EXPERIMENTAL DEMONSTRATION/REPORT
REPORT Number 0001
Eagle Eye, Inc.
30 June 2004
AFSOR SBIR Phase I contract FA8750-04-C-0147
Contract title “A DNA Taggant Watermarking System “.
Report on Initial EXPERIMENTAL DEMONSTRATION Studies
Summary.
We refined our initial goals to provide exponential DNA amplification without thermal
cycling and without refrigeration of reagents. We developed a protocol to do this and
began initial experimental testing.
Summary of Work Initiated on the following Tasks:
- Initiated Design of Taggants and Refine Protocols for Task 1.
- Studied various listed approaches to Task 2 & 3 and plan next month to chose one for
each task.
- Initiated studies of Tasks 4 & 5.
Summary of Tasks Executed:
Task 1a: Experimental Demonstration of a Taggant Detection System.
***Please see below for details of our critical recent work on “Details of our Recent
Work on Improved DNA Taggant Amplification & Detection”.
(i) Further Scaling.
(iii)Software Development Tasks. Existing software is being adapted for design of the
primer and probe sequences.
Task 1b: Improvements to Provide Enhanced Performance of Molecular Barcode
Taggant System
The following are being studied:
(i) Sequence Design Improvements.
(ii) Use of Taqman probes for Detection.
(iii) Improved Multiplexing.
(iv) Environmental Surface Testing and Optimization.
Task 2a: Development of Improved DNA Taggants to Protect DNA Taggants from
Environmental Degradation.
To mitigate risks inherent in the research of these largely unexplored areas, we are
currently making in these two months a preliminary investigation of various methods for
hardening amplicon taggants to environmental degradation:
(i) 'Hardened' Taggants: Amplicon Taggants with Terminal Modifications.
(ii) DNA Cyclization
(iii) Secondary Structure Formation
We plan in the following three months we will narrow our investigations to one of the
above methods.
Task 2b: Development of Improved DNA Taggants to Protect DNA Taggants from
Unintended Detection, and Avoidance of Detection.
To mitigate risks inherent in the research of these largely unexplored areas, we are
making in these first two months a preliminary investigation of various methods for
avoidance of detection: denial and deception:
(i) DNA-based Steganographic Techniques Using Obscuring DNA.
Other DNA Techniques for Avoidance of Detection of Molecular Taggants.
(ii) Bury the primer binding sites in secondary structure
(iii) Mask the ends by cyclization
(iv) Use of Oligonucleotide Taggants
In the following three months we will narrow our investigations to one of the above
methods.
Task 4: Experimental Methods for Taggant Dispersal and Capture
We will test the methods mentioned in section 2.
Task 5: Improved Portability of the Detection Apparatus. We are testing a
modification of rolling amplification[Dolinnaya et al, 1993] to see if we can dispense
with the thermal-cycling apparatus required for conventional PCR amplification of
taggants, thus making the taggant detection apparatus more transportable. Please see
below for details of our critical recent work on Improved DNA Taggant Amplification &
Detection.
Details of our Recent Work on Improved DNA Taggant Amplification & Detection
Our Goals for Improved DNA Taggant Amplification & Detection:
Goal 1: The detection of specified DNA strands needs to be exquisitely sensitive. This
entails the use of a protocol providing exponential DNA amplification, as for example
realized in PCR.
Goal 2: Ideally, we require that the DNA detection apparatus be very compact and
transportable, and with a low energy cost.
Goal 3: Ideally, we require that the DNA detection chemicals not require refrigeration.
Why of our Goals for DNA Amplification & Detection are key to the Commercial
Viability of our Project:
Achieving these goals will allow a DNA amplification and detection apparatus that is far
more transportable and provide us with a powerful commercial advantage over other
more conventional DNA amplification and detection methods.
This advantage would hold both for DNA taggant detection applications for government
customers including military intelligence, CIA, FBI, homeland defense and police. This
advantage would hold in the much larger biotechnology industry, where DNA
amplification and detection is of central importance for many applications.
Prior Approaches to DNA amplification:
Conventional PCR Methods.
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. After
amplification, there are many known detection methods including fluorescence,
chemiluminescence, as well as molecular beacons.
Rolling circle amplification.
Rolling circle amplification (see [Dolinnaya et al, 1993], as [Nallur, et al, 2001] and
[Dean, et al 2001]) is a DNA amplification technique which uses circularization and
primer extension, to provide amplification of specified DNA strands. This method results
in 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. However, it has
the advantage over PCR of not requiring thermal cycling, and thus making the
amplification apparatus far more transportable.
References for DNA Circularization:
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.
Beck and Dickerson, 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 protein restriction enzyme that
requires refrigeration to avoid the degradation. Their detection methods include
fluorescence polarization, chemiluminescence, or colorimetric signals.
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
Our New DNA Amplification Protocol
Isothermal Circular PCR (proprietary to Eagle Eye, Inc)
A key goal for Eagle Eye’s improved protocols for DNA amplification that they be
isothermal, that is not requiring thermal cycling, This will allow the amplification and
detection apparatus to be far more transportable and provide us with a commercial
advantage over other more conventional detection methods based on PCR.
Eagle Eye, Inc recently developed a new isothermal method for doing exponential DNA
amplification without the use of PCR thermo-cycling, which is exquisitely sensitive (up
to a few molecules), and uses a DNA-based restriction enzyme rather than temperaturesensitive protein restriction enzymes, and will an appropriate polymerase may require no
refrigeration for these enzymes. The apparatus required is extremely compact (just a card
roughly the size of a playing card).
We are in the process of experimentally testing the new method as an initial experimental
study for our AFSOR SBIR phase I grant to Eagle Eye, Inc.
Here we sketch a protocol that produces a number of products growing exponentially
with time but requires no thermal cycling and is hence isothermal and autonomous.
Figure 1 illustrates the basic design of the protocol.
Inputs:
- An exponential number of circular single strand DNA templates (C).
- The restriction enzyme recognition sites are methylated and hence the restriction
is inhibited.
- An exponential number of short linear single strand DNA containing the
restriction site (E). An initial primer (X).
Output. A number of linear products (X) growing exponentially with time.
Step 1. The primer X hybridizes with the circular template C, and a polymerase starts
extending the primer.
Step 2. After finishing one full circle, the linear strand L extends due to geometrical
constraint and strand displacement by polymerase, in a similar fashion as the standard
circular PCR.
Step 3. Next, E hybridizes with the linear product L and the duplex is cut into short linear
primers X using a DNA restriction enzyme. Then each new primer X can serve as a new
further starting point for a new round of synthesis. As such the reaction goes on in an
exponential way.
Step 3. Detection of the exponentially amplified product X by various possible methods,
including fluorescence polarization, chemiluminescence, or colorimetric signals.
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 E, we only require a tiny
amount of X which will be synthesized in numbers growing exponentially in time
by the above protocol.
Further Work Plans for June 2004.
We are refining the above Isothermal Circular PCR protocol, and will begin
experimentally testing the protocol in early June 2004 for a single specified DNA strand.
Subsequent experimental tests June 2004 will provide demonstration of scalability to
allow amplification and detection of multiple distinct specified DNA strands.
Personnel at BD Technologies have expressed interest in collaboration with Eagle Eye,
Inc in joint development (and possible SBIR pase II fast track support) of improved
isothermal DNA detection systems. We are intending to visit BD Technologies to learn
more of their existing DNA amplification and detection methods and possible
collaborations.