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)

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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

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www.pharmaceutical-int.com/images/companies/8www.hemorrhoidinformationcenter.com/wp-conten

www.sciencedaily.com/.../05/050513102615.jpgwww.medicues.com/.../bacteriasalmonella-copy.jpg

Raman Spectroscopy

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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

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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

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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

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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

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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.

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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

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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

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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

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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

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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

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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.

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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

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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

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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

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DNA Sequence Detection Using Surface-Enhanced Resonance Raman Spectroscopy in a Homogeneous Multiplexed Assay

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Alexandra MacAskill, David Crawford, Duncan Graham, and Karen Faulds

Anal. Chem. 2009, 81, 8134-8140

Goal

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Detect different strains of hospital-acquired infections (HAI) based on specific

DNA identification using a SERRS-based assay

Surface-Enhanced Raman Spectroscopy (SERS)

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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

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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

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“The patient in the next bed is highly infectious. Thank God for these curtains.”

http://postmanpatel.blogspot.com/2008_07_27_archive.html

Experimental

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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

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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)

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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

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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

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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

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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

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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)

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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?

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Quantum Dot Probes SERRS

Acknowledgements

Dr. Robin L. McCarleyDr. Steven Soper

Soper Research GroupNational Science

FoundationSeminar Audience

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