james stanek, oms ii jocelyn powell, oms ii
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James Stanek, OMS II
Jocelyn Powell, OMS II
Learning Objectives:
1) What are the current limitations to viral detection and why do we need to develop new methods?
2) What are morpholino probes and why are they beneficial over DNA probes?
3) How can the morpholino technique be applied to improve viral detection methods?
What are the current techniques for detecting viruses?
Polymerase Chain Reaction(PCR)
Cell Culture
Antibody/Antigen Testing
Limitations:
• Time
• Equipment
• Laboratory
• Training
Example: Past Ebola Outbreaks
Better method of viral detection would include:
• The ability to test outside of a laboratory and in the field
• Low cost for use in rural and underserved areas
• Detection even with very small amounts of DNA
• Ex. Saliva, low viral count, incubation period
• Limited training needed
• Fast
• Without need for transport of samples
Morpholino Anatomy
Dye Sequence
complementary
to target DNA
A B
Molecular structure of phosphorodiamidate morpholino oligomer (PMO) probes.
Target DNA made based on
conserved sequence of Ebola
genome.
Two morpholinos differ in the following ways:
1) Attached dye(donor, acceptor)
2) Pairing Sequence (Region A vs B)
3) Orientation of binding (upstream vs downstream)
Excitation: 470 nm
Emission for Alexa488(donor): 520 nm
Emission for TMR(acceptor): 575 nm
Donor Morpholino(Alexa 488)
Excitation @
470 nmEmission @
520 nm
Excitation @
470 nm
Acceptor Morpholino(TMR)
Donor Morpholino(Alexa 488) and Acceptor(TMR)
Excitation @
470 nmEmission
@ 575 nm
Target DNAAcceptor
Target
DNA
Excitation @ 470 nm
Emission Wavelength
Inte
nsit
y
Donor
600 700
Donor & Acceptor
Emission @
575 nm
Figure2: Fluorescence resonance energy transfer caused by PMO probes binding to target DNA sequence.
Blue line: without target DNA(Control)Red line: with target DNA
0 200 400 600 800 1000
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
R
Target Concentration (pM)
Adjusted R2 = 0.97
Morpholino probes detected as low as 20pM target sequence.
0.0
0.2
0.4
0.6
0.8
1.0
500 550 600 650
-0.2
0.0Flu
ore
scence
Wavelength (nm)PMO probes detection of target DNA doped in human saliva.
500 550 6000.00
0.25
0.50
0.75
1.00
500 550 600
B. SalivaA. Buffer
filename is Figure3BufferSaliva
Wavelength (nm)
Flu
ore
scence
500 550 600 650
0.00
0.25
0.50
0.75
1.00
500 550 600 650
0.00
0.25
0.50
0.75
1.00
500 550 600 650
0.00
0.25
0.50
0.75
1.00
DNA Probes
Hight SaltFlu
ore
sce
nce
Wavelength (nm)
C
PMO Probes
Low Salt
B
Wavelength (nm)
PMO Probes
Hight Salt
A
Wavelength (nm)
PM
O+d
sTar
get/T
ES
PM
O+s
sTar
get/T
ES
PM
O+d
sTar
get/T
E
PM
O+s
sTar
get/T
E
DNA+d
sTar
get/T
ES
DNA+s
sTar
get/T
ES
0.0
0.2
0.4 dsTarget
ssTargetD
R
PMO probes detection of hybridized double strand target.
500 550 6000.00
0.25
0.50
0.75
1.00
500 550 600500 550 600
ds target ds target
ss target
DNA Probe Pair
High Salt
Flu
ore
sce
nce
A
D E
ds target
ss targetss target
PMO Probe Pair
Low SaltC
PMO Probe Pair
High SaltB
Wavelength (nm)
0.00 0.05 0.10 0.15 0.20 0.25
filename is Morpholinos/Figure6ModifiedC
high salt
high salt
low salt
PMO probe pair
PMO probe pair
DNA probe pairds target
high salt
low salt
R 0.00 0.05 0.10 0.15 0.20 0.25
high saltss target
R
500 550 600 650
0.00
0.25
0.50
0.75
1.00
500 550 600 650
0.00
0.25
0.50
0.75
1.00
500 550 600 650
0.00
0.25
0.50
0.75
1.00
500 550 600 650
0.00
0.25
0.50
0.75
1.00
-Urea +Urea
Low
Salt
A
Flu
ore
sce
nce
High
Salt
B
C
Wavelength(nm)
D
Morpholino probes bind to target in denaturing buffer.
500 550 600 650
0.00
0.25
0.50
0.75
1.00
500 550 600 650
0.00
0.25
0.50
0.75
1.00
500 550 600 650
0.00
0.25
0.50
0.75
1.00
500 550 600 650
0.00
0.25
0.50
0.75
1.00
A
Flu
ore
sce
nce
(a
.u.)
B
High
Salt
Low
Salt
+Urea
C
Wavelength(nm)
-Urea
D
DNA probes bind to target in denaturing buffer.
• Morpholino probes bind to DNA and emit FRET in denaturing
conditions in which DNA probes cannot.
• Morpholino probes provide a rapid and efficient method for
detecting pathogens in human saliva or blood.
Stanek, J. MS, Powell, J. BS, Mata, J. PhD, McQuistan, T. MS, Smythe, C. BS, Summerton, J. PhD, Squier, T. PhD, Xiong, Y. PhD
Authors and affiliations:
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