Single Molecule Sequence Detection Via Microfluidic Planar Extensional Flow
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1) 164 transcription factors (TFs) are responsible for 277 human diseases or syndromes. 2) The document suggests detecting TF binding sites on DNA using microfluidic planar extensional flow at a stagnation point to stretch DNA for single-molecule detection of TFs. 3) Three additional suggestions for stretching DNA for TF detection include using nanochannels, laminar flow, or stretching DNA in a casting film.
Single Molecule Sequence Detection Via Microfluidic Planar Extensional Flow
- 1. Single-molecule sequence detection via microfluidic devicesBiochips and LOCWoohyuck Choi
- 2. OutlineMotivationMicrofluidic planar extensional flow at stagnation pointBioconjugatedflourescentnanoparticlesLimitations from developed devicesSuggestion 1Suggestion 2Suggestion 3
- 3. MotivationTranscription Factor(TF)sKey cellular components that control gene expressionTheir activities determine how cells function and respond to the environmentDetecting locations of widely spaced target sequences across genomic lengths of DNA while preserving information about orderTF binding sitesSingle-nucleotide polymorphismsAssociated with human diseaseCancers
- 5. 164 TFs are directly responsible for 277 diseases or syndromes
- 6. 25 TFs that were expressed exclusively in disease samples but not in healthy tissuesJ. M. Vaquerizas, S. K. Kummerfeld, S. A. Teichmann and N. M. Luscombe, "A census of human transcription factors: function, expression and evolution," Nature Reviews Genetics, vol. 10, pp. 252-263, 2009R. Dylla-Spears, J. E. Townsend, L. Jen-Jacobson, L. L. Sohn and S. J. Muller, "Single-molecule sequence detection via microfluidic planar extensional flow at a stagnation point," 31st March, 2010
- 7. Locations of transcription factor clusters in human genomeDistribution of gene expression levels for transcription factors (TFs) (red) and non-TFs (blue) in different tissue typesThere are 23 chromosomal loci that contain a high density of transcription factor (TF) genes (red boxes)Blue bars: Hox clusters are present on chromosomes 2,7, 12 and 17Green bars: zinc-finger clusters on chromosome 19MT: mitochondrial DNAJ. M. Vaquerizas, S. K. Kummerfeld, S. A. Teichmann and N. M. Luscombe, "A census of human transcription factors: function, expression and evolution," Nature Reviews Genetics, vol. 10, pp. 252-263, 2009
- 8. Traditional MethodMeniscus method generate stretched DNA moleculesImage of broken loops of fragments of E. coli DNA on silanated surfacesComparison between Untreated glass & polystyrene coated surface
- 9. Stained with YOYO-1 visualize DNA molecules H. Labit, A. Goldar, G. Guilbaud, C. Douarche, O. Hyrien and K. Marheineke, "A simple and optimized method of producing silanized surfaces for FISH and replication mapping on combed DNA fibers," BioTechniques, vol. 45, pp. 649-658, 2008.
- 10. DNA combing of λ phage DNAλ phage DNA stained with YOYO-1 was combed onto silanizedcoverslips and immediately visualized by fluorescence microscopyStained DNA was diluted in 50 mM MES buffer at different pH: (A) 5.74, (B) 6.04, and (C) 6.2 and combed. (D) Size distribution of 149 λ DNA; bin size, 2 μm.H. Labit, A. Goldar, G. Guilbaud, C. Douarche, O. Hyrien and K. Marheineke, "A simple and optimized method of producing silanized surfaces for FISH and replication mapping on combed DNA fibers," BioTechniques, vol. 45, pp. 649-658, 2008.
- 11. fluorescent in situ hybridization(FISH)FISH on combed human rDNA sequences (HeLa cell DNA)Panel shows map of the rDNA repeat unit and the probe (gray rectangle)histogram of measured probe sizes in kb
- 12. Visualisation of labels on combed lambda moleculesAligned shorter molecules (23.5±25.5 µm).aligned longer (26±27.5 µm) moleculesDNA was stained with YOYO-1 (green)labeled DNA fragments contained Alexa Fluor 546 (red); they therefore appear as yellow spots. The bars represent 5 mm
- 13. limitations of traditional methods Surface immobilization Prevents molecules from being available to downstream processes for integrated lab-on-a-chip applicationsMake some sites stericallyinaccessible, Inhibit the binding of certain probes or markersResult in overstretching of the DNA
- 14. Cross-slot trapping schemeTagged l-DNA complexes are introduced through one channel, opposed by buffer containing no fluorescent particlesFlow exits through the two outlet channels to one fixed-height and one variable height reservoir, adjustment of which permits trapping at stagnation point. Device depth is 130 mmImage area for DNA studies is 80-mm square centered at stagnation pointStreak image showing stagnation point and particle path lines arising from planar extensional flowStrain rate map for flow in 800-mm-wide cross slot, calculated using 2D fluid dynamics simulationsR. Dylla-Spears, J. E. Townsend, L. Jen-Jacobson, L. L. Sohn and S. J. Muller, "Single-molecule sequence detection via microfluidic planar extensional flow at a stagnation point," 31st March, 2010
- 15. EcoRI markerSchematic representation of the mutant EcoRI marker bound to stained ds DNAX-ray structure of the wild-type EcoRIspecific complex J. R. Taylor, M. M. Fang and S. Nie, "Probing specific sequences on single DNA molecules with bioconjugated fluorescent nanoparticles," Anal. Chem., vol. 72, pp. 1979-1986, 2000.
- 16. fluorescence images of markerRepresentative fluorescence images of marker–DNA complexes trapped at the stagnation pointStretched in planar extensional flow (De = 3.9), with corresponding fluorescence intensity profilesPeaks in intensity indicate marker positions. Dashed vertical lines denote expected target locations for EcoRI on fully extended l-DNAScale bar is 5 mm
- 17. Comparison of binding distributionsComparison of binding distributions(a) N = 130 markers bound to DNA with no mixing during incubation (b) N = 133 markers bound to DNA with continuous mixing during incubationSolid lines depict Gaussian best fits to the data, while dashed vertical gray lines indicate expected target locations of EcoRI on fully extended l-DNA
- 18. Comparison of best Gaussian fitsComparison of best Gaussian fits from binding distributions obtained from slide-stretched DNA Solid gray: 79% extension, broken gray: 92% extension Flow-stretched DNA (broken black: De = 1, solid black: De = 3.9)Dashed vertical gray lines indicate expected target locations of EcoRI on fully extended l-DNA
- 19. Suggestion 1 (Nanochannel)Top view micrograph of the central area of an array of nanochannels. The channels are 10 µm wide (light cyan), except in two constricted areas (arrows) of 350nm width. Scale bar: 50 µmScheme of detecting single molecules flowing in the nanochannelFluorescence correlation spectroscopy (FCS) is used to detect moleculesVelocities as high as 0.1 m/s were reached, corresponding to average molecular residence times in observation volume as short as 10 µs.M. Foquet, J. Korlach, W. Zipfel, W. Webb and H. Craighead, "Focal volume confinement by submicrometer-sized fluidic channels," Anal. Chem., vol. 76, pp. 1618–1626, 2004.
- 20. Limitation of nanochannelDetect single molecule not DNAFlowing force is required be very high by narrow channel areaLow velocity is limitationShort observation time is disadvatageProcessing is challenging by sacrificial layer fabricationStretching ability can be given using fixating one end of double-stranded DNA to detect FTs
- 21. Suggestion 2 (laminar flow)Using laminar flow which shows different velocity between center and edge of channel DNA could be stretched with two point fixating on wallDNA anchoring technology requiredSophisticated flow manipulation is required to obtain stable signals I. Papautsky, J. Brazzle, T. Ameel and A. B. Frazier, "Laminar fluid behavior in microchannels using micropolar fluid theory," Sensors and Actuators A: Physical, vol. 73, pp. 101-108, 1999
- 22. Suggestion 3 (DNA casting film)DNA stretching by film expansionDeposited filmUsing extension of film (ex. Parylene C has stretching ability)DNA could be stretched with increasing volume of filmDNA anchoring on film will give stretching DNA shape“Flow and Stop Process” is required to obtain stable signals On surface measurement (vs.Fluidics Devices are inside measurement of devices)Y. Okahata, T. Kobayashi, K. Tanaka and M. Shimomura, "Anisotropic electric conductivity in an aligned DNA cast film," J. Am. Chem. Soc., vol. 120, pp. 6165-6166, 1998
- 23. Summary164 TFs are directly responsible for 277 diseases or syndromesMicrofluidic planar extensional flow at stagnation pointDNA Transcription Factor(TF)s detectionNanochannelLaminar flowStretching DNA casting Film
- 24. References[1] D. Bensimon, A. Simon, V. Croquette and A. Bensimon, "Stretching DNA with a receding meniscus: experiments and models," Phys. Rev. Lett., vol. 74, pp. 4754-4757, 1995. [2] H. Labit, A. Goldar, G. Guilbaud, C. Douarche, O. Hyrien and K. Marheineke, "A simple and optimized method of producing silanized surfaces for FISH and replication mapping on combed DNA fibers," BioTechniques, vol. 45, pp. 649-658, 2008. [3] J. M. Vaquerizas, S. K. Kummerfeld, S. A. Teichmann and N. M. Luscombe, "A census of human transcription factors: function, expression and evolution," Nature Reviews Genetics, vol. 10, pp. 252-263, 2009. [4] P. J. Farnham, "Insights from genomic profiling of transcription factors," Nature Reviews Genetics, vol. 10, pp. 605-616, 2009. [5] R. Dylla-Spears, J. E. Townsend, L. Jen-Jacobson, L. L. Sohn and S. J. Muller, "Single-molecule sequence detection via microfluidic planar extensional flow at a stagnation point," 31st March, 2010. [6] I. Papautsky, J. Brazzle, T. Ameel and A. B. Frazier, "Laminar fluid behavior in microchannels using micropolar fluid theory," Sensors and Actuators A: Physical, vol. 73, pp. 101-108, 1999. [7] J. R. Taylor, M. M. Fang and S. Nie, "Probing specific sequences on single DNA molecules with bioconjugated fluorescent nanoparticles," Anal. Chem., vol. 72, pp. 1979-1986, 2000. [8] T. Wu and D. C. Schwartz, "Transchip: Single-molecule detection of transcriptional elongation complexes," Anal. Biochem., vol. 361, pp. 31-46, 2007. [9] G. Jimenez-Sanchez, B. Childs and D. Valle, "Human disease genes," Nature, vol. 409, pp. 853-855, 2001. [10] M. Xiao, E. Wan, C. Chu, W. C. Hsueh, Y. Cao and P. Y. Kwok, "Direct determination of haplotypes from single DNA molecules," Nature Methods, vol. 6, pp. 199-201, 2009. [11] A. Bensimon, A. Simon, A. Chiffaudel, V. Croquette, F. Heslot and D. Bensimon, "Alignment and sensitive detection of DNA by a moving interface," Science, vol. 265, pp. 2096, 1994. [12] R. Dylla-Spears, J. E. Townsend, L. L. Sohn, L. Jen-Jacobson and S. J. Muller, "Fluorescent Marker for Direct Detection of Specific dsDNA Sequences," Anal. Chem., pp. 1137-1146, 2009.