA amplification protocols. We will also in this task
extend this colorimetric DNA detection method to detect the AND of a pair of
distinct DNA strands chosen from the library.
SubTask IIIa (Months 6-24): Combine a commercially available
colorimetric DNA detection system based on colloidal gold, from Nanosphere
Inc., with our proprietary isothermal DNA amplification protocols and optimize
performance.
Milestone (end of Month 7): Demonstration of colorimetric DNA
detection method for detection of a chosen DNA strands.
SubTask IIIb (Months 7-24): Extend this colorimetric DNA detection
method to detect the AND of a pair of distinct DNA strands chosen from the
library and optimize performance.
Milestone (end of Month 9): Demonstration of extended colorimetric
DNA detection method for detection of AND of two distinct DNA strands.
Task IV (Months 8-24): Transfer the detection protocols and assays of Task II
to a lateral flow (dipstick) format on membrane strips. We don’t envision this to
be a significant hurdle as Nanosphere’s detection format is already membranebased. However it will involve a key manufacturing step, and subsequent
automation step, involving the use of a computer controlled mechanical spotter.
SubTask IVa (Month 8): Assembling the detection reagents on the lateral
flow (dipstick) format on membrane strips. This involves, as a key-manufacturing
step, the use of a computer controlled mechanical spotter to deposit the various
reagents along the length of the membrane strips of the dipstick.
SubTask IVb (Month 9): Testing the liquid flow rate and re-hydration
rates.
SubTask IVc (Month 9-10): Improved use of programming of the
mechanical spotter to provide automation of the manufacture of the lateral flow
(dipstick) format on membrane strips.
Milestone (end of Month 10): Demonstration of automation of the
manufacture of the lateral flow (dipstick) format on membrane strips. This will
demonstrate that the key-manufacturing step of the DNA detection apparatus can
be automated.
SubTask IVd (Month 10-24): Determination of reagent concentrations to
ensure optimized performance.
Milestone (end of Month 12): Demonstration of execution of the
detection assays of Task II in a lateral flow (dipstick) format on membrane strips.
Task V (Month 12-24): Extensive (artificial, without application demonstrations)
tests of the DNA Taggant system, and repeated optimization of the detection
assays and colorimetric readout in the lateral flow (dipstick) format. (Here we will
employ a commercial portable optical reader for increased quantization and
sensitivity of colorimetric readout of the membrane strips.)
SubTask Va (Month 12): Testing for commercial portable optical reader
for increased quantization and sensitivity of colorimetric readout of the
membrane strips. A number of handheld fluorescent systems, already on the
market, can be modified for colorimetric detection. (The optical reader will mostly
be used to optimize performance; it in not intended for normal use in the actual
product where the output will be observed by the eye.)
Milestone (end of Month 13): Demonstration of use of commercial
portable optical reader for increased quantization and sensitivity of colorimetric
readout of the membrane strips.
SubTask Vb (Month 12-24): Repeated optimization of the assays and
colorimetric DNA detection method for the DNA Taggant library, using quantified
results from optical reader.
Milestone (end of Month 13): Demonstration of optimized assays and
colorimetric DNA detection method for the DNA Taggant library.
Task VI (Month 14-24): Demonstrations of use of full-scale prototype for various
applications of commercial interest.
SubTask VIa (Months 16-18): Demonstrations
protection:
- For a machine part in a material distribution system,
- For a copyrighted high cost product (e.g., a watch), and
- For an administration system (e.g., a document).
of
counterfeiting
Milestone (end of Month 18): Demonstration of counterfeiting protection
for above scenarios.
SubTask VIb (Months 19-20): Demonstrations of tamper detection to
determine if a particular container or object has been opened or tampered, for:
- For a shipping container,
- For a laptop, and
- For a restricted room.
Milestone (end of Month 20): Demonstrations of tamper detection for
above scenarios.
SubTask VIc
(Months 21-24):
Demonstrations of intelligence
applications:
- Demonstrations of surveillance of suspects,
- Demonstrations of mapping contact networks, and
- Demonstrations of use of DNA Taggants for agent authentication.
Milestone (end of Month 24): Demonstrations of intelligence applications
for above scenarios.
(Optionally performed if all above are on schedule) Task VII (Month 12-24):
Test improved DNA Taggants (designed in Phase I) consisting of DNA loops
and/or nanostructures to provide protection to environmental degradation and
detection. This will require some minor modifications of our detection assays for
these improved DNA Taggants. Test and optimize performance of detection
assays for these improved DNA Taggants.
(Optionally performed if all above are on schedule) Milestone (end of Month
24): Demonstration of use of improved hardened DNA Taggants on our portable
DNA detection apparatus. Our development of hardened taggants and their use
on portable detection devices will provide a secondary element of technology
innovation (beyond the improved portability of the detection apparatus) that will
allow our molecular taggant system to be superior to other commercial taggant
systems in the commercial markets.
(3B.2) Interrelation of Tasks and Subtasks
It is intended that the Tasks be sequentially initiated, in the order listed, and at
the dates indicated. However the optimization portion of the work of each for
Tasks II-V, once initiated, will be continued to the end of the contract to ensure
optimization of assays and performance will continue through the entire period of
the contract. In particular, SubTasks IIa, IIb, IIIa, IIIb and Vb will be repeated
many times, as we continue to optimize the performance of the system.
(3B.3) Locations: Where we plan to achieve stated objectives of Phase II
All tasks will be executed in the laboratory space to be leased by Eagle Eye, Inc.
4 Related Work
4A Skills Required
4B Strengths & Prior work of our Company
Prior Work by Eagle Eye, Inc.
National Reconnaissance Office Contract No. NRO 000-00-C-0056 Titled: "A
BIOMOLECULAR SYSTEM FOR ULTRA-SCALE ASSOCIATIVE SEARCH"
(i) Description of Prior Project: We developed an ultra-compact storage using
DNA, that supports highly parallel associative searches within very large
databases of the order of over 1000 terabytes. In particular, we made use of
DNA as the media for ultra-compact information storage.
(ii) Client for which work was performed: National Reconnaissance Office
(NRO)
(iii) Date of completion: 5/1/2002
National Reconnaissance Office Contract No. NRO 000-01-C-0204 Titled: "A
PEDA-OP BIOCHEMICAL SYSTEM FOR PROCESSING DATA BASE
QUERIES WITH AFM IMAGE OUPUT ".
(i) Description of Prior Project: We developed a biochemical system for storing
an image database, processing logical database queries on properties of these
images, and output of these queries. The system was unique in its capability to
process, within a few minutes, complex logical queries in a database with at least
1015 elements. The total mass of the storage scales linearly with database size,
and a pedabyte database requires less than 1/10 of a gram of DNA (in solution
within approximately 20 milliliters of water). Furthermore, we developed a method
for the rendering of the images selected by queries to be done at the molecular
scale by a self-assembly process.
(ii) Client for which work was performed: National Reconnaissance Office
(NRO)
(iii) Date of completion: 4/1/2001
Key Personal of Company:
PI Scott Norton, Ph.D., Chief Scientist of Eagle Eye, Inc.
Responsibilities: Overall Management of Project.
Scott Norton has a Ph.D. in Optical Engineering, post-doctoral experience in
immunology, and 6 years of biotech and nanotechnology corporate experience in
the fields of biomarker discovery at SurroMed, Inc. and nanotechnology applied
to product/brand security. Scott Norton has more than 10 years of experience in
the biotechnology and nanotechnology field, specifically in the areas of assay
development and product/brand security. Scott Norton has developed taggants
consisting of particles with submicrometer metallic strips known that
Nanobarcodes; these particles have patterns of metallic stripes that relect light in
predetermined patterns. His specialty in instrumentation design in the fields of
diagnostics and research has led to the development of a novel high-throughput
cytometer as well as a portable imaging system for nanotaggant detection. In
addition, he has explored numerous engineering and marketing opportunities in
the field of brand/product security.
Also, his skills in software algorithm development and optical instrumentation
design are beneficial to tasks in software design, lateral flow assay development,
and potential field-portable colorimetric reader. He has 4 years of experience in
the marketing aspects of brand/security, specifically the use of nanotaggants in
covert labeling, as well the experience in designing and leading a team to
develop a high-resolution portable microscope for field detection of metal
nanotaggants. See his vita for a detailed description of this work.
John Reif, President of Eagle Eye, Inc
Responsibilities: Project Administration and Direction of Assay Design.
Prior Work(other than recent SBIR Pase I work described above):
Reif has considerable experience leading large interdisciplinary research projects
both in the industry and in academia. In prior experimental tests on DNA
taggants by John Reif supported by an increment to a DARPA contract to Duke
Univ., small scale DNA taggant systems were demonstrated using molecular
barcodes, with rapid detection capability. Our experiments demonstrated very
rapid (20 minutes) and exquisitely sensitive taggant detection methodologies
using the capabilities of compact, stand-alone, off-the-shelf commercial hardware
(the Cepheid SmartCycler™ system).
In prior work in DNA computing most relevant to this proposal(outside of the
Phase I BIR), the Reif group developed [Reif, et al 2000] and then experimentally
tested [Reif at al, 2001] the synthesis of very large DNA databases DNAencoded databases with the capability of storing vast amount of information in
very compact volumes. We tested [Reif at al, 2001] the use of DNA hybridization
to do fast associative searches within these DNA databases. [Reif, 1995] also
developed theoretical DNA methods for executing more sophisticated data base
operations on DNA data such as the data base join operations and various
massively parallel operations on the DNA data. Also, Reif’s group investigated
[Gehani, 1997] methods for executing DNA-based computation using microfluidics technologies, and a number of methods for DNA-based cryptography
[Gehani, et al 1999] and counter-measures for DNA-based steganography
systems as well as discuss various modified DNA steganography systems which
appear to have improved security. We also investigated [Gehani, et al 1998]
methods for executing DNA-based computation using micro-fluidics technologies.
A survey of the entire field of DNA-based computation is given in [Reif, 1998].
The Reif group also executed prior work in DNA nanostructures.DNA selfassembly can be used to execute massively parallel computations at the
molecular scale, with concurrent assemblies that may execute computations
independently. Due to the very compact form of DNA molecules, the degree of
parallelism (due to distinct tiling assemblies) may be up to 10 15 to possibly 1018.
We developed various DNA tiling assemblies that execute various basic
computational tasks, such as sequences of arithmetic and logical computations
executed in massively parallel fashion. We have investigated various assembly
techniques for patterned 1D and 2D DNA lattices using techniques of
unmediated algorithmic self-assembly, step-wise assembly, and directed
nucleation assembly. We developed, in collaboration with Ned Seeman’s group
at NYU, a new family of DNA nanostructures known and TX tiles, and methods
for self-assembly of these TX tiles into 2D lattices [LWR00, LYK+00]. We have
also demonstrated, for the first time, computations using self-assembly of these
TX tiles into 1D lattices [MLRS00]. The actual computations executed were a
sequence of logical bit-wise XOR operations. We have achieved visualization of
self-assembled DNA nanostructures by transmission electron microscopy (TEM)
following platinum rotary shadowing, yielding higher resolution images of DNA
lattice than any previously available from atomic force microscopy (AFM),
including the ability to visualize individual tiles. We have begun to study the use
of complementary DNA annealing to organize materials (e.g. gold nanoparticles)
displaying useful electrical characteristics onto DNA tile lattices. We also
investigated extensions of these computational methods to 3D DNA tiling lattices
and to assemblies that hold state. Surveys of DNA nanotechnology are given in
[Reif, LaBean and Seeman 2001a] and [Reif, 2002].
4C Additional Skills Provided by Consultants
Consultant(Biochemist): Optimization of Enzymic Reactions for 1st protocol:
Thomas LaBean, Assistant Research Professor, Department of Computer
Science Duke University. Phd , Biochemistry. Expert on optimization of enzymic
reactions for DNA amplification and detection.
We also intend to hire the following addition consultants (to be determined):
Consultant(Programmer): design of DNA Taggant library:
Consultant(Chemist): Optimization of DNA Nanostructures for 2nd protocol
Consultant(Bioengineer): manufacture lateral flow format on membrane strips
Consultant(Chemist): Optimization of colorimetric DNA detection method
Consultant(Bioengineer): Optimization of lateral flow (dipstick) format on
membrane strips
Consultant(Computer Automation Engineer): program computer control of
mechanical spotter
Consultant(ex Secret Service): Demonstrations of counterfeiting protection
Consultant(ex Customs): Demonstrations of tamper detection
Consultant(ex CIA): Demonstrations of Intelligence Applications
5 Applications & Indications of Commercial Potential
5A Government & Commercial Applications
Government
Overview of Market Survey: Existing and Potential Markets for DNA Taggants:
Markets include: material distribution and administration systems (potential
customers: the US Armed Forces and commercial manufactures of copyrighted
goods), tamper detection (potential customers: Dept Homeland Security, the
Armed Forces, shipping and pharmaceutical companies), and to intelligence
(potential customers: CIA, FBI, Secret Service, and military intelligence).
Company Customers of DNA Taggants:
There is a major existing market for authentication of copyright merchandise by
commercial manufacturers in the private sector. From known data, we (see
below) determine the existing Market Size all commercial sector applications of
DNA Taggants to be over 100 million dollars/Year and the potential Market size
to be over 300 million dollars/Year. Even 10% market penetration by Eagle Eye
could provide sales of over 30 million dollars/year.
Potential Government Customers of DNA Taggants
Consumers of Eagle Eye’s taggant technology include the following US
government agencies:
(i) Federal Intelligence and Law Enforcement Agencies:
CIA, FBI, DoE, and Homeland Security (Customs & Secret Service),
as well internal security branches of other Federal agencies that deal with
sensitive material,
(ii) Armed Forces (Military Intelligence groups within branches of Armed Forces).
(iii) Foreign Service offices, and
(iv) State and Local Police.
The branches of the Armed Forces might be expected to have multiple uses for
such taggants systems, as might various agencies at all levels of state, county,
municipal, and local law enforcement. Of these applications, we estimate (see
below) the potential market for all government applications of DNA Taggants to
be at least 105 million dollars/year. Again, even 10% market penetration by
Eagle Eye could provide sales of over 10 million dollars /year.
Detailed list of Applications of DNA Taggants.
There are a large number of potential commercial and military applications of
DNA Taggants. DNA Taggants systems with rapid and exquisitely sensitive
detection capabilities can provide potentially high impact and revolutionary
improvements to material distribution systems, counterfeit protection,
administration systems, and to intelligence tasks. We enumerate these
applications below:
(i) Application of DNA Taggants as Watermarks to Thwart Counterfeiting in
Material Distribution Systems and Administration Systems
Potential Customers: the US Armed Forces (US Air Force, Army, Navy) and
commercial manufactures of copyrighted goods.
Market Size: At least $1000M/Year for all Taggants, as determined by known
market size determinations quoted below, and at least $120M/Year for DNA
Taggants
Projected Size of the Market by Sectors:
$100M/year for commercial manufactures of copyrighted goods
$20M/year for the US Armed Forces
Many part distribution systems and administration systems, in both the military
and commercial sectors, are plagued with a vast number of materials and
documents, without adequate methods for certification of their origin
(watermarking). The interjection of counterfeit parts and/or documents is
extremely disruptive to these military and commercial systems. The materials of
concern include ammunition and weapon parts. In addition, there are wide
varieties of copyrighted commercial products that are vulnerable to
counterfeiting. For example, there is a substantially growing market for using
DNA Taggants to mark sports memorabilia and other valuable objects. Also,
explosives are frequently tagged with specific chemical compounds in order to
detect them or determine their provenance. Furthermore, various governments
are actively considering the use of taggants applied to currency and passports.
According to Insight on the News - World, 11/11/03
(see
http://www.insightmag.com/global_user_elements/printpage.cfm?storyid=539999
), "FBI and customs and border agents estimate sales of counterfeit goods are
lining the pockets of criminal organizations to the tune of about $500 billion in
sales per year. By midyear for fiscal 2003, the Department of Homeland Security
already had reported 3,117 seizures of counterfeit branded goods including
cigarettes, books, apparel, handbags, toys and electronic games with an
estimated street value of about $38 million - up 42 percent from last year. "
According to the same article, "The International AntiCounterfeiting Coalition
(IACC) estimates that counterfeiting results in more than $200 billion a year in
lost jobs, taxes and sales. Fortune 500 companies spend an average of between
$2 million and $4 million a year each to fight counterfeiters." Thus, just counting
the Fortune 500 companies, the market size for all taggants use for
antiCounterfeiting is at least 1 billion dollars/year. Of this commercial market,
over 10% is for DNA taggants, which is at least 100 million/year. The military
market for counterfeit protection of weapons and ammunition could be
approximately 20 million/year.
(ii) Tamper Detection
Potential Customers: Dept Homeland Security (specifically, US Customs), the
Armed Forces, and commercial shipping and pharmaceutical companies.
Market Size: Potential for At Least $120M/Year, as estimated in part by US
Customs determination of yearly imports.
Estimated Size of the Market by Sectors:
$100M/year for commercial shippers, and pharmaceutical companies
$20M/year for US Customs, US military and intelligence agencies
DNA Taggants can be used by agencies such as the Dept of Homeland
Security’s US Customs that need to know if a particular container or object has
been opened or tampered. The tamper detection protocol takes advantage of
the fact that molecular DNA Taggants are invisible and can be transferred by
contact. An example of tamper detection involves determining whether a 50gallon drum has been opened and its contents exposed. In this case, the inside
of the drum and perhaps a layer of plastic covering the contents are sprayed with
a unique DNA Taggant and the outside of the drum is sprayed with a different
unique DNA Taggant. If someone does open the drum their hands have come in
contact with the outside DNA Taggant and will transfer it to the inside of the
drum. They will also transfer the inside taggant to the outside of the drum when
they reseal the container. Both types of DNA Taggants can be detected upon
investigation. In addition since two DNA Taggants were used, even if the
perpetrator wipes down the outside of the drum to remove their ‘fingerprints’,
taggant is still present and detectable inside the drum. Customs Service or
diplomatic personnel could use DNA Taggants in this way to determine if a
shipment, package, or document pouch has been opened by determining if a
DNA Taggants which was originally sealed into the container has made its way to
the out side of the vessel. This approach can be easily extended to monitor the
tampering on many objects including laptops and restricted rooms using DNA
Taggants.
By US customs data, over 1 trillion dollars of imports arrive in US ports per year.
Even if 0.1% of this cost were used for Tamper Detection by Taggants, the total
market for tamper detection of imports using Taggants would be over 1.2 billion
dollars a year. Of this, over 10% of the submarket can be expected to be used for
DNA Tagants, which are far less detectable, resulting in a potential market of
over 120 million dollars a year for Tamper Detection by DNA Taggants. There
would also be a moderately large market (approximately 20 million dollars a year)
for total market tamper detection in the US military and intelligence agencies.
(iii) Applications of DNA Taggants to the Intelligence Sector
Potential Customers: CIA, FBI, Secret Service, and the military intelligence units
within the Armed Forces.
Market Size: Approximately 75 Million Dollars/Year.
Projected Size of the Market by Sectors:
$30M/year for the CIA
$30M/year for the FBI,
$5M/year for the Secret Service
$10M/year for military intelligence units within the Armed Forces
TAGINT is a well-established subarea of military intelligence involved with covert
tagging of objects. DNA taggant systems employing rapid and exquisitely
sensitive detection capabilities can provide potentially high impact and
revolutionary improvements to intelligence-gathering and surveillance
capabilities. Intelligence applications include: (i) determination of networks of
personal contacts and flow of material within and between targeted
organizations, (ii) verification of human assets within such organizations and also
(iii) internal monitoring and barcoding of various materials (e.g., internal
document distribution).
Surveillance of Suspects (for CIA, FBI, Homeland Security, Police)
As a simple example, DNA Taggants can be introduced into or onto a suspected
terrorist in order to follow the path or associations of that terrorist. An individual
need not even be specified; an entire area could be covered with a molecular
taggant, and everything that moved into and out of that area could be accounted
for via detection of the taggant.
There are also numerous other related applications:
- Border patrol agents could perform quick wipe-tests of shoes to determine if
pedestrians in sensitive areas have passed through areas, which are forbidden
and have been flagged with taggant.
- Law enforcement agencies could use Eagle Eye taggant systems for labeling
suspects via aerosol spraying for later positive identification of individual
suspects by taggant analysis using quick assay methods. (DNA Taggants on
clothing should persist until laundering and on skin even through mild washing
procedure. )
- The military can apply molecular taggants at checkpoints and verify that
vehicles and personnel are traveling within allowed corridors and to corroborate
drivers’ stories of the vehicles’ recent travel history.
Mapping Contact Networks (for CIA and FBI)
A typical application of such a DNA Taggant system might target an organization
consisting of a network of a few hundred individuals. The monitoring of such a
large number of individuals by conventional intelligence methods would be
extremely costly (or impractically asset-intensive). For example, DNA Taggants
can be used in this way to map interactions among groups and individuals
suspected of plotting to carry out terrorist activities. Certain individuals can be
tagged with particular sequences, and then assays can be performed to detect
the appearance of other known sequences in mixtures with the first by which
agents can infer that, for example, two individually tagged suspects are
interacting with one another. In this scenario, individuals would be targeted for
monitoring, and DNA Taggants would be covertly placed on them or objects they
regularly touch (e.g., documents, doorknobs of buildings and autos). The DNA
Taggants would be distributed to other individuals and places by contact with the
labeled individuals or objects. Cycles of detection would provide a timely
monitoring of contacts between these individuals. After some time duration,
samples would be covertly collected from suspected network members (or
objects likely to be touched by them) for testing. In this way, DNA Taggants can
be utilized to map contact networks without alerting suspects to the surveillance.
Positive identification of a DNA Taggants would indicate the necessity for further
monitoring of the suspect, while negative results might release assets for
monitoring other suspects. Contact networks can thus be mapped without the
risk and expense of continuous visual surveillance. Several subareas of a contact
network can be labeled with different DNA Taggants, which can be distinguished
from one another during analysis.
Similarly, molecular taggants can be introduced into or onto a potential
bioweapon, and used to identify the provenance of that bioweapon or to identify
people or places that have come in contact with the bioweapon.
Taggants for Agent Authentication. The combinatorial complexity of DNA
Taggants and their concealed nature makes the technology an ideal candidate
for authenticating both people and objects. In a typical authentication scenario,
the trust of a particular object (or individual) is unknown, however it is known to
the challenger that a trusted object will be tagged in a particular location with a
pre-designated taggant. The challenged object can then be sampled and
checked for the trusted taggant—confirming the status of trust. Likewise, the
trust of an agent could be inconspicuously confirmed by sampling a hand (or
other physical location) after taggant-transferring contact. There are also related
methods for anonymous and discreet communications using DNA Taggants.
5B Phase III Plans
Significance of anticipated Phase II results to Phase III
The development of a full scale DNA Taggant system with highly portable DNA
detection will transition the technology into a commercial product. The extreme
portability of our DNA taggant detection apparatus provides it a unique and
critical commercial advantage over any other DNA detection method with similar
sensitivity. The demonstration of our detection apparatus includes (see detailed
work schedule) demonstration of manufacturing via automation (the spotting
machine used), which will provide a key demonstration of our ability to
manufacture the product using automation. Our planned application
demonstrations will provide dramatic demonstrations in key application market
areas that will be of critical use for marketing (and they will also substantially aid
and foster partnering with companies that produce products in these markets).
With these demonstrations, our discussions begun during Phase I SBIR with
Potential Phase III Company and Institutional Partners should now culminate in
agreements for Phase III funding and joint development.
Venture Capital Plans. Prior to and during Phase II, we intend to solicit venture
capital from a variety of sources, including In-Q-Tel (which is a non-profit that
provides venture capital to small companies with technology relevant to
intelligence applications relevant to the CIA) and the NC Biotechnology Center.
In-Q-Tel, Langley, VI.
This is a venture capital nonprofit that partners with small companies on
development of technology with applications of interest to CIA. In 2003 they
reviewed a preliminary business plan by Eagle Eye on DNA Tagants. We are
submitting in December, 2004 a complete business plan. Contact Person: Steve
Stassinos
NC Biotechnology Center, RTP, NC.
They partner and provide start up loans to emerging NC small biotech
companies. They have reviewed this Phase II proposal.
Contact person: Ken Tindell, Senior VP, Science and Business Development.
Please see support letter included in Phase II proposal.
Potential Phase III Company Partners: Companies that have expressed
interest in Partnering
Beck and Dickerson(BD, Inc), RTP, NC.
Personnel at BD Technologies in RTP have expressed considerable interest in
possible collaboration with Eagle Eye, Inc in joint development of improved
isothermal DNA detection systems. In early July we have visited BD
Technologies to learn of their existing DNA amplification and detection methods
and have made steps of our intended technical collaborations. BD Technologies
also discussed with us the feasibility and applications of the demonstration of an
extremely compact device of executing these isothermal detection protocols.
Contact Persons: J Bruce Pitner, Chief Technologist, BioSense Group, DB
Technologies, RTP, NC and James G. Nadeau and Bob Sallitt, DB Technologies,
Sparks Maryland, Hunt Valley, Maryland
Please see support letter from J Bruce Pitner. included in Phase II proposal.
Nanosphere, Inc., Northbrook, IL 60062
Uwe R. Müller visited our company November 11, 2004 to discuss possible joint
development of improved DNA detection systems using colorimetric output.
Contact Persons: Uwe R. Müller, Vice President, Applied Science, Nanosphere,
Inc., 4088 Commercial Avenue, Northbrook, IL 60062
Syngenta Inc, Research Triange Park, NC.
This company and its owner have extensive activities in biotechnology and their
divisions have multiple applications for DNA Taggants and portable DNA
detection assays. Xun Wang has contacted us to arrange demonstrations of our
technology.
Contact Persons: Dr. Janis McFarland, Manager, Novartis Inc subdivision of
Syngenta Inc,, Research Triangle Park, NC and
Xun Wang, Ph.D., Section Head, Trait Research and Technologies Syngenta
Biotechnology Inc., Research Triangle Park, NC.
Ongoing Relationships with Other Potential Phase III Institutional Partners
CIA, Langley, VI.
A presentation by Reif in 2002 at Directorate of Science and Technology(DS&T),
CIA Langley, VI led to detailed discussions of DNA Taggants with CIA senior
operatives and demonstrations to them of our early DNA Tagant technology.
Contact person: Norm Kahn, Directorate of Science and Technology(DS&T),
ITIC: Innovative Technology Intelligence Center, CIA Langley, VI
MITRE, Arlington, VI.
Jorden Feidler has arranged several demonstrations of our Taggant
Technologies and have linked us with various potential Government customers.
Contact person: Jorden Feidler
Virginia Institute of Forensic Science and Medicine, Richamond, VI.
They partner with emerging small companies developing products for police and
federal law enforcement agencies. Members of this organization have discussed
with us possible joint demonstrations for police applications of DNA Taggants.
Contact person: Linda Carne, Executive Director and Mona Thiss, Director,
Grants and Research
Department of Homeland Security, Homeland Security Institute/Threats Division,
Arlington, VA.
US Customs within this organization are promising potential customers of
portable DNA Taggant systems, and we have made demonstrations to a US
Customs technology officer.
HS administration: Jane I. Alexander, Deputy Director, Homeland Security
Advanced Research Projects Agency, Department of Homeland Security,
Washington, DC 20528
FBI. Forensic Science Research Center.
They are also promising potential customers of portable DNA Taggant systems.
Contact Person: Mona Thiss, Director, Grants and Research
North Carolina Board of Science and Technology, RTP, NC
They have various programs that encourage emerging small companies in NC.
Contact person: Robert McMahan, Senior Advisor to the Governor of North
Carolina for Science and Technology and the Executive Director of the North
Carolina Board of Science and Technology
Letters indicating Interest in Technology and/or Partnering
6 Key Personnel
6A Key Individuals within Firm
Key Personnel.
SCOTT NORTON, Phd: EAGLE EYE, Inc., Chief Scientist,
PI of DNA Taggant AFSOR Phase I SBIR: April 2004 - current
VITA of SCOTT NORTON, Phd Citizenship: USA
SUMMARY OF QUALIFICATIONS
Six years of industrial experience in image processing, data analysis, and
prototype biomedical instrumentation design, specifically in the areas of
fluorescent-based assays and mass spectrometry-based proteomic system
development. Adept at developing novel algorithms for preprocessing, feature
extraction, and data analysis in mass spectrometry, cell cytometry, and
fluorescence image analysis.
Expert in optically-based biomedical and
diagnostic instrument design.
Extremely skilled in image processing,
bioinformatics, chemometrics, and software programming. Extensive track
record in developing novel IP in the areas of mass spectrometry analysis, laser
scanning microvolume cytometry, and image analysis. Successful in leading
staffs that are oriented towards rapid product development.
PROFESSIONAL EXPERIENCE AND ACCOMPLISHMENTS
PI of Phase I SBIR contract
EAGLE EYE, Inc., Chief Scientist, PI of DNA Taggant AFSOR Phase I SBIR:
April 2004 - current
TRELLIS BIOSCIENCE, INC. Engineering/Informatics Consultant, Mountain
View, California July, 2003 – Dec. 2003 Consultant to the Engineering
Department of Trellis Bioscience, Inc., a biotech company looking to identify
protein translocation pathways to aid in the discovery of novel drug targets.
Analyze and report on instrumentation as well as develop novel image
processing algorithms.
NANOPLEX TECHNOLOGIES, INC. (wholly-owned subsidiary of SurroMed, Inc.)
Engineering/Informatics Consultant, Mountain View, California July, 2003 –
present. Consultant to the Engineering Department of Nanoplex Technologies.
Develop and advise on algorithms for image processing software development,
perform fluorescent data analysis of Nanobarcode® particles used in novel
biological solution arrays, and advise on the development of a portable
microscope reader .
NANOPLEX TECHNOLOGIES, INC. (wholly-owned subsidiary of SurroMed, Inc.)
Engineering and Informatics Manager, Mountain View, California July, 2002 –
present
Managed software and instrumentation operations in the Engineering
Department of Nanoplex Technologies, a biotechnology offshoot of SurroMed,
Inc.
Developed proprietary algorithms for image and data analysis of
Nanobarcode® particles used in novel biological solution arrays. Authority on all
business decisions concerning imaging, detection, and analysis of
Nanobarcode® particle tags. Accomplishments:
-Modeled multiple optical system designs (scanning and wide-field imaging) to
determine the company’s focus for a high-throughput, high-sensitivity detection
system for fluorescence-based immunoassays using the company’s novel
Nanobarcode (NBC) Particles®.
-Developed prototype image processing package to recognize NBC’s in
microscope images, classify, and quantify fluorescence patterns.
-Investigated and championed a probability distribution method to more
accurately quantify fluorescence leading to improved sampling on small
fluorescence data sets.
-Introduced novel ridge detection methods to Nanoplex’s image analysis package
resulting in both increased accuracy and throughput in Nanobarcode® particle
classification.
SURROMED, INC. Senior Bioinformatics Scientist, Mountain View, California
April, 2000 –July, 2002 Primary lead on developing a proteomic mass
spectrometric data analysis system which creates a pipeline from instrument to
Oracle database. Responsible for development of all algorithms leading to
accurate protein quantification for differential phenotyping.
Managed two
software engineers in bioinformatics group. Provided hardware and software
support for 4 different mass spectrometers: MicroMass LCT, Finnigan LCQDeca, PE Biosystems Voyager DE-Pro MALDI-TOF, and Bear Instruments Triple
Quad. Reported directly to Director of Informatics.
Systems Engineer, Mountain View, California August, 1998-April, 2000
Critical contributor to the development of the company's microvolume fluorimetry
and laser scanning cytometry technology, SurroScanTM.
Key lead in
development of an image processing package to identify and quantify
fluorescently labeled cell images. Co- designed and responsible for the
prototyping of a next-generation multispot laser-scanning cytometer. Role as
primary support on all hardware and software issues concerning use and
maintenance of the SurroScanTM System.
EDUCATION
Post Doc in Immunology, “The role of VDJ rearrangement in the production of
systemic lupus erythematosus autoantibodies,” University of Rochester Strong
Medical Center (June 1997-August 1998)
Ph.D. in Optical Science, "Resonant Grating Structures: Theory, Design, and
Fabrication,” University of Rochester (December 1997)
M.S. in Optical Engineering, University of Rochester (1992)
B.S. in Physics, Clarkson University (1990) with High Distinction 3.91/4.00
PUBLICATIONS
Weixun Wang, Haihong Zhou, Hua Lin, Sushmita Roy, Thomas A. Shaler,
Lander R. Hill, Scott Norton, Praveen Kumar, Markus Anderle, and Chrisopher H.
Becker, “Quantification of Proteins and Metabolites by Mass Spectrometry
without Isotopic Labeling or Spiked Standards,” Anal. Chem., Vol. 75, p 4818-26
(2003)
Curtis A. Hastings, Howard J. Cohen, and Scott M. Norton, “Method for filtering
chemical noise in ESI mass spectra,” Currently in submission to Rapid Commun.
Mass Spectrom.
Scott M. Norton, Arjuna Balasingham, Jared Schettler, and Curtis A. Hastings
“Chromatographic Alignment Techniques in LC/MS,” Currently in submission to
Spectroscopy journal
Curtis A. Hastings, Scott M. Norton, Sushmita Roy, “New algorithms for
processing and peak detection in liquid chromatography/mass spectrometry
data,” Rapid Commun. Mass Spectrom., Vol. 16, No. 5 (2002)
Scott M. Norton, Pierre Huyn, Curtis A. Hastings, Jonathan C. Heller, “Data
mining of spectroscopic data for biomarker discovery,” Current Opinion in Drug
Discovery and Development, Vol. 4, No.3 (2001)
Scott M. Norton, Jim Winkler, Louis J. Dietz, "Cell enumeration and
characterization in microvolume laser scanning cytometry: a multicolor image
processing package," SPIE Proceedings Vol 3921, San Jose, CA (2000)
Ian D. Walton, Louis J. Dietz, Gary Frenzel, Jerry Chen, Jim Winkler, Scott M.
Norton, Aaron B. Kantor, "Microvolume laser scanning cytometry platform for
biological marker discovery," SPIE Proceedings 3926, San Jose, CA (2000)
Scott M. Norton, G. Michael Morris, Turan Erdogan, "Experimental investigation
of resonant-grating filter lineshapes in comparison to theoretical models," J. Opt.
Soc. Am. A, Vol. 15, No. 2 (1998).
Scott M. Norton, Turan Erdogan, G. Michael Morris, "Coupled-mode theory of
resonant-grating filters," J. Opt. Soc. Am. A, Vol. 14, No.3 (1997).
Scott M. Norton, G. Michael Morris, "Grating resonant filters: An overview," SPIE
Proceedings, Vol. 2532, (1995).
Daniel H. Raguin, Scott M. Norton, G. Michael Morris, "Subwavelength structured
surfaces and their applications," SPIE Proceedings Vol. CR49, San Diego, CA
(1993).
VITA of JOHN H. REIF
Citizenship: USA
President of Eagle Eye, Inc. Responsibility in this Project: Project Administrator. and Scientist.
Also Professor, Dept. of Computer Science, Duke University, Durham, N.C. 277080129
Tel. (919) 493-7978, Fax (919) 660-6519,
Email: reif@cs.duke.edu Homepage: http://www.cs.duke.edu/~reif
Research Homepage URL:
http://www.cs.duke.edu/~reif/BMC/Reif.BMCproject.html
Education. Ph.D. Applied Mathematics (1977), Harvard Univ. M.S. Applied
Mathematics(1975), Harvard Univ., B.S. Applied Math & CS (1973 magna cum
laude) Tufts Univ.
Qualifications: As President of Eagle Eye, Inc., Reif has experience in administration of industrial
contracts and leading applied research projects. At Duke, John Reif has been a Full Professor in
the Computer Science Department for 17 years (previously, Associate Professor, Harvard
University). Starting Sept 1, 2003, he has been awarded A. Hollis Edens
Distinguished Professor of Computer Science. He is the author of over 180 papers (see
http://www.cs.duke.edu/~reif/HomePage.html ) and has been awarded Fellow of the ACM and
IEEE. John Reif is the PI of multiple large DARPA, AFSOR and NSF contracts and Grants. Reif
also has extensive experience in many applied research areas, and has previously had
numerous other large government contracts, including to NASA in satellite image processing and
image compression, and from NSF, ONR and DARPA in massively parallel algorithms and
architectures. He has previously been President of a company, which developed massively
parallel hardware for data compression under NASA and DARPA contracts for constructing
special purpose VLSI processor chips.
Academic Appointments.
Hollis Edens Distinguished Professor in Trinity College of Arts and Sciences at
Duke University,2003.
Professor of Computer Science at Duke University, since 1986.
Associate Professor, Harvard University, Cambridge, Mass. 1/83 - 5/86.
Visiting Scientist, M.I.T. Lab. for Computer Science, Camb., MA 1/84 - 5/84.
Assistant Professor, Harvard University, Cambridge, MA 8/79 - 5/83.
Assistant Professor, University of Rochester, Rochester, NY. 8/78 - 8/79.
Research Associate, University of Rochester, Rochester, NY. 8/77-8/78.
Honors: Fellow, Association for the Advancement of Science (AAAS), 2003,
Fellow of IEEE, 1993, Fellow of ACM, 1996. Fellow of Inst. of Combinatorics,
1991.
Research Areas. Reif is and has been the PI of a number of DARPA and NSF
contracts in DNA computing and DNA nanostructures. Reif began seven years
pursuing research in molecular-scale computing; he was a PI on a collaborative
NSF (jointly funded by DARPA) project in this area, and has written a number of
papers in the new field of biomolecular computing (also known as DNA
computing). With LaBean and Seeman, he has designed DNA nanostructure tiles
that self-assemble into linear lattices that form computational structures to
implement an XOR computation. Previous Areas of Research: Although originally
primarily a theoretical computer scientist, he also has made a number of
contributions to practical areas of computer science including parallel
architectures, data compression, robotics, and optical computing. He has also
worked for many years on the development and analysis of parallel algorithms for
various fundamental problems including the solution of large sparse systems,
sorting, graph problems, data compression, etc. He has written papers on
parallel algorithms, particularly randomized parallel algorithms. Reif edited two
books on parallel algorithms and implementations, one of which is: Synthesis of
Parallel Algorithms, Kluwer Academic Publishers, 1993. He has extensive
experience working with systems and hardware designers both as a consultant to
the industry and as also as a P.I. on Industrial and University Contracts.
Publications.
(195
papers
by
Reif
are
listed
at
http://www.cs.duke.edu/~reif/vita/papers.html)
Selected Publications (John Reif)
Papers
can
be
downloaded
from
http://www.cs.duke.edu/~reif/vita/topics/biomolecular.html
1) Gehani, A., T. H. LaBean, and J.H. Reif, DNA-based Cryptography, Invited
Chapter for text "Aspects of Molecular Computing - Essays dedicated to Tom
Head on the occasion of his 70th birthday", Springer Verlag series in Natural
Computing (edited by N. Jonoska, Gh. Paun and G. Rozenberg) LNCS 2950
Festschrift,
Springer
(2004).
http://www.cs.duke.edu/~reif/paper/DNAcrypt/dna.pdf
2) J.H. Reif and T. H. LaBean, Computationally Inspired Biotechnologies:
Improved DNA Synthesis and Associative Search Using Error-Correcting Codes
and Vector-Quantization, Sixth International Meeting on DNA Based Computers
(DNA6), DIMACS Series in Discrete Mathematics and Theoretical Computer
Science, Leiden, The Netherlands, (June, 2000) ed. A. Condon. To be published
by Springer-Verlag as a volume in Lecture Notes in Computer Science, (2001).
http://www.cs.duke.edu/~reif/paper/Error-Restore/Error-Restore.pdf
3) J. H. Reif, T. H. LaBean, M. Pirrung, V. Rana, B. Guo, K. Kingsford, and G.
Wickham, Experimental Construction of Very Large Scale DNA Databases with
Associative Search Capability, Seventh International Meeting on DNA Based
Computers (DNA7), Tampa, FL, June 11-13, 2001. Lecture Notes in Computer
Science, Springer-Verlag, New York, Volume 2340, pages 231-247, (2002).
http://www.cs.duke.edu/~reif/paper/DNAsearch/DNAsearch.pdf
4) Hao Yan, Sung Ha Park, Gleb Finkelstein, John H. Reif, and Thomas H.
LaBean, DNA-Templated Self-Assembly of Protein Arrays and Highly Conductive
Nanowires, Science, Vol. 301, pp. 1882-1884, Sep 26 2003.
http://www.cs.duke.edu/~reif/paper/hao/pub.DNAtemplate.pdf
5) Hao Yan, Thomas H. LaBean, Liping Feng, and John H. Reif, Directed
Nucleation Assembly of Barcode Patterned DNA Lattices, Proceedings of the
National Academy of Science(PNAS), Volume 100, No. 14, pp. 8103-8108, July
8, (2003).
http://www.cs.duke.edu/~reif/paper/DNAbarcode/DNAbarcode.pdf
6) Liping Feng, Sung Ha Park, John H. Reif, and Hao Yan, A Two State DNA
Lattice Actuated by DNA Motors, Angewandte Chemie [International Edition],
Vol. 42, pp. 4342-4346, Sept. 2003.
http://www.cs.duke.edu/~reif/paper/hao/pub.2statelattice.pdf
7) Hao Yan, Liping Feng, Thomas H. LaBean, and John Reif, Parallel Molecular
Computation of Pair-Wise XOR Using DNA "String Tile, Accepted to Journal of
American Chemistry Society(JACS), 2003.
http://www.cs.duke.edu/~reif/paper/hao/stringtile/stringtile.pdf
8) C. Mao, LaBean, T.H. Reif, J.H., Seeman, Logical Computation Using
Algorithmic Self-Assembly of DNA Triple-Crossover Molecules, Nature, vol. 407,
Sept. 28 2000, pp. 493-495.
http://www.cs.duke.edu/~reif/paper/SELFASSEMBLE/AlgorithmicAssembly.web.
pdf
9) LaBean, T. H., Yan, H., Kopatsch, J., Liu, F., Winfree, E., Reif, J.H. &
Seeman, N.C., The construction, analysis, ligation and self-assembly of DNA
triple crossover complexes, Journal of American Chemistry Society(JACS) 122,
1848-1860 (2000).
http://www.cs.duke.edu/~reif/paper/DNAtiling/tilings/JACS.pdf
10) J. H. Reif, DNA Lattices: A Method for Molecular Scale Patterning and
Computation, special issue on Bio-Computation, Computer and Scientific
Engineering Magazine, IEEE Computer Society, Vol. 4, No. 1, February 2002, pp
32-41.
http://www.cs.duke.edu/~reif/paper/DNAlattice/DNAlattice.pdf
11) J. H. Reif, Molecular Assembly and Computation: From Theory to
Experimental Demonstrations, plenary paper, 29-th International Colloquium on
Automata, Languages, and Programming(ICALP), Málaga, Spain (July 8, 2002).
Lecture Notes in Computer Science, New York, Volume 2380, pages 1-21,
(2002). http://www.cs.duke.edu/~reif/paper/ICALPassemble/ICALPassemble.pdf
12) J. H. Reif, The Emergence of the Discipline of Biomolecular Computation in
the US, invited paper to the special issue on Biomolecular Computing, New
Generation Computing, edited by Masami Hagiya, Masayuki Yamamura, and
Tom Head, Vol. 20, No. 3, pp. 217-236, (2002).
http://www.cs.duke.edu/~reif/paper/NGCsurvey/NGCsurvey.pdf
13) Text Book: Synthesis of Parallel Algorithms. 22 chapters, 1011 pages.
Published by Morgan Kaufmann, San Mateo, California, 1993.
6B VITA of Key Consulting Individuals
Thomas LaBean, Assistant Research Professor, Department of Computer Science
Duke University. Phd , Biochemistry. Expert on optimization of enzymic reactions
for DNA amplification and detection.
7 Facilities, Equipment, Usage Fees and Supplies
Here we provide justification for the total cost for costs for Facilities, Equipment,
Usage Fees and Supplies.
The below indicates that the Facilities, Equipment, Usage Fees and Supplies
over the two year period of the Phase II SBIR are:
$52,500 for facility leasing (office and laboratory space)
$41,104 for equipment and instrumentation
$4,800 for Internet connection & Internet telephone usage fees
$63,000 for consumable laboratory and computer supplies
7A Justification of Proposed Purchases of instrumentation to be used
(Used) Equipment and Instrumentation.
We have determined that $71,708 would have to be spent on lab equipment, if
purchased new. This will be broken down in DNA Purification ($13,507.), DNA
Manipulation ($20,573.), DNA Characterization ($27,910.), and General Lab
Equipment ($9718.). Detailed breakdown and vendor information for new
equiptment is given below. However, we intend to purchase used lab equipment,
which we estimate to be 50% of the cost when new. Hence the total cost for used
lab equipment will be $35,854.
In addition, there will be three computer workstations purchased for computer
design, simulations of the protocols, and computer automation control. The cost
per workstation preloaded with software will be $1,750, with a total $5,250
expenditure for these three workstations.
Hence the total cost for equipment and instrumentation will be $41,104.
DNA Purification:
Instrument
Quantit Amount
y
4
4x
$1,020
Vendor or
manufacturer
Amersham
Pharmacia
Biotechnology
2
2
2 x $995
2x
$5,000
DNA110 DNA SpeedVac System
1
$4,945
Dual Intensity UV
Transilluminators, Ultra-Lum
FisherBiotech White Light
1
$1,360
1
$337
BioRad
Amersham
Pharmacia
Biotechnology
Thermo Savant
Corporation
VWR Scientific
Products
Fisher Scientific
Electrophoresis Gel System,
Vertical Electrophoresis, Hoefer
SE 600 Series, Dual Cooled
Vertical Gel Units (for 18x16 cm
plates)
PowerPac 1000 Power Supply
MutliTemp III Thermostatic
Circulator
Transilluminator
Temperture Prob
Hoefer HE 33 Mini Submarine
Electrophoresis Unit
1
2
$195
2x$300
Total
$13,507.
1
1
$7,995
$2,350
2
2 x $290
VWRbrand Mini Vortexer
1
$260
Dri-Bath Incubators and Modular
blocks, thermolyne
VWRbrand LowTemperature/B.O.D. Incubator,
Model 2005
Eppendorf 2000 Pipettor with Tip
ejector, Brinkmann, 0.5—10 μl
Eppendorf 2000 Pipettor with Tip
ejector, Brinkmann, 10—100 μl
Eppendorf 2000 Pipettor with Tip
ejector, Brinkmann, 100—1000
μl
VWRbrand Orbital Shaker
4
4 x $320
Bio-Rad
VWR Scientific
Products
VWR Scientific
Products
VWR Scientific
Products
VWR
1
$ 1,820
VWR
4
4 x $305
4
4 x $305
4
4 x $305
VWR Scientific
Products
VWR Scientific
Products
VWR Scientific
Products
2
VWR
Total
2x
$1,314
$20,573.
1
$20,000
1
$6,990
Thermo
Spectronic
VWR Scientific
Products
4
4 x $230
Total
$27,910.
1
$3,563
VWR Scientific
1
$390
VWR Scientific
DNA Manipulation:
PCR, iCycler Thermal Cycler
Eppendorf Micro Centrifuge,
Model 5415C, Brinkmann
VWRbrand Mini Centrifuge
DNA Characterization:
Fluorometer
DU 530 UV/Vis
Spectrophotometer, Beckman
coulter
Spectrophotometer Semi-micro
Cell with lid and black walls
General Item:
FI-Steem III 4 Liter Glass Stills,
Barnstead
VWRbrand Benchtop
BioRad
Amersham
Pharmacia
Biotechnology
Amersham
Pharmacia
Biotechnology
pH/Temperature Meter, Model
8000
VWRbrand pH Electrode Arm
Low-Profile Hot Plate Stirrers,
Corning, Model PC-220
VWRbrand Utility Oven, Model
1305U
Bransonic Ultrasonic Cleaner,
Branson
Analytical Balance, Basic Series,
Sartorius Number: BP61S
Top-Loading Balance, B-600,
Acculab (0.1-600 gram)
Dry ice machine
Products
1
$70
5
5 x $290
1
$618
1
$532
1
$2,145
1
$200
1
$750
Total
$9718.
VWR Scientific
Products
VWR Scientific
Products
VWR Scientific
Products
Fisher Scientific
VWR Scientific
Products
VWR Scientific
Products
VWR Scientific
Products
7B Justification of Proposed Purchases of Lab Reagents and Other Lab
Materials to be used
Lab Reagents and Other Lab Materials. A total of $63,000 will be required for
consumable lab reagents, other lab materials, and computer supplies (including
$58,000 for consumable laboratory supplies (see detail justification below), and
an additional $5,000 for computer supplies).
A major laboratory supply cost is oligonucleotide synthesis and purification,
which costs about $0.60/base for the scale required thus the oligos for a single
experiment cost approximately $200 - $300. Testing a variant of an oligo would
cost $50 - $90 depending on the length. Modified DNA (-amino,-thiol, -fluorescent
label) costs an additional $40 - $60 per oligo. We have been spending
$1000/month on oligonucleotide synthesis. Other supplies needed will be
biochemical supplies such restriction enzymes and other reagents, including
radioactive label, membrane filters, organic chemicals, pipet tips and eppendorf
tubes, glassware, disposable pipets, acrylamide and bisacrylamide, tris and urea,
electrophoresis plates and chambers. Typical enzymes cost $80 - $100 per
tube.
Reagents, Supplies, DNA strands, markers and enzymes:
DNA oligo strands
1
$24,000
IDT
Size markers and biological DNA 1
$1,000
Amersham
Pharmacia
Enzymes (DNA polymerase ,
1
$10,000
New England
kinase, ligase, restriction
Biolab
endonucleases, phosphatase)
Glassware
$9500
Plastics (tube, tip)
Chemicals
Total
$7500
$6000
$58,000
VWR Sci.
Products
Various suppliers
Sigma-Aldridge
7C Justification of Usage Fees
In the RTP area, the leased spaces listed below charge a monthly $200 Fee for
Internet Connections and Internet Telephone using Roadrunner’s High Speed
Internet cable connections. This implies a cost of $2400/year Fee for these
(internet connection & internet telephone) usage fees, or a total of $4,800 for the
24 months of the SBIR Phase II contract duration.
7D Justification of Physical Facilities to be Leased
The physical facilities are planned to be leased with equal amount lab and office
space. The office spaces will be used for administration, and the lab spaces will
be used to conduct the planned experiments.
There are two locations that have quoted prices:
BD Technologies, 21 Davis Drive, RTP, NC 27709 has Incubator Space that
rents for a blended yearly rate of $35/Sq Ft; their Labs are approx. 12x 30 or 360
Sq Ft; The labs are set up for biochemical experiments. The offices are about
12x 12 or 144 Sq Ft. This facility will provide approx. 750 Sq Ft to a total cost of
$26,250/year, or a total of $52,500 for 2 years.
- BioPharm Properties, RTP, NC is another opportunity providing leasing
arrangements for Lab/Offices of about 1100 Sq Ft. at a yearly rate which is no
more than above.
The total cost for leasing at either location will be at most $26,250/year, or a total
of $52,500 for the 24 months of the SBIR Phase II contract duration.
7D Government furnished equipment and facilities
None.
8 Consultants and Subcontract Support
8A Consultants
We intend to hire the following consultants(most, to be determined):
Consultant(Programmer): design of DNA Taggant library
Consultant(Biochemist): Optimization of Enzymic Reactions for 1st protocol
Consultant(Chemist): Optimization of DNA Nanostructures for 2nd protocol
Consultant(Bioengineer): manufacture lateral flow format on membrane strips
Consultant(Chemist): Optimization of colorimetric DNA detection method
Consultant(Bioengineer): Optimization of lateral flow (dipstick) format on
membrane strips
Consultant(Computer Automation Engineer): program computer control of
mechanical spotter
Consultant(ex Secret Service): Demonstrations of counterfeiting protection
Consultant(ex Customs): Demonstrations of tamper detection
Consultant(ex CIA): Demonstrations of Intelligence Applications
For Consultant(Biochemist): Optimization of Enzymic Reactions for 1st protocol,
we intend to use:
Thomas LaBean, Assistant Research Professor, Department of Computer
Science Duke University. Phd , Biochemistry. Expert on optimization of enzymic
reactions for DNA amplification and detection
8B Subcontracts
None.
9 Pending Support
None.
Appendix: Additional References of Cited Papers
Agrawal, S. and Zhang, R. (1997). Pharmacokinetics of oligonucleotides. Ciba
Found Symp 209:60-75.
Andreola, M.-L., Calmels, C., Michel, J., Toulme, J.-J., and Litvak, S. (2000).
Towards the selection of phosphorothioate aptamers. Eur J Biochem 267:50325040.
Cook, P.D. (1993). Medicinal chemistry strategies for anti-sense research. In
Antisense Research and Applications, Crooke, S.T. and Lebleu, B., eds, pp. 149189. CRC Press, Ann Arbor.
Dean, F.B., Nelson, J.R., Giesler, T.L. & Lasken, R.S. Rapid amplification of
plasmid and phage DNA using phi29 DNA polymerase and multiply primed rolling
circle amplification. Genome Res. 11, 1095-1099 (2001).
Dolinnaya et al, Oligonucleotide circularization by template-directed chemical
ligation. N.A. Res 21, 5403-5407 (1993) .
Gehani, A., T. H. LaBean, and J.H. Reif, DNA-based Cryptography, Proc. DNA
Based Computers V: Cambridge, MA, June 14-16, 1999. Published in DIMACS
Series in Discrete Mathematics and Theoretical Computer Science, Volume 54,
edited by E. Winfree and D.K. Gifford, American Mathematical Society,
Providence, RI, pp. 233-249, (2000). URL:
Gehani and J.H. Reif, Microflow Bio-Molecular Computation, 4th DIMACS
Workshop on DNA Based Computers, University of Pennsylvania, June 15-19,
1998. DNA Based Computers, IV, DIMACS Series in Discrete Mathematics and
Theoretical Computer Science, (ed. H. Rubin), American Mathematical Society,
1999. Also appeared in a special issue of Biosystems, Journal of Biological and
Informational Processing Sciences, Vol. 52, Nos. 1-3, (ed. By L. Kari, H. Rubin,
and D. H. Wood), pp 197-216, (1999).URL:
Green, L.S., Jellinek, D., Bell, C., Beebe, L.A., Feistner, B.D., et al. (1995).
Nuclease-resistant nucleic acid ligands to vascular permeability factor / vascular
endothelial growth factor. Chem Biol 2:683-695.
Harrington, J.J., Sherf, B., Rundlett, S., Jackson, P.D., Perry, R., et al. (2001).
Creation of genome-wide protein expression libraries using random activation of
gene expression. Nat Biotechnol 19:440-445.
Horlacher, J., Hottiger, M., Podust, V.N., Hubscher, U., and Benner, S.A. (1995).
Recognition by viral and cellular DNA polymerases of nucleobases bearing
bases with nonstandard hydrogen bonding patterns. Proc Natl Acad Sci USA
92:6329-6333.
Jhaveri, S., Olwin, B., and Ellington, A.D. (1998). In vitro selection of
phosphorothiolated aptamers. Bioorganic Med Chem Lett 8:2285-2290.
Kuske, C.R., Barns, S.M., and Busch, J.D. (1997). Diverse uncultivated bacterial
groups from soils of the arid southwestern United States that are present in many
geographical regions. Appl Environ Microbiol 63:3614-3621.
T. H. LaBean, E. Winfree, and J.H. Reif, Experimental Progress in Computation
by Self-Assembly of DNA Tilings, Gehani, A., T. H. LaBean, and J.H. Reif, DNAbased Cryptography, Proc. DNA Based Computers V: Cambridge, MA, June 1416, 1999. Published in DIMACS Series in Discrete Mathematics and Theoretical
Computer Science, Volume 54, edited by E. Winfree and D.K. Gifford, American
Mathematical Society, Providence, RI, pp. 123-140, (2000).
LaBean, T. H., Yan, H., Kopatsch, J., Liu, F., Winfree, E., Reif, J.H. & Seeman,
N.C., The construction, analysis, ligation and self-assembly of DNA triple
crossover complexes, Journal of American Chemestry Society 122, 1848-1860
(2000). URL:
Levy, M. and Ellington, A.D. (2001). Selection of deoxyribozyme ligases that
catalyze the formation of an unnatural internucleotide linkage. Bioorg Med Chem
9:2581-2587.
Lutz, M.J., Held, H.A., Hottiger, M., Hubscher, U., and Benner, S.A. (1996).
Differential discrimination of DNA polymerase for variants of the non-standard
nucleobase pair between xanthosine and 2,4-diaminopyrimidine, two
components of an expanded genetic alphabet. Nucl Acids Res 24:1308-1313.
C. Mao, LaBean, T.H. Reif, J.H., Seeman, Logical Computation Using
Algorithmic Self-Assembly of DNA Triple-Crossover Molecules, Nature, vol. 407,
Sept. 28 2000, pp. 493–495; C. Erratum: Nature 408, 750-750(2000).
Nallur, G. et al. Signal amplification by rolling circle amplification on DNA
microarrays. Nucleic Acids Res. 29, e118 (2001).
Pandolfi, D., Rauzi, F., and Capobianco, M.L. (1999). Evaluation of different
types of end-capping modifications on the stability of oligonucleotides towards 3'and 5'-exonucleases. Nucleosides Nucleotides 18:2051-2069.
Peyman, A. and Uhlmann, E. (1996). Minimally modified oligonucleotides combination of end-capping and pyrimidine protection. Biol Chem Hoppe-Seyler
377:67-70.
Piccirilli, J.A., Krauch, T., Moroney, S.E., and Benner, S.E. (1990). Enzymatic
incorporation of a new base pair into DNA and RNA extends the genetic
alphabet. Nature 343:33-37.
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