1. HIV detection by DNA cascade amplification at point-of-care setting
Samantha Bodner (B.S.), Chad Coarsey (M.S. Bioengineering), and Dr. Waseem Asghar
College of Engineering, Department of Electrical Engineering and Computer Science
Asghar-Lab: Micro and Nanotechnology in Medicine
http://faculty.eng.fau.edu/asghar/
Cascade Amplification
Introduction
Human Immunodeficiency Virus (HIV) is a
progressive retrovirus that causes the immune
system to deteriorate. Upon direct contact, HIV
particles integrate themselves into the host cell.
This viral RNA is then converted to DNA, which
transports to the nucleus inside the cell, as it
becomes part of the human DNA. The host cell
begins to die out, which ultimately weakens the
immune system, causing HIV, and eventually,
Acquired Immunodeficiency Syndrome (AIDS).
The body is unable to fend off any infection.
Current Detection Method
Polymerase Chain Reaction (PCR) is the gold
standard method to detect and quantify viruses,
but PCR is time consuming, expensive, and not
suitable for POC settings. By designing probes
that detect the nucleic acids rapidly, we can
ultimately reduce the cost and long time
required for PCR-based virus detection.
Potential New Detection Method
By developing tools that give people easier
access to the detection of this fatality, HIV can
be detected at POC settings and can help in
disease management. Single stranded DNA
(ssDNA) is able to form self-complementary
secondary structures and form hairpin stem-
loop structures that are able to interact with the
HIV-1 target sequence. Cascade amplification is
important when analyzing the interactions of the
probes. We can create two hybridization probes
such that the hairpin loop of first probe opens
only when it binds with target HIV sequence;
second probe will then bind with opened first
probe and its hairpin loop opens; first probe will
bind with second probe and this cascade
reaction continues. Using gold nanoparticles
attached to probes will ultimately develop
different color contrast due to aggregation of
gold particles. Success in using this surrogate
DNA of the virus will lead us to in vitro testing.
Method
It is important to first design a set of probes that
are complementary to a given target sequence
of HIV-1. First, the probes are analyzed for free
energy and high melting temperature.
We then synthesize thiol-modified sequences
from Integrated DNA Technologies, Inc. After
reduction and purification, the DNA probes are
conjugated to functionalized gold nanoparticles
through centrifugation. We then bind the target
sequence to the probes and observe for
aggregation. Color change determines positive
or negative results.
Discussion
Although experimentation has currently not
been done for this nucleic acid detection, it is
projected that the results will be successful.
Through careful trials, it is possible to use
human DNA samples for point-of-care diagnosis
with reduced logistic and financial burden.
References
1.SantaLucia, John. Hicks, Donald. The
Thermodynamics of DNA Structural Motifs.
2004.
2.Yu, Xu. Zhang, Zhi-Ling. Zheng, Si-Yang.
Highly sensitive DNA detection using cascade
amplification strategy based on hybridization
chain reaction and enzyme-induced
metallization. 2014.
3.Noor, Mohammud. Goyal, Swati. Christensem,
Shawn. Iqbal, Samir. Electrical detection of
single-base DNA mutation using functionalized
nanoparticles. 2009.
4.Li, Feng, Hongquan Zhang, Brittany Dever,
Xing-Fang Li, and X. Chris Le. "Thermal Stability
of DNA Functionalized Gold Nanoparticles."
Bioconjugate Chemistry 24.11 (2013): 1790-797.
Web.
Undergraduate Research Symposium, April 6, 2012
Functionalization of Gold
Nanoparticle
Aggregation
Figure 1
The target sequence binds to
the highlighted blue region in
probe one, opening up the
hairpin. The open first probe
then becomes vulnerable for
binding, so it’s single stranded
side binds to the second
probe. This opens the second
probe, which has a
complementary side to the
target sequence, continuing
the cascade effect.
Probe 1 Probe 2
HIV-1 Target sequence
Add
Target
DNA
Figure 3
Color change caused by success in
cascade amplification and AuNP
aggregation.
Image reference: "Nanotechnology: A Brief Overview." N.p.,
n.d. Web. <http://barrett-group.mcgill.ca/tutorials/nanotechnology/nano07.htm>.
Figure 2
Thiolated oligonucleotides can be
easily conjugated to gold
nanoparticles for thermal stability and
proper binding.