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Transcript of Novel Methods for the Detection of Nucleic Acid Sequences Katrina Battle Literature Presentation...
Novel Methods for the Detection of Nucleic Acid Sequences
Katrina BattleLiterature Presentation
March 22, 2010
Objective
Provide a comparison of alternative methods for the detection of
hybridized DNA sequences
2
3
• Introductiona) DNA and importance of detectionb) Raman Spectroscopyc) the Quantum Dotd) Comparison of Fluorophores to Quantum Dots
• Detection Approachesa) Use of Quantum dots (QDs) b) Use of Surface-Enhanced Resonance Raman Spectroscopy (SERRS)
• Comparison of methods
• Critiques
• Acknowledgements
• Questions
Deoxyribonucleic Acid (DNA)
4
www.bio.miami.edu/.../gene/c16x6base-pairs.jpghttp://writersforensicsblog.wordpress.com/2009/11/Library.thunkquest.org/18617/data/types/dna.html
•Oligonucleotides are short nucleic acid polymers, typically with twenty or fewer bases
•Readily bind to their respective complementary nucleotide
r respective complementary nucleotide
Importance of Detection
Gene detection
Potential biomarkers
Screening for various infections
Diagnosis and treatment
5
www.pharmaceutical-int.com/images/companies/8www.hemorrhoidinformationcenter.com/wp-conten
www.sciencedaily.com/.../05/050513102615.jpgwww.medicues.com/.../bacteriasalmonella-copy.jpg
Raman Spectroscopy
6
3210
Lowest excited electronic state
ΔE
ΔE
Virtual states
Ground electronic states
E = hνexE = hνex
321
0
Rayleigh scatteringE = hνex
Raman scatteringE = hνex ± ΔE
Stok
es, E
= h
ν ex -
ΔE
anti-
Stok
es, E
= h
ν ex +
ΔE
Resonance Raman Spectroscopy
• Excited electron immediately relaxes into a vibrational level of the ground electronic state, giving up a Stokes photon, νs
• Excitation with wavelengths that closely approach that of the electronic absorption band of an analyte
• Increased selectivity
• Tunable laser required
7
νsνex
Δνr
νflνex
Resonance Raman Fluorescence
Quantum Dots (QDs)• Semiconductor nanocrystals whose
excitons are confined in all three spatial dimensions.
• Nanometer-scale atom clusters comprising a core, shell, and coating.
• Characteristics are closely related to the size and shape of the individual crystal.
• Possess great tunability
• Give narrow emission profile
www.invitrogen.com
Tunability of Quantum Dots
• Possible to have very precise control over the conductive properties of the material
• Available with a choice of surface reactivities
• Can be made biocompatible and can be functionalized for binding specificity (i.e. avidin/biotin)
9Medintz, I. et al., Nature Materials, 2005, 4, 435-446
ΓCdSe core (Å)
λmax em. (nm)
Absorption and Emission Profiles
10Medintz, I. et al., Nature Materials, 2005, 4, 435-446
11
Property Fluorophores Quantum DotsAbsorption Spectra Variable/narrow generally a
mirror of the emission spectraBroad spectra, steadily
increases toward the UV from the first absorption band edge
Emission Spectra Broad, asymmetric red-tailed emission
Narrow-full width at half maximum 25-20 nm for CdSe
core materials
Effective Stokes Shift Generally <100 nm >200 nm possible
Tunable Emission NA Unique to QDs/can be sized tuned from the UV to the IR
Quantum Yield Variable, low to high Generally high, 0.2 to 0.7 in buffer (surface coating)
Fluorescent Lifetime Short < 5ns Long ~10-20 ns or greater
Photostability Variable to poor Excellent, strong resistance to photobleaching
Multiphoton cross section Variable to poor Excellent > 2-3 orders of magnitude that of dyes
FRET capabilities Variable, mostly single donor-single acceptor configurations
Excellent donors (size tuning)
Multi - valent attachment Rare-mostly bis-functional Can attach several molecules
Sapsford, K. et al., Sensors, 2006, 6, 925-953
12
Sun Hee Lim, Philippe Buchy, Sek Mardy, Moon Sik Kang, and Alexey Dan Chin Yu
Anal. Chem. 2010, 82, 886-891
Specific Nucleic Acid Detection Using Photophysical Properties of Quantum Dot Probes
Goal
13
Introduce an approach for single-step labeling and separation free target detection with only
quantum dot (QD) probes using a custom-made portable system and report this application to
avian influenza virus A (H5N1).
Investigate the affect of oligonucleotide density and length on the lifetime and quenching-based
hybridization detection.
Avian Influenza• Influenza virus that occurs
naturally among birds. Wild birds worldwide carry the viruses in their intestines
• Most cases of avian influenza infection in humans have resulted from contact with infected poultry (e.g., domesticated chicken, ducks, and turkeys) or surfaces contaminated with secretions/excretions from infected birds.
14
www.ciriscience.org/thumbimage.php?id=85http://www.ehow.com/way_5697386_herbal-cure-bird-flu.htmlhttp://www.cdc.gov/flu/avian/gen-info/facts.htm
DNA Sequences
15
5’ CGGGAGTTCCCTAGCACT 3’18-mer
Probe Sequences
5’ AGTGCTAGGGAACTCGCC 3’18-mer
5’ AGTGCTAGGGAACTCGCCACTGTAG 3’25-mer
5’ CTACAGTGGCGAGTTCCCTAGCAC 3’25-mer
Complementary Target Sequences
Noncomplementary Sequences
18-mer 5’ GTAATACGACTCACTATA 3’25-mer 5’ GTAATACGACTCACTATAGGGCGAA 3’
Experimental
16
17
H3C N
C
N NH
CH3
CH3Cl-
COOH
COOH
COO
H
COO
H
COOH
COOH
COOH
COOH
H3C N N NH
OO
PMMA
CH3
CH3
COO
H
COOHCOOH
COOH
COO
COO
H
COOH
COOH
N
o
o
OH
N
o
o
H3N R
N
o
o
COOH
COO
H
COOH COOH
C
COOH
COOH
COOH
NH
Preparation of QD Probes
R=
C18 Spacer Oligonucleotide
Hybridization of QD Probes and Target DNA
18Lim, S. et. al., Anal. Chem., 2010, 82, 886-891
Portable Detection Device
19
Filter
Detector
Sample Holder
Lens 2
Pulse Generator
Blue LED Lens 1
Lim, S. et. al., Anal. Chem., 2010, 82, 886-891
Dye Intercalation
• Involves the insertion of a planar fused aromatic ring system between DNA base pairs, leading to significant π-electron overlap
• This mode of binding is stabilized by stacking interactions and is thus less sensitive to ionic strength relative to other binding modes
20
www.photobiology.com/.../pierard/intintercal.jpghttp://commons.wikimedia.org/.../DNA_intercalation.jpeghttp://commons.wikimedia.org/wiki/File:PicoGreen_(topological_formula).png
PicoGreen
Verification of Hybridization• 1 µL of 25-mer target DNA was
hybridized with 3 µL of 0.15 nM OD probes in PBS buffer (total volume 50 µL)
• Shaken at 1000 rpm for 3 hrs. at 25 ° C
• Stained with PicoGreen
• Calibration curve obtained for quantitative analysis
• Calculated hybridization efficiency
21Lim, S. et. al., Anal. Chem., 2010, 82, 886-891
Probe Sequence
Target Sequence
Results
22
QD Probe Conjugation Analysis
23Lim, S. et. al., Anal. Chem., 2010, 82, 886-891
QD Probe and Target DNA Hybridization Analysis
24Lim, S. et. al, Anal. Chem. 2010, 82, 886-891
Determination of Hybridization Time
• Performed verification of QD probe and hybridized DNA by using microfiltration to remove unhybridized DNA
• Shows an increase in hybridization of the 25-mer target DNA and 25-mer QD probes
• Hybridization efficiency changed very slightly
25
0.45 nm QD3 µM target DNA
Lim, S. et. al., Anal. Chem., 2010, 82, 886-891
Quantitative Analysis of QD Probe and Target DNA Hybridization
• Obtained a calibration curve by measuring fluorescence of the QD probe/target DNA hybrid after staining (PicoGreen)
• Fluorescence intensity increased rapidly with target concentration.
26
0.45 nm QD3 µM target DNA
Lim, S. et. al., Anal. Chem., 2010, 82, 886-891
Effect of Oligonucleotide Density• Density of conjugated oligonucleotides
on the QDs using 500, 1000, and 2000 pmol of oligonucleotides were 3.5, 15.6, and 71 molecules/QD
• Change in photophysical properties of the QDs was negligible when oligonucleotide density was low
• With an increase in oligonucleotide density, target capturing rate also increased.
• Hybridization efficiency dropped with very high probe density
27
0.45 nm QD400 µM target DNA
Lim, S. et. al., Anal. Chem., 2010, 82, 886-891
Effect of Probe Length
• 18-mer QD probes show a 40% lifetime reduction and 50 % fluorescence quenching when hybridized with the 25-mer complementary target DNA
• However, the 25-mer QD probes show a 30% lifetime reduction and a 40% fluorescence quenching
28
0.45 nm QD400 target DNA µM
Lim, S. et. al., Anal. Chem., 2010, 82, 886-891
Quantitative Analysis of QD Probes and DNA Hybridization in a Portable
Device
29Lim, S. et. al., Anal. Chem., 2010, 82, 886-891
Conclusions
• Single-step hybridization for detection• Simple and fast compared to conventional methods• Portable target detection system• Single-label and wash-free platform using the QD
photophysical properties of fluorescence lifetime and quenching
• QD probes were proved to be stable and homogenous• Applicable to biological samples
30
DNA Sequence Detection Using Surface-Enhanced Resonance Raman Spectroscopy in a Homogeneous Multiplexed Assay
31
Alexandra MacAskill, David Crawford, Duncan Graham, and Karen Faulds
Anal. Chem. 2009, 81, 8134-8140
Goal
32
Detect different strains of hospital-acquired infections (HAI) based on specific
DNA identification using a SERRS-based assay
Surface-Enhanced Raman Spectroscopy (SERS)
33
Corrugated metal surface
PlasmaResonance
Pump
Molecule
SERS Signal
Combining Resonance Raman and SERS(Surface-Enhanced Resonance Raman)
• Increase in signal intensity is roughly the product of the intensity produced by each of the techniques
• Extends Raman Spectroscopy into a wide variety of interfacial systems that were previously inaccessible due to lack of sensitivity
• Because the technique has the same capabilities as conventional Raman Spectroscopy, structural information can also be provided
34
Hospital Acquired Infections (HAIs)
• The Centers for Disease Control and Prevention estimates that HAIs roughly, cause or contribute to 99,000 deaths each year.
• Can cause severe pneumonia, infections of the urinary tract, and the bloodstream
35
“The patient in the next bed is highly infectious. Thank God for these curtains.”
http://postmanpatel.blogspot.com/2008_07_27_archive.html
Experimental
36
Silver Nanoparticle (NP) Synthesis
• EDTA was added to 2000mL of distilled H2O and heated on a hot plate. NaOH was added prior to boiling.
• At 100 °C, silver nitrate was added and the solution boiled for 30 minutes
• NP solution allowed to cool at room temperature and was analyzed using UV-Vis
• Concentration of EDTA-reduced silver NPs was calculated to be 1.16 x 10-10 mol/L and average particle size was 36.8 nm
37
EDTA/H2O
+ NaOH
At 100 °C
+ AgNO3
UV-Vis
DilutionCool at room temp.
MacAskill, A. et al., Anal. Chem. 2009, 81, 8134-8140
Locked Nucleic Acid
• Modified RNA nucleotide in which the ribose moiety has been modified with an extra bridge connecting the 2’ oxygen and 4’ carbon
• The bridge “locks” the ribose in the 3’-endo conformation
• Enhances base-stacking and backbone preorganization
• Increase in hybridization specificity
38http://www.renatamusic.it/LNA.gif
Polymerase Chain Reaction (PCR)
39
Preparation of PCR Product
• PCR products for femA-SA, femA-SE, and mecA were prepared using 4 µL of template DNA and a master mix of enzyme polymerase, nucleotides, and hybridization buffer, 4 µL each of 100 nM forward and reverse primers, and 38 µL of PCR grade water.
• Repeated for 30 cycles 1 x 109 copies of the DNA template
40MacAskill, A. et al., Anal. Chem. 2009, 81, 8134-8140
ASSAY with PCR Products
• Dilution series prepared by varying the concentration of complementary PCR product to a fixed concentration of probe.
• 1.8 µL of 1 µM probe was added to 18.2 µL of 100 nM PCR product (complementary or noncomplementary)
• PCR product was further diluted with PCR product that contained no template DNA to obtain the dilution series.
• Performed a triplex reaction using femA-SA FAM, femA-SE TAMRA, and mecA HEX LNA probes
• Stock solutions of the LNA probes and complementary DNA sequences were prepared at concentrations of 1.0 x 10-6 mol dm-3
• The multiplexing samples were prepared using 10 µL of each of the 3 dye-labeled probes
• 12 µL of each of the complementary sequences was then added or replaced with distilled H2O if absent.
• 34 µL of PBS hybridization buffer was added
41
Multiplexing
MacAskill, A. et al., Anal. Chem. 2009, 81, 8134-8140
Enhancing the Surface
• Silver NPs are compatible with solution-based approaches to DNA detection by SERRS and can be readily prepared by the reduction of the corresponding metal salt using a reducing agent (i.e. citrate or EDTA)
• Results in overall negative charge on the silver NPs
42
Silver Nanoparticle
Citrate or EDTA
surface layer
Sperminesurface layer
MacAskill, A. et al., Anal. Chem. 2009, 81, 8134-8140
SERRS Detection Assay
43MacAskill, A., et al., Anal. Chem. 2009, 81, 8134-8140
Results
44
Producing the SERRS-active Probe
• Evaluated fluorescently labeled DNA sequences where every fourth base was modified to be an LNA residue
• 3 different sequences used, either as DNA only or with the LNA
• 3 different labels, FAM, HEX, and TAMRA were used for the identification of the sequences
45
LNA and DNA Sequences Used and the Corresponding Labels
Raman Shift, cm-1
SERR
S Pe
ak In
tens
ity
LNA Probe
LNA Probe + Nonsense DNA
LNA Probe + Complementary DNA
MacAskill, A. et al., Anal. Chem. 2009, 81, 8134-8140
EDTA-reduced NPs• Citrate-reduced NPs gave more intense signals, however, the difference in
signal between complementary and noncomplementary DNA was more marked when EDTA-reduced silver NPs were used.
• Hybridization buffer had to be PBS-based to obtain the highest SERRS signal while maintaining quantitative and reproducible detection of the target DNA
• The silver NPs were diluted to 50% within the SERRS sample
• Tween 20 was also added to minimized nonspecific adsorption of the labeled single-stranded probes.
46MacAskill, A. et al., Anal. Chem. 2009, 81, 8134-8140
(mol/L)
Discrimination of Target DNA (femA-SA)
• Comparison of complete DNA probe and LNA probe
• Discrimination of target DNA was examined a function of concentration
• LNA probe showed greater discrimination between complementary (Red Line) and noncomplementary target (Blue Line)
47MacAskill, A. et al., Anal. Chem. 2009, 81, 8134-8140
LNA
DNAConc. Of DNA (mol/L)
Conc. Of DNA (mol/L)
Versatility and Applicability to Biological Samples
• femA-SA, femA-SE, and mecA were used and have 142, 162, and 99 base pairs, respectively
• PCR products were mixed with appropriate probe and underwent hybridization cycle
• FAM-labeled and LNA and DNA probes containing the same base sequence were used for comparison
48MacAskill, A. et al., Anal. Chem. 2009, 81, 8134-8140
LNA DNA
Red-NC-PCRBlue-Com-PCR
Conc. of PCR Product (mol/L) Conc. of PCR Product (mol/L)
Multiplexing
• Authors wanted to detect species commonly present in an HAI swab.
• DNA sequences used correspond to the femA gene in S. aureus (femA-SA) which will identify the presence of S. aureus, and the mecA gene, which codes for methicillin resistance in MRSA.
• The sequence for the femA-SE gene in methicillin resistant S. epidermidis (MRSE) was also detected.
49MacAskill, A. et al., Anal. Chem. 2009, 81, 8134-8140
Spectra of Labeled LNA Probe Sequences
50
femA-SE TAMRA
Triplex Spectrum
femA-SA FAM
mecA HEX
1649 cm-1
645 cm-1
747 cm-1
MacAskill, A. et al., Anal. Chem. 2009, 81, 8134-8140
Hybridization Assay
• Detect three types of DNA sequences simultaneously, by using threes different dye-labeled probes.
• Eight samples were prepared in total
• Peaks that correspond to each of the probes were monitored: femA-SA FAM (645 cm-1), mecA HEX (747 cm-1), and femA-SE TAMRA (1649 cm-1)
51MacAskill, A. et al., Anal. Chem. 2009, 81, 8134-8140
Results of Hybridization Assay
52MacAskill, A. et al., Anal. Chem. 2009, 81, 8134-8140
Conclusions
• Assay gives high discrimination between nonsense and complementary DNA when the probe sequence contains LNA bases
• Shows high selectivity and affinity due to the use of the LNA bases
• Discrimination is very well observed after a change in the dye label of the probe
• Capable of multiplex detection• Applicable to biological samples• Identification can be confirmed within a few hours (vs. 48 hrs
plus required for culturing methods)
53
Assay Comparison
+ Fast
+ Applicable to biological samples
+ Selective (QD Probe very specific for target DNA)
+ QD probes are very stable
+ Single-step hybridization
+ Separation free
+ Portable target detection device
+ Applicable to biological samples
+ Very sensitive (detection limits in pmol range)
+ Very selective (Raman signal low when target is present)
+ Separation free
+ Fast (compared to methods that require culturing)
+ Hybridization of long oligonucleotide sequences
+ Simultaneous detection54
Quantum Dot Probes SERRS
Critiques
+ Questionable concentration used for hybridization kinetics
+ No degradation studies shown
+ Device used for detection was not compared to any other devices
+ Failed to show how they calculated the amount of oligonucleotides per QD
+ Dynamic Range
+ Assay requires the use of PCR, the actual time for the PCR is not included
+ Degradation of probes
+ How homogeneous is the silver NP surface?
+ How long is the laser in contact with the sample?
55
Quantum Dot Probes SERRS
Acknowledgements
Dr. Robin L. McCarleyDr. Steven Soper
Soper Research GroupNational Science
FoundationSeminar Audience
56
57www.themillionairesecrets.net/.../questions.gif