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Computational Model of Asymmetric

Amplification to Enable HIV Drug


Resistance Testing in Low-Resource
Settings
PRINCIPAL INVESTIGATOR: DR. BARRY LUTZ, PHD
MENTOR: NUTTADA PANDRADIST, PHD CANDIDATE

ABSTRACT
The Oligonucleotide Ligation Assay (OLA) specifically detects point mutations in the HIV polymerase gene
conferring drug resistance. Screening patients for drug-resistant HIV is a necessary step in determining the
proper treatment. However, the traditional OLA is complex and lengthy, making it non-ideal for low-
resource settings. We are exploiting a novel approach to adapt the OLA into a point-of-care, paper-based
format. To enable downstream isothermal ligation and paper-based detection steps, the amplification step
of the OLA must be designed to preferably generate many copies of single-stranded HIV DNA (ssDNA).
Typically, this can be achieved using a Linear-After-The-Exponential Polymerase Chain Reaction (LATE-
PCR), which uses an uneven ratio of forward and reverse primers. Though the LATE-PCR is a useful tool in
producing large amounts of ssDNA, it involves a plethora of parameters whose contributions to the reaction
output are not well understood. Thus, the LATE-PCR is often optimized via trial and error and may not
yield the most desirable outcome. In order to minimize wasted resources from unnecessary trials and
achieve a high-efficiency amplification, we aim to understand the behavior, predict the outcome, and
develop a platform for LATE-PCR optimization. To accomplish this, we have observed the behavior of
LATE-PCR reactions by measuring ssDNA output when altering key reaction parameters such as the
number of cycles run, primer concentration ratios, enzyme concentrations, enzyme rates, salt
concentrations, and primer sequences. Analysis of these results will allow us to understand the effects of
these parameters on the thermodynamics and kinetics of the LATE-PCR. This will form the basis of a
computational model which will accurately predict reaction behavior, and provide specific LATE-PCR
reactions given user-defined reaction parameters and desired ssDNA output. The success of this project will
enable a higher sensitivity OLA test and will form a basis for ssDNA amplification for various applications
in the realm of molecular diagnostics.

Annapurni Sriram
402 TRACK | RESEARCH CAPSTONE PROPOSAL | 5 JUNE 2017
TABLE OF CONTENTS

Background & Significance .................................................................................................... 2


Background .................................................................................................................................. 2
Figure 1 .......................................................................................................................................... 3
Figure 2 .......................................................................................................................................... 2
Statement of Problem and Need ................................................................................................ 4
Solution and Prior Art ................................................................................................................. 4
Preliminary Work ........................................................................................................................ 5
Equation 1.......................................................................................................................................5
Figure 3 .......................................................................................................................................... 6
Consequences of Success ............................................................................................................6
Ethical and Social Constraints ................................................................................................... 7
Economic Constraints ................................................................................................................ 7
Legal and Regulatory Issues ....................................................................................................... 7
Overview of Specific AIMS ....................................................................................................... 7
Figure 4 .......................................................................................................................................... 8
Figure 5 .......................................................................................................................................... 8
Design and Research Strategy ............................................................................................... 9
AIM 1 ............................................................................................................................................9
AIM 2 ......................................................................................................................................... 10
AIM 3 ......................................................................................................................................... 13
Key Personnel ............................................................................................................................ 13
Equipment and Facilities .......................................................................................................... 13
References .................................................................................................................................. 14
Appendix 1: Request for Proposal ....................................................................................... 15
Appendix 2: Concept Sheet ....................................................................................................16
Appendix 3: Gantt Chart......................................................................................................... 17
Appendix 4: Figures & Tables ............................................................................................... 17

A. Sriram Background & Significance |1


Background & Significance

Background| Human Immunodeficiency Virus (HIV) weakens the immune system and eventually
causes progression to Acquired Immunodeficiency Syndrome (AIDS). Since its discovery in the 1980s,
over 75 million people have been infected with HIV [1]. HIV is commonly treated through anti-
retroviral therapy (ART) which is not as effective against drug resistant strains of HIV. The prevalence
of transmitted drug resistant HIV is increasing worldwide [2], [3]. In high-resource settings, HIV drug
resistance is screened using Sanger consensus sequencing which costs 200-700USD [4]. This method of
sequencing is financially inaccessible for most people living in low- to middle- income countries which
have the highest HIV prevalence and the fewest resources to adequately address the burden of disease
[1]. This disparity is shown in Figure 1, where the dark red regions represent areas with the highest
prevalence of HIV and coincide with low resource settings. To improve patient treatment outcomes,
preserve the efficacy of first-line ART, and limit further transmission of drug resistant HIV, screening
for drug resistant strains is necessary in both high- and low-resource settings [4], [5].

Figure 1. HIV Prevalence is highest in low resource settings. Regions colored dark red have the highest HIV
prevalence. Many low resource settings, such as sub-Saharan Africa, have the highest burden of HIV. Figure from WHO 2016.

The Oligonucleotide Ligation Assay (OLA) offers a modality to detect Single Nucleotide
Polymorphisms (SNPs), or point mutations, in the HIV genome which confer drug resistance [4, 5]. The
traditional OLA is a laboratory assay which has four main steps: sample preparation, amplification,
ligation, and detection. In the assay, DNA is extracted from a patient blood sample, amplified using
symmetric PCR, run through a ligation reaction to detect the presence of drug resistance mutations, and
the results of the assay are reported to the user via an enzyme-linked immunosorbent assay (ELISA).
A. Sriram Background & Significance |2
This workflow is complex, lengthy, and relies heavily on expensive laboratory instrumentation which
makes it non-ideal for low-resource settings. To address the need for HIV drug resistance screening in
low resource settings, we are adapting the OLA into a point-of-care, paper-based format to enable cost-
effective HIV drug resistance testing in low-resource settings.

Our adapted OLA workflow includes the same four main steps as traditional OLA but is
simplified, shorter, and less reliant on expensive instrumentation which is optimal for use low resource
settings. In our adapted OLA workflow, nucleic acid is extracted and purified from the patient’s blood
sample and is then amplified using PCR [5]. The subsequent ligation step checks for mutations in the
DNA and the detection step reports the test results to the user using a paper-based lateral flow assay.
The ligation step of OLA relies on the binding of a single strand of HIV DNA to short DNA fragments
called probes to form ligated product. The presence of ligated product is reported to the user. Currently,
OLA uses a symmetric Polymerase Chain Reaction (PCR) reaction to generate many copies of double
stranded DNA (dsDNA). During the ligation step, the complementary strand of single stranded HIV
DNA (ssDNA) competes with DNA probes for binding. This results in the formation of less ligated
product. Previous work in our lab by Annie Wong-On-Wing has shown that only 2% of probes in this
workflow were converted into ligated product. The less ligated product generated in the ligation step
impacts the results of the detection step, thereby reducing the overall sensitivity of the OLA test. By
preferentially amplifying single stranded HIV DNA which binds to DNA probes in the subsequent
ligation step, it is possible to eliminate binding competition from the complementary HIV DNA,
generate more ligated product, and thus increase the overall sensitivity of the OLA test as seen in Figure
2.

Figure 2. Amplification of ssDNA in OLA will improve the test. The top diagram illustrates the traditional OLA
workflow which is lengthy, complex, and reliant on expensive instrumentation. Instrumentation is needed to cycle temperature
in the ligation step. The bottom diagram illustrates the adapted OLA workflow with the implementation of LATE-PCR
highlighting its reduced reliance on expensive instrumentation because the use of ssDNA eliminates the need for temperature
to be modulated in the ligation step.

A. Sriram Background & Significance |3


Linear-after-the-exponential PCR (LATE-PCR) is an asymmetric PCR reaction which uses an
unequal ratio of forward and reverse primers to generate excess amounts of ssDNA [6]. This reaction
has two distinct phases. It begins with exponential amplification of dsDNA, similar to symmetric PCR,
and then switches to linear amplification of ssDNA. However, LATE-PCR does not efficiently amplify
PCR products. We will be using computational models combined with wet lab experimentation to build
a predictive model for LATE-PCR optimization. We hope to implement LATE-PCR in OLA via a LATE-
PCR optimization tool to enable the efficient amplification of ssDNA during the amplification step of
the OLA workflow which consequently enables high efficiency ligation and detection steps.

Statement of Problem and Need| The contribution of LATE-PCR reaction parameters (such as
primer concentration, salt concentration, and enzyme rate) to ssDNA output is not well understood.
Thus, the LATE-PCR reaction is unable to be used efficiently. To exploit LATE-PCR to its maximum
efficiency for use in an OLA test in low resource settings there is a need for a predictive model of LATE-
PCR which optimizes ssDNA output.

Solution & Prior Art| Outcomes derived from PCR theory do not match experimental outcomes of
LATE-PCR, rendering PCR theory a non-ideal basis for a predictive model of LATE-PCR. Theoretical
PCR concepts do not account for preferential amplification of ssDNA and assume a constant reaction
efficiency. An ideal model of LATE-PCR would accurately predict ssDNA amplification, including the
effects of declining efficiency. Mathematical models which match or predict experimental outcomes do
not exist for LATE-PCR or other asymmetric PCR reactions. However, models of symmetric PCR
reactions do exist and are important in understanding the behavior of LATE-PCR. There are two broad
categories of PCR mathematical models. The first accounts for a decline in PCR efficiency over time [7].
The efficiency of each cycle is calculated based on the thermodynamics of DNA binding during the
distinct stages of PCR. The more accurate cycle efficiency is used in combination with PCR theory to
produce a more accurate prediction of PCR output. The second model uses an asymmetric sigmoidal
curve-fit to accurately predict final fluorescence of a PCR reaction [8]. Both models improve upon
theoretical practices of quantifying PCR output and effectively model exponential amplification of
dsDNA. However, the linear phase of LATE-PCR is critical for the amplification of ssDNA and is not
accounted for in these models. More work is needed in this area to model and then optimize LATE-PCR
output.

We aim to understand the behavior, predict the outcome, and develop a platform for
optimization of LATE-PCR. Our model will include a mathematical connection between fluorescence
observed during PCR and the DNA amplified, account for DNA binding kinetics, and optimize ssDNA
output through modulation of key reaction parameters such as number of cycles run, primer
concentration ratios, enzyme concentrations, enzyme rates, salt concentrations, and primer sequences.

A. Sriram Background & Significance |4


This will be achieved through a combination of MATLAB programming and wet lab experimentation
where algorithms will be developed to replicate and optimize experimental outcomes.

Preliminary Work| Work done in the Lutz research group has underscored the need for a LATE-PCR
optimization tool. Undergraduate researcher Annie Wong-On-Wing has shown that only 2% of probes
were converted into ligated products in our current OLA workflow which uses symmetric PCR to
amplify dsDNA in the amplification step. This highlights inefficiencies in the ligation step which are
partially attributed to the competition of complementary DNA with probes. This work motivates the
development of a LATE-PCR optimization tool which will be used to amplify ssDNA, thus enabling
higher efficiency ligation.

We have performed fundamental research which characterized the melting temperature (Tm) of
HIV primers, established a connection between experimental outcomes and equations from PCR
theory, and began establishing the effect of primer concentrations on LATE-PCR dsDNA output. The
first experiments we performed explored the melting temperature of primers used to amplify HIV DNA.
The melting temperature is the temperature at which 50% of primers are bound to a complementary
DNA. This has important implications in determining reaction efficiency. The Tm equation, shown in
Equation 1, is derived from thermodynamic equations and DNA binding theory.

dH
T = 1
m dS - R*ln( )
([Pr]0−0.5[DNA]0)

Equation 1. Tm equation. This equation describes the Tm, or temperature at which 50% of primers are bound to a
complementary DNA. This equation only works for a bimolecular DNA binding reaction. R is the gas constant, dS and dH are
the entropy and enthalpy of the primer respectively, [Pr]0 represents the initial concentration of primer DNA, and [DNA]0
represents the initial concentration of primer DNA.

We performed experiments in which the melting temperature of HIV primers at different


concentrations was determined using a melt curve. Using the Tm equation we predicted that melting
temperature would decrease with concentration. This was confirmed in experimental results, see Figure
3. The Tm, indicated by the ‘x’ of each melt curve (Figure 3), decreases as concentration of primer
decreases. As a PCR reaction progresses, primer concentration decreases as primers are used up. This
data implies that a reduction in primers would decrease the primer Tm decreases which results in less
primer bound to template DNA during the annealing phase. This suggests that less product is amplified
in later cycles than in later cycles and which implies a declining reaction efficiency. Traditional PCR
theory assumes a constant reaction efficiency over time. It is necessary to further explore how a
declining efficiency effects LATE-PCR output.

Additionally, the effect of primer concentration on LATE-PCR dsDNA output was studied by
running LATE-PCR and symmetric PCR reactions with varying primer concentrations. Results from
these experiments showed similar reaction profiles but further assessment of the changes in
A. Sriram Background & Significance |5
amplification efficiency and absolute DNA quantification from relative fluorescence units (RFU) is
needed to mathematically analyze these results and quantify the effect of primer concentration on
LATE-PCR output.

Figure 3. Tm decreased with decreasing concentration as predicted using Equation 1. A melt curve assay using
HIV primers and their reverse complements was used to determine the effect of primer concentration on Tm. Results show that
Tm decreased between 1-2 degrees Celsius as primer concentration was decreased from 400µM to 100µM. This decrease was
predicted using the derived TM equation.

Consequences of Success| The successful development of a predictive model of LATE-PCR would


enable a more sensitive OLA test for low-resource settings and could serve as a platform for optimizing
isothermal asymmetric reactions which are necessary in later iterations of our paper-based OLA test.
These isothermal asymmetric reactions will further reduce the instrumentation necessary to carry out
the OLA test, allowing it to be deployed at the point of care. In the larger scope of the OLA, the success
of this project would aid in increasing global access to rapid and cost-effective HIV drug resistance
screening thereby improving patient outcomes, and limiting the spread of drug resistant HIV.

A. Sriram Background & Significance |6


Additionally, LATE-PCR can be applied across the field of molecular diagnostics. ssDNA is
commonly used as a template molecule in genetic sequencing and isothermal reactions, and as a
capture molecule in lateral flow diagnostics. A predictive model of LATE-PCR could prove useful
beyond the scope of the OLA project.

Ethical and Social Constraints| The use of HIV derived from patient blood samples introduces
safety and health hazards. A shortened HIV template which does not pose a health risk will be used to
mitigate safety concerns. Apart from this, there are no ethical or social constraints surrounding the
development and use of a mathematical model of LATE-PCR.

Economic Constraints| The predictive model of LATE-PCR outlined in this project will be used to
optimize reactions in a point-of-care HIV drug resistance test. The reaction should be low-cost to keep
the overall cost of the OLA device low. The polymerase enzyme is the most expensive reagent in the
PCR reaction and the extent of its use serves as the primary economic constraint in this project.

Legal and Regulatory Issues| There are no legal and regulatory issues surrounding the
development and use of a LATE-PCR optimization tool.

Overview of Specific AIMs


Design and Hypothesis| We hypothesize that the implementation and optimization of LATE-
PCR reactions in the amplification step of the OLA workflow will improve the sensitivity of a point-of-
care OLA test for HIV drug resistance [9]. We will design and build a LATE-PCR optimization tool
which will accept user-inputted reaction parameters and return reaction conditions to enable optimized
ssDNA production. This will be in the form of graphic user interface (GUI) which will serve as a
platform for designing LATE-PCR reactions to optimize the generation of excess ssDNA. This project
will executed via three specific AIMs:
AIM 1: Modelling exponential amplification phase of LATE-PCR| The purpose of AIM
1 is to create a predictive model of the amplification of LATE-PCR. This will involve absolutely
quantifying dsDNA output of the exponential amplification phase of PCR and subsequently analyzing
experimental data to mathematically determine expressions which can be used to accurately predict
dsDNA output of a LATE-PCR reaction.
AIM 2: Modelling linear amplification phase of LATE-PCR| The purpose of AIM 2 is to
design a predictive model of the linear amplification phase of LATE-PCR which allows for user
optimization of reaction conditions via the development of a Graphic User Interface (GUI).
AIM 3: Validation of LATE-PCR optimization Tool| The purpose of AIM 3 is to validate
the efficacy of the LATE-PCR optimization tool.

A. Sriram Background & Significance |7


This project is designed to be completed within 3 academic quarters. Figure 4 illustrates a
timeline of the tasks necessary to complete each AIM within a three quarter time constraint. Figure 5
depicts these tasks in the order that they will completed to maximize the efficiency of this project.

Figure 4. Timeline of specific AIMs and corresponding tasks. Each AIM is assigned a separate color. Within this scheme,
each task is assigned a dark color which represented the ideal timeline for the task and a light color which represents an extended
timeline which accounts for any unanticipated obstacles or problems.

Figure 5. Flowchart depicting project workflow. Each box houses a separate task which is color coded according to its
corresponding AIM. AIM 1 is purple, AIM 2 is blue, and AIM 3 is green. This workflow represents an ideal progression through the
project AIMs. Otherwise, iteration of several tasks may be required.

Design and Research Strategy


AIM 1: Modelling exponential amplification phase of LATE-PCR
A. Sriram Background & Significance |8
Approach| In order to understand and predict the behavior of ssDNA output in LATE-PCR, it
is necessary to dissect the LATE-PCR amplification curve, starting with the exponential phase of
amplification. The purpose of this AIM is to model the exponential amplification phase of LATE-PCR.
This AIM will involve 2 sub-AIMs: the determination of (1) a mathematical link between EvaGreen
fluorescence observed and amount of dsDNA amplified at any point in the reaction and (2) a predictive
model of the exponential amplification phase of LATE-PCR derived from PCR theory and experimental
data.
AIM 1.1| To report the amount of dsDNA amplified during a PCR reaction, the quantitative
PCR (qPCR) machine reports fluorescence emitted by fluorescent dye. The Lutz Research Group uses
EvaGreen dye to monitor the presence and amplification of dsDNA [10][11]. EvaGreen is an
intercalating dye produced by Biotium which fluoresces when bound to dsDNA. Quantitative
fluorescence data reported in relative fluorescence units (RFU) by the qPCR machine is currently used
to quantify dsDNA amplification but only provides relative quantification of dsDNA production rather
than exact amount of dsDNA at any point in the reaction. The goal of this sub-AIM is to develop a
mathematical link between fluorescence reported by the quantitative PCR (qPCR) machine and dsDNA
amplified throughout the reaction which will allow for the absolute quantification of dsDNA
production.
To establish this mathematical relationship, a LATE-PCR reaction will be run using Roche PCR
reagents and FastStart Taq polymerase [12]. Optimal reagent quantities have been determined through
previous work in the Lutz Research Group by Nuttada Panpradist. It is important that the reaction is
primer limited so that fluorescence plateaus due to a reduction in amplification rather than the optical
saturation of the qPCR machine. This ensures that amplification occurs in the dynamic range of the
machine so RFU data will be valuable. A non-primer limited reaction, which will reach machine
saturation, will be run in addition to the primer limited PCR reaction to verify that optical saturation is
not reached in the primer limited reaction. In parallel with the LATE-PCR reactions, a reaction with
known amounts of dsDNA template in high concentrations (close to 1012 copies/microliter which is
relevant to concentrations of amplified DNA) will be run with without the addition of polymerase. In
this reaction, amplification will not occur, but fluorescence of the dsDNA will be tracked throughout the
course of the reaction. At the end of these experiments, we will have data which connects RFU to known
concentrations of dsDNA. This relationship will be expressed mathematically. Using MATLAB, the
expression will be applied to the previously run LATE-PCR reactions to create a plot of the absolute
amount of DNA amplified vs. PCR cycles. This information will enable the execution of AIM 1.2.
AIM 1.2| LATE-PCR experiments will be designed to change key parameters including enzyme
concentration, enzyme rate, number of cycles run, salt concentration, primer sequence, and primer
ratios. Differences in dsDNA copies generated will be analyzed to determine how each of the reaction
parameters contributes to the dsDNA output. This analysis may include a combination of fitting
A. Sriram Background & Significance |9
equations from PCR theory to experimental data, using a 5-parameter sigmoidal fit, or using regression
analysis techniques.
Deliverables| AIM 1.1 will result in a mathematical equation which relates RFU from
EvaGreen tracking to dsDNA copies generated. AIM 1.2 will result in a model, involving a set of
mathematical expressions and a MATLAB program which effectively predicts dsDNA output from the
exponential amplification phase of LATE-PCR. This is summarized in Table 1 which lists the major
milestones for each AIM, see Appendix 4.
Anticipated Outcomes| The formation of a primer dimer during amplification,
contamination of experiments, and machine wait times pose risks to AIM 1. The primers used for the
HIV population have an affinity for forming primer-primer duplexes, called primer dimers, during the
annealing phase, rather than binding with the template DNA. This primer dimer is then amplified
rather than the template DNA strands resulting in non-specific amplification and unwanted reaction
products. To mitigate this risk, a higher annealing temperature, above the melting temperature of the
primers will be used during PCR reactions to ensure primer dimers do not form during the annealing
phase of the reaction. Additionally, contamination of PCR experiments by template strands is a re-
occurring issue in PCR reactions. To address this risk, the Lutz Research Group has policies in place
which establish pre- and post- PCR spaces so that template contamination is minimized. We will
continue to adhere strictly to these policies to mitigate the risk of contamination.

Engineering Design Standards| Experiments necessary to complete AIM 1 will involve


working in the Lutz Research Group’s Biosafety Level 1 (BSL1) lab space [13]. The Center for Disease
Control (CDC) has issued guidelines which detail appropriate lab practice in BSL1 facilities which will
be adhered to when in the BSL1 lab space. Additionally, many of the experiments required in AIM 1
involve the use of synthetic nucleic acids. Though these are from the HIV genome, they do not pose an
infection risk. The NIH has issued guidelines for research involving recombinant or synthetic nucleic
acid molecules which will be followed to maintain lab safety [14].

AIM 2: Modelling linear amplification phase of LATE-PCR


Approach| The purpose of this AIM is to model the linear amplification phase of LATE-PCR.
This AIM will involve 3 sub-AIMs: the determination of (1) a mathematical link between fluorescence
generated by Elitech single stranded probes and amount of ssDNA amplified at any point in the
reaction, (2) a predictive model of the linear amplification phase of LATE-PCR, and (3) the
development of a GUI for the LATE-PCR optimization tool.
AIM 2.1| In AIM 1.1, EvaGreen intercalating dye was used to monitor dsDNA production in the
exponential amplification phase of LATE-PCR. Because EvaGreen fluoresces when bound to dsDNA, it
cannot be used to report the presence of ssDNA. In order to monitor the production of ssDNA in the
A. Sriram Background & Significance |10
linear amplification phase of LATE-PCR, single stranded fluorescent probes manufactured by Elitech
will be used [15][16]. These probes are short pieces of DNA complementary to the amplified ssDNA
template which fluoresce when bound to ssDNA. The binding affinity and signal intensity of the Elitech
single stranded probes are different than those of the EvaGreen dye. Furthermore, the Elitech probes
are custom manufactured for our applications so emitted fluorescence and binding properties are not
documented in literature and are hard to predict. Thus, it is necessary to determine a mathematical link
between RFU generated from the Elitech probes and ssDNA copies generated during LATE-PCR to get
absolute quantification of ssDNA produced. We will achieve this using a similar procedure used in AIM
1.1. A primer limited LATE-PCR reaction will be run in addition to a non-primer limited reaction. This
will ensure that amplification of the primer limited reaction will occur in the dynamic range of the
qPCR machine. Additionally, a PCR reaction with known amounts of ssDNA template without
polymerase will be run in parallel to the LATE-PCR reactions. In this reaction, amplification will not
occur, ssDNA will be tracked throughout the course of the reaction using Elitech probes. This will result
in data which connects RFU to known concentrations of ssDNA. This relationship will allow for the
mapping of absolute concentration of ssDNA to RFU. Using MATLAB, this relationship can be
expressed mathematically and related to the previously run LATE-PCR reactions to create a plot of the
absolute amount of ssDNA amplified vs. PCR cycles. The ability to absolutely quantify ssDNA
production will enable the execution of AIM 2.2.
AIM 2.2| The goal of this sub-AIM is to produce a mathematical representation of the linear
amplification phase of LATE-PCR which can be used to predict ssDNA output. In order to achieve this,
the contribution of various reaction parameters to ssDNA output will be understood. These parameters
include enzyme concentration, enzyme rate, number of cycles run, salt concentration, primer sequence,
and primer ratios. Experiments will designed which modulate these key parameters and absolute
ssDNA output will measured given the mathematical relationship developed in AIM 2.1. For each
parameter, a mathematical expression will be determined which summarizes the impact of the reaction
parameter on the ssDNA output. Once a set of mathematical expressions are determined, they can be
integrated into one program or equation which has a basis amplification equations from PCR theory.
This equation should be able to predict the absolute quantity ssDNA produced in the linear
amplification phase of LATE-PCR. It may be necessary to iterate on the mathematical analysis of
experimental results. This analysis may include fitting equations from PCR theory to experimental data
and using linear regression analysis techniques.
AIM 2.3| The model of the exponential amplification phase of LATE-PCR generated in AIM 1.2
and the model of the linear amplification phase of LATE-PCR generated in AIM 2.2 will be combined to
create a holistic model of LATE-PCR. This model will entail combining mathematical expressions so
that all key reaction parameters are addressed. Then, an optimization algorithm will be created in

A. Sriram Background & Significance |11


MATLAB which optimizes ssDNA output of LATE-PCR reactions. This optimization algorithm will be
translated into a GUI in MATLAB which will allow for user input and greater accessibility [17].
Deliverables| AIM 2.1 will result in a mathematical equation which relates RFU from Elitech
sequence specific probes to ssDNA copies generated. AIM 2.2 will result in a model of ssDNA
amplification which will be a series of mathematical expressions. AIM 2.3 will result in a GUI which
allows for optimization of ssDNA output of LATE-PCR reactions, illustrated in Figure 6, see Appendix
4. Additionally, deliverables for AIM 2 are summarized in Table 1 which lists the major milestones for
each AIM, see Appendix 4.
Anticipated Outcomes| All three of the anticipated outcomes of AIM 1 are applicable to AIM
2. Additionally, obtaining known concentrations of ssDNA template strands will poses a risk to AIM 2.1.
To mitigate this risk, we can routinely order new template ssDNA, which is expensive. Alternatively, we
can buy one set pf phosphate labelled primers for use in LATE-PCR reactions. The LATE-PCR products
can be purified into ssDNA template strands through the use of the enzyme lambda endonuclease which
will chew up the complementary strand of DNA labelled with phosphate.
Engineering Design Standards| The engineering standards for AIM 1 are applicable for
AIM 2. Additionally, there are no engineering design standards for the creation of a MATLAB GUI, but
usability and program efficiency will be considered throughout the design and development process.

AIM 3: Validation of LATE-PCR optimization Tool


Approach| The purpose of this AIM is to validate the efficacy of the LATE-PCR optimization
tool created in AIM 2.3. This will be done through experimentation and analysis. AIM 3 can be done in
in parallel with each AIM and then appropriate iterations can be made on algorithm and model design.
When the LATE-PCR optimization tool, specified in AIM 2.3, is completed, a series of LATE-PCR
reactions can be run using the optimization tool. The experimental output of these reactions will be
compared to the outcomes generated by the LATE-PCR model detailed in AIM 1.2, AIM 2.2 and AIM
2.3. The efficacy of the model will be evaluated based on percent deviation of the experimental
outcomes from the expected outcomes. To properly judge the efficacy of the optimization tool, a large
number of reactions should be run and various test cases should be evaluated.
Deliverables| AIM 3 will result in graphs or tables, and a numerical analysis of outcomes
which compare expected and experimental LATE-PCR ssDNA output from reactions designed using the
LATE-PCR optimization tool outlined in AIM 2.3. These deliverables are summarized in Table 1 which
lists the major milestones for each AIM, see Appendix 4.
Anticipated Outcomes| All three of the anticipated outcomes of AIM 1 are applicable to AIM
3. Additionally, if experimental and expected ssDNA output is not comparable using the LATE-PCR
optimization tool, iterations of mathematical expressions determined in AIMs 1 and 2 may be
necessary.
A. Sriram Background & Significance |12
Engineering Design Standards| The engineering standards for AIM 1 are applicable for
AIM 3.

Key Personnel| Nuttada Panpradist is my graduate student mentor and is in charge of the OLA
project. She will provide me with guidance and support when necessary. Dr. Barry Lutz is the Principal
Investigator in the Lutz Lab and will guide high level direction of the project. Additionally, the OLA
Team in the Lutz Lab and collaborators in the Lai Lab and Seattle Children’s Research Institute are
essential for development of the OLA project and will provide guidance where necessary. These
stakeholders are outlined in Table 2, sees Appendix 4. Additionally, the needs associated with each of
these stakeholders is outlined in Table 3, se Appendix 4.

Equipment and Facilities| For this project, I will be working in the Lutz Lab spaces on the second
floor of Foege. I will need access to a CFX qPCR machine, NanoDrop reader, and post- and pre-PCR
spaces all of which are currently available to the Lutz Lab at no additional cost. I will require Roche PCR
reagents, HIV templates, and HIV primers which are available for purchase. Additionally, MATLAB and
other software required for this project is available for purchase or is licensed through the UW
Department of Bioengineering. My work is currently funded through the Mary Gates Research
Scholarship 2017 and the APF Student Prize 2016-2017 awarded to Nuttada Panpradist.

A. Sriram Background & Significance |13


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B. Hallett, C. A. B. Boucher, T. F. Rinke De Wit, and D. A. M. C. Van De Vijver, “Increasing the use of
second-line therapy is a cost-effective approach to prevent the spread of drug-resistant HIV: A
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format for detecting HIV drug resistance in clinical specimens by oligonucleotide ligation,” PLoS One,
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[5] I. A. Beck, M. Mahalanabis, G. Pepper, A. Wright, S. Hamilton, E. Langston, and L. M. Frenkel,
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Appendix 1: Request for Proposal
This proposal responds to a request from the Lutz Research Group. The Lutz Research Group seeks to
model asymmetric amplification techniques to enable HIV drug resistance testing using the
Oligonucleotide Ligation Assay (OLA). The Lutz Research Group requests (1) a quantitative method for
analyzing amount of amplicon generated throughout PCR and (2) a model of late amplification which
will allow for user-optimization of the reactions.

PI Approval of RFP:

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Appendix 2: Concept Sheet

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Appendix 3: Gantt chart

Gantt Chart. Timeline of specific AIMs and corresponding tasks. Each AIM is assigned a separate color. Within this
scheme, each task is assigned a dark color which represented the ideal timeline for the task and a light color which represents an
extended timeline which accounts for any unanticipated obstacles or problems.

Appendix 4: Tables and Figures

Table 1. Major milestones in the project workflow are organized according to their corresponding AIM.
Table 1. Research Milestones for each phase of project.
AIM 1 AIM 2 AIM 3
Equation relating EvaGreen Equation relating seuqnce specific Data which assesses the
RFU to dsDNA copies generated probe RFU to ssDNA copies efficacy of the LATE-PCR
during exponential amplification generated during linear optimization tool.
phase of LATE-PCR. amplification phase of LATE-PCR.
Equation describing absolute Equation describing absolute Integration of optimizes
amounts of dsDNA generated in amounts of dsDNA generated in LATE-PCR reactions into
exponential amplification phase exponential amplification phase of OLA workflow.
of LATE-PCR. LATE-PCR.
LATE-PCR optimization
algorithm.
MATLAB GUI for LATE-PCR
optimization tool.

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Table 2. Ranked needs for all stakeholders. Stakeholders and their associated needs are
highlighted for this project. Each need is ranked in importance on a scale of 1 to 5, with 1 assigning
the highest priority.
Need # Stakeholder Need Rank 1-5
(1=highest importance to
stakeholder)

1 Seattle Children’s LATE-PCR optimization tool is 1


Research Institute + effective at amplifying ssDNA.
Nuttada Panpradist +
OLA end user
2 Seattle Children’s Reactions optimized with LATE-PCR 2
Research Institute + optimization fit into OLA workflow.
Nuttada Panpradist
3 Seattle Children’s LATE-PCR optimization tool is 3
Research Institute + intuitive and easy to use.
Nuttada Panpradist
+OLA end user
4 OLA end user Accessibility of optimization tool. 1

Table 3. Needs-Metric Table. Metrics and evaluation criteria are identified for needs identified
in Table 2.
Metric Need # Specification (metric description) Units Target Overall
# (# from Values Rank
Needs (1=most
important, etc.)
Table)
1 1 Experimental ssDNA output % 5-10 1
deviation from expected ssDNA
output.
2 2 Feedback from Nuttada Panpradist feedback positive 2
and researchers at Seattle Childrens
Research Institute.
3 3 Feedback from Nuttada Panpradist feedback positive 3
and researchers at Seattle Childrens
Research Institute.

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Figure 6. Prototype Model and Functional Decomposition. Part A shows the vision of what the LATE-PCR
Optimization Tool will look like. Part B describes the consequences and subsystems of the optimization tool.

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