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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| 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.
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.
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.
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.
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.
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.
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.
PI Approval of RFP:
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.
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.
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.