James Stanek, OMS II Jocelyn Powell, OMS II
Transcript of James Stanek, OMS II Jocelyn Powell, OMS II
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James Stanek, OMS II
Jocelyn Powell, OMS II
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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?
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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
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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
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Morpholino Anatomy
Dye Sequence
complementary
to target DNA
A B
Molecular structure of phosphorodiamidate morpholino oligomer (PMO) probes.
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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)
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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
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Target DNAAcceptor
Target
DNA
Excitation @ 470 nm
Emission Wavelength
Inte
nsit
y
Donor
600 700
Donor & Acceptor
Emission @
575 nm
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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
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Target Concentration (pM)
Adjusted R2 = 0.97
Morpholino probes detected as low as 20pM target sequence.
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Wavelength (nm)PMO probes detection of target DNA doped in human saliva.
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DNA Probes
Hight SaltFlu
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Wavelength (nm)
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PMO Probes
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Wavelength (nm)
PMO Probes
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PMO probes detection of hybridized double strand target.
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Wavelength (nm)
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filename is Morpholinos/Figure6ModifiedC
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Morpholino probes bind to target in denaturing buffer.
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DNA probes bind to target in denaturing buffer.
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• 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.
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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: