WO2017220637A1 - Procédés de cartographie optique de polynucléotides - Google Patents

Procédés de cartographie optique de polynucléotides Download PDF

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WO2017220637A1
WO2017220637A1 PCT/EP2017/065200 EP2017065200W WO2017220637A1 WO 2017220637 A1 WO2017220637 A1 WO 2017220637A1 EP 2017065200 W EP2017065200 W EP 2017065200W WO 2017220637 A1 WO2017220637 A1 WO 2017220637A1
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nucleic acid
dye
seconds
range
acid molecule
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PCT/EP2017/065200
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Rodolphe Charly Willy MARIE
Anders Kristensen
Jonas Nyvold PEDERSEN
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Danmarks Tekniske Universitet
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention relates to methods for the optical mapping and subsequent analysis, such as sequencing of polynucleotides, such as genomic DNA, in microchannel devices.
  • Optical maps generated by partial denaturation and renaturation (D-R) of intercalating dye (ID) stained single genomic DNA molecules which become patterned according to their AT/GC content can be read at diffraction limited resolution using a flow- stretch device and epifluorescence microscopy.
  • the high degree of stretching and the near immobilization of the molecule in the cross-flow allow high resolution imaging of D-R patterns. This has enabled identification of structural variations relative to a human reference genome in single molecules of megabase lengths.
  • the present invention provides for optimized and more convenient methods for the analysis of nucleic acid molecules, e.g. single molecules, such as a genomic sequence from a single cell.
  • photonic dyes used to stain single nucleic acid molecules may also be used in a rational and controlled approach to introduce nicks in the nucleic acid molecule to fragment the string of nucleic acids, such as a long genomic sequence of DNA in a controlled manner to a predetermined size within a certain range.
  • the present invention relates to a method for visualising or analysing a single nucleic acid molecule, the method comprising the steps of
  • nucleic acid analysis such as optical melting mapping with the viewing of said molecule in an imaging device.
  • the present invention relates to a method for visualising or analysing a single nucleic acid molecule, the method comprising the steps of a) staining a nucleic acids molecules with an photonic dye;
  • the present invention relates to a method for fragmenting a nucleic acid molecule, the method comprising the step of a) photonicking a nucleic acid molecules with a predetermined dose of light suitable to provide a predefined fragment length, and at a wavelength suitable for specific photonicking by a photonic dye; optionally introducing a reagent to prevent further photonicking.
  • the present invention relates to a single cell processing device for analysing a single nucleic acid molecule by a method according to the invention, the processing device comprising an organic polymer matrix enabling fixation of a nucleic acid molecule and having an inlet for introduction of a liquid solution.
  • the matrix is made of a material transparent for the light at the wavelength used, such as glass or a plastic polymer, such as a material transparent for UV light. In some embodiments the matrix is made of a material transparent for only the light at the wavelength used. In some embodiments the device has an opening or part of the device made of a material transparent for the light at the wavelength used.
  • the device comprises a photonic dye configured to collecting light energy and transfer the light energy to the nucleic acid molecule fixated in the matrix.
  • the device comprises an inlet or a container for the photonic dye.
  • Fig. 1 Protocol for extracting and imaging genomic DNA from a single cell.
  • a cell is trapped, and (B) lysed by a detergent.
  • C The nucleus remains in the cell trap and DNA is stained with an intercalating dye.
  • D DNA is fragmented by photonicking, and (E) released into a microfluidic channel by proteolysis.
  • F A denaturation-renaturation pattern is created on the DNA with a heat cycle.
  • G DNA fragments are elongated by entropic confinement in a nanofluidic device, and the DR patterns in on individual molecules are imaged.
  • Fig. 2 Overview of the device with nanochannel array.
  • a cell is trapped and lyzed (Fig. 3A-E).
  • the proteins are removed with proteolysis so the DNA can pass through the trap (Fig. 3F-J), and enter the region with the meandering channel (green box and Fig. 3K).
  • the DNA molecules enter the nanochannel array (red box).
  • A-E Fluorescence time-lapse imaging of the lysis of the cytoplasm with loss of the calcein signal (A to B) followed by staining of the DNA by 0.1 ⁇ YOYO-1 (B through E).
  • steps A-E The duration of steps A-E is 10 minutes.
  • F-J Proteolysis of the chromatin. The arrow shows the flow direction through the cell trap (delineated by the dash lines). Scale bar is 10 ⁇ . The duration of steps F-J is 7.5 seconds.
  • K Genomic DNA extracted from a single cell in the meandering channel (green box in Fig.2). The DNA forms a plug as observed at five different positions separated by 4700 ⁇ (i.e., containing a volume of 4.2 nL). Most of the DNA is contained within three meanders corresponding to a volume below 10 nL under the flow conditions of the lysis.
  • Fig 4. Mean intensity of the cells during lysis and staining at 5uM (red, 4 cells), luM (yellow, 5 cells averaged), 0.5uM (cyan, 5 cells) and O. luM (blue, 5 cells) in 0.5xTBE + 0.5% v/v triton-X100.
  • Fig. 5 Size distribution and exponential fit of fragment lengths. Lengths of DNA extracted from a single cell exposed to a photonicking dose of 1 ⁇ /( ⁇ )2 for 210 seconds.
  • Fig. 6 Device with nanochannel array for DNA fragment sizing.
  • A Bright field image of the meander channel and
  • B the connection between micro channel and nanochannel array of the sealed polymer device.
  • C-F The four main steps of the device operation including the trapping of a cell, the lysis of the cytoplasm, the proteolysis and the introduction of DNA fragments to the nanochannel array for sizing.
  • Fig. 8. Device for optical mapping with cross-shaped nanoslit (a) An all-polymer lab-on-a-chip device with 14 connectors comprises a cell trap (blue), a meandering channel (green) and a flow-stretch device (red), (b) A single cell is captured by hydrodynamic trapping, DNA is extracted and patterned according to AT/GC composition by a heating-cooling cycle, and genomic DNA is stretched and visualized. The flow-stretch device allows imaging the patterned DNA at 85-100% stretching, (c) A single cell trapping device can also be used to extract and amplify DNA before sequencing, (d) The device in (a-b) is used to prepare genomic DNA for optical mapping and structural variation detection.
  • Fig. 9. (A) Single field-of-view of a segment of a stretched DNA molecule (scale bar is ⁇ ). (B) Full D-R map of a molecule is stitched together from five field-of-views. (C) Match between the D-R map and a position on chromosome 7 of computer-simulated whole genome melting map.
  • matrix refers to any chemical, such as an organic polymer with a fixed structure enabling the fixation of a nucleic acid molecule, while allowing the introduction of liquid solutions.
  • single nucleic acid molecule refers to one single polymer of a nucleic acids molecule including DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), in any form such as genomic DNA.
  • single cell processing devices refers to micro- and nano-fluidic devices suitable for the processing at single cell level known to the person skilled in the art. Included within this definition e.g. single-use polymer devices fabricated, e.g. by injection molding, lab-on-a-chip devices, as well as integrated devices which couple single cell handling with single cell genome extraction and other means of handling the DNA.
  • photonic dye refers to any dye capable of collecting a light energy and transferring the light energy to the nucleic acid molecule; and thereby nicking the nucleic acid molecule to produce fragments of the nucleic acid molecule.
  • Photonicking refers to the process wherein a photonic dye collects light energy and transferring the light energy to the nucleic acid molecule; and thereby nicking the nucleic acid molecule to produce fragments of the nucleic acid molecule. This is preferably performed at room temperature.
  • predetermined dose of light is a combination of time for illumination and intensity of the light suitable to provide a predefined fragment length.
  • the time and intensity may be varied within wide ranges. However the skilled person will know how to vary these two parameters to obtain a desired length within a range.
  • wavelength suitable for specific photonicking by said photonic dye refers to any wavelength of the light used to nick the nucleic acid with a particular dye.
  • light with a wavelength from 10 nm to 700 nm is used.
  • YOYO-1 is a green fluorescent dye with an absorption wavelength around 450-490 nm what would be a suitable wavelength.
  • optical melting mapping refers to a well-established method used to visualise single nucleic acid molecules.
  • the term refers to an optical map formed through a heat cycle that creates partial denaturation and renaturation, which takes advantage of the sequence-dependent melting temperature of double-stranded DNA bonds.
  • DNA from the single cell stained with an intercalating dye and fragmented are stretched in e.g. nanochannels and imaged by fluorescence microscopy and from their melting pattern the molecules are mapped to their origin in the human reference genome.
  • reagent to prevent further photonicking refers to any agent that may work as an antioxidant or oxygen scavenging agent to stop the process of photonicking by the photonic dye.
  • Suitable reagents may include 2-Mercaptoethanol (BME), glucoxidase, dithiothreitol (DTT), mercaptoethylamine, Tris[2-carboxyethyl] phosphine (TCEP), (N- acetylcysteine), Nacystelyn, dornase alfa, thymosin ⁇ 4, guaifenesin, or any combination thereof.
  • BME 2-Mercaptoethanol
  • DTT dithiothreitol
  • TCEP Tris[2-carboxyethyl] phosphine
  • TCEP Tris[2-carboxyethyl] phosphine
  • Nacystelyn dornase alfa, thymosin ⁇ 4,
  • proteolysis refers to the complete or partial removal of protein components of surrounding or attached to nucleic acid molecules, such as to genomic DNA molecules. Any suitable non-specific protease may be used, such as proteinase K or pepsin.
  • the present invention comprises the photonicking of a nucleic acid molecules with an photonic dye. This photonic dye is capable of collecting a light energy and transferring the light energy to the nucleic acid molecule; and thereby nicking the nucleic acid molecule to produce fragments of the nucleic acid molecule.
  • the dye collects a light energy that illuminates on it and is being excited to an excited state by the light energy. The dye then transfers the light energy to the nucleic acid molecule.
  • 5-hydroxytryptamine acridines
  • Alexa Fluor® type ATTO
  • BODIPY® boron-dipyrromethene
  • photonic dyes are generally available from commercial sources such as Sigma-Aldrich Co. LLC, USA, Life Technologies, New York, USA, ATDBio Ltd., UK, ATTO-TEC GmbH, Germany, Hoechst AG, Germany, and SETA BioMedicals, IL, USA.
  • ATTO type of photonic dyes include, but are not limited to, ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 520, ATTO 532, ATTO 550, ATTO 565, ATTO 590, ATTO 594, ATTO 610, ATTO 61 IX, ATTO 620, ATTO 633, ATTO 635, ATTO 637, ATTO 647, ATTO 647N, ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO 725 and ATTO 740.
  • Acridines include, but are not limited to, N,N,N',N'-tetramethylacridine-3,6-diamine (Acridine orange), 2,7-dimethylacridine-3,6-diamine (acridine yellow), 9-bromoacridine, 9- chloroacridine, 2-hydroxy-10H-acridin-9-one, 9-aminoacridine, 9,10-dihydroacridine, 9- amino-l,2,3,4-tetrahydroacridine, 6,9-dichloro-2-methoxyacridine, 9-acridinecarboxylic acid, l,3-dihydroxy-9-acridinecarboxylic acid, 9-hydroxy-4-methoxyacridine, l,2,3,4-tetrahydro-9- acridinecarboxylic acid, 2-methyl-9-acridinecarboxaldehyde, 6,9-diamino-2-e
  • Alexa Fluor type of photonic dyes include, but are not limited to, Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 500, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700 and Alexa Fluor® 750.
  • BODIPY® type of photonic dyes include, but are not limited to BODIPY® 493/503, BODIPY® FL-X, BODIPY® FL, BODIPY® R6G, BODIPY® 500/510, BODIPY® 530/550, BODIPY® TMR- X, BODIPY® 558/568, BODIPY® 564/570, BODIPY® 576/589, BODIPY® 581/591, BODIPY® TR-X, BODIPY® 630/650-X, and BODIPY® 650/665-X.
  • CY type of photonic dyes include, but are not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5, including NHS esters and azides.
  • Hoechst type of photonic dyes include, but are not limited to, Hoechst 33342, Hoechst 33258, and Hoechst 34580.
  • Oregon Green type of photonic dyes are fluorinated analogs of fluoresceins, which include, but are not limited to, Oregon Green 488, Oregon Green 500 and Oregon Green 514.
  • Rhodamine type of photonic dyes include, but are not limited to, rhodamine 6G, rhodamine 101, rhodamine 110, rhodamine 123, rhodamine B, 5(6)-carboxy-X- rhodamine, 5(6)- carboxy-X-rhodamine N-succinimidyl ester, 5(6)- carboxytetramethylrhodamine, 5(6)- carboxytetramethylrhodamine N-succinimidyl ester, 5- carboxy-X-rhodamine N-succinimidyl ester, 5-carboxy-tetramethylrhodamine N-succinimidyl ester, 5- carboxytetramethylrhodamine, 6-carboxy-tetramethylrhodamine N-succinimidyl ester, 6- carboxytetramethylrhodamine, N-(2-aminoethyl
  • Compounds comprising Ru(bpy) 3 2+ include, but are not limited to, Ru(bpy) 3 CI 2 ,
  • Compounds comprising Pi 2 (P 2 0 5 H 2 ) 4 ⁇ (Pt 2 (pop) 4 4" ) include, but are not limited to, K 4 Pt 2 (pop) 4 , and (NH 4 ) 4 Pt2(pop) 4 .
  • YOYO type of photonic dyes are dimeric cyanine compounds which include, but are not limited to, YOYO-1 and YOYO-3.
  • SeTau type of photonic dyes include, but are not limited to, SeTau-647, SeTau-655, SeTau- 665, SeTau-380-NHS, SeTau-404-NHS, SeTau-405-NHS, and SeTau-425- NHS.
  • the photonic dye is 5-hydroxytryptamine, ATTO 565, ATTO 655, Acridine Orange, Acridine Yellow, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 680, BODIPY® 500/510, BODIPY® 530/550, BODIPY® FL, BODIPY® TR-X, Cascade Blue®, (pyrenyloxytrisulfonic acid), Coumarin 6, CY2, CY3B, CY3, CY3.5, CY5, CY5.5, Dansyl, 5-(dimethylamino)naphthalene-l - sulfonamide, DAPI, l,6-diphenyl-l,3,5-hexatriene (DPH), 2-(6- hydroxy-2,4,5,7-tetraiodo-3- oxo-xanthen-9-yl)
  • the photonic dye is fluorescein, a fluorescein salt or a fluorescein derivative.
  • Salts of fluorescein include, but are not limited to, the sodium salt and disodium salt.
  • fluorescein derivatives There are many fluorescein derivatives.
  • fluorescein derivatives For example, l-(0'- methylfluoresceinyl)piperidine-4- carboxylic acid, 2',7'-dichlorofiuorescein diacetate, 5(6)- carboxyfiuorescein, 5(6)- carboxyfiuorescein diacetate, 5(6)-carboxyfiuorescein diacetate N- succinimidyl ester, 5- (bromomethyl)fluorescein, 5-(iodoacetamido)fiuorescein, 5-([4,6- dichlorotriazin-2- yl]amino)fluorescein hydrochloride, 5-carboxy-fiuorescein diacetate N- succinimidyl ester, 5- carboxyfiuorescein, 5-carboxyfiuorescein N-succinimidyl ester, 6-carboxy- fluorescein diacetate
  • Additional fluorescein derivatives include 2',7'-difiuorofiuorescein (OREGON GREENTM), 5-[4-benzoic acid]-10,15,20-tris[3,5- di-tert-butylphenyl]-2 IH,23H-porphyrin, and 9-[2-(3-carboxy-9, 10-diphenyl)anthryl]-2,7- difiuoro-6-hydroxy-3H-xanthen-3-one, Taku Hasobe, et al., Chemical Physics, 319 (2005) 243- 252. Specific embodiments of the invention
  • the present invention relates to a method for visualising or analysing a single nucleic acid molecule, the method comprising the step of
  • nucleic acid analysis such as optical melting mapping with the viewing of said molecule in an imaging device.
  • this matrix is an organic polymer, such as a gel, such as an agarose or polyacryalamide gel.
  • nucleic acid molecule is fragmented to provide sub- fragments approximating to megabase-lengths, such as more than about 1 Mb length.
  • proteolysis is performed in the presence of proteinase K or pepsin.
  • the photonic dye is selected from 5-hydroxytryptamine, an acridine, an Alexa Fluor® dye, an ATTO dye, a BODIPY® dye, Coumarin 6, a CY dye, DAPI, an ethidium compound, a Hoechst dye, Oregon Green, rhodamine, a compound comprising Ru(bpy)32+, a compound comprising (Pt2(pop)44-, a YOYO dye, a TOTO dye, a BOBO dye, or a SeTau dye.
  • the photonic dye is a fluorescent intercalating dye such as one selected from the list consisting of YOYO-1, YOYO-3, BOBO-1, BOBO-3, TOTO-1, and TOTO-3.
  • the nucleic acid molecule is double-stranded DNA, such as genomic DNA.
  • the nucleic acid molecule is double-stranded DNA with length 100 kbp-1 Mbp. In some other embodiments the nucleic acid molecule is double-stranded DNA with length 1 Mbp-10 Mbp.
  • the nucleic acid molecule is double-stranded DNA with length 10 Mbp- 100 Mbp.
  • the nucleic acid molecule is double-stranded DNA with length 100 Mbp-1000 Mbp.
  • the method is performed in a nanochannel of a nanofluidic device.
  • the method is performed in a device selected from a microfluidic device or a nanofluidic device. In some other embodiments the method is performed in an organic polymer.
  • the method comprises the removing of non-nucleic acid molecules by contacting the processing device or matrix comprising the nucleic acid molecule with a proteinase, an elastase, a collagenase, a lipase, a carbohydratase, a pectinase, a pectolyase, an amylase, an RNase, a hyaluronidases, a chitinase, a gluculase, a lyticase, a zymolyase, a lysozyme, a labiase, an achromopeptidase, or a combination thereof.
  • a proteinase an elastase, a collagenase, a lipase, a carbohydratase, a pectinase, a pectolyase, an amylase, an RNase, a hy
  • the method comprises the removing of non-nucleic acid molecules by contacting the processing device or matrix comprising the nucleic acid molecule with a non-enzymatic proteolysis.
  • the method comprises moving said fragmented nucleic acid molecules from a processing device or matrix, such as a micro channel device to a device capable of stretching the fragmented nucleic acids, such as a microchannel, a nanochannel, a pillar, or pore, or a nanoslit of a flow-stretch device optionally using electrophoresis by applying a voltage difference across the nanoslit.
  • the method comprises moving said fragmented nucleic acid molecules from a processing devices or matrix, such as a micro channel device to a separate instrument for nucleic acid analysis or amplification, optionally with an analysis of the amplification product.
  • the reagent to prevent further photonicking is selected from 2- Mercaptoethanol (BME), trolox, glucoxidase, dithiothreitol (DTT), mercaptoethylamine, Tris[2-carboxyethyl] phosphine (TCEP), (N-acetylcysteine), Nacystelyn, dornase alfa, thymosin ⁇ 4, guaifenesin, or any combination thereof betamercaptoethanol.
  • BME 2- Mercaptoethanol
  • trolox glucoxidase
  • DTT dithiothreitol
  • TCEP Tris[2-carboxyethyl] phosphine
  • Nacystelyn dornase alfa, thymosin ⁇ 4, guaifenesin, or any combination thereof betamercaptoethanol.
  • the visualisation is done in an imaging device.
  • the dose of light represent an intensity in the range of 0.1- 10 ⁇ /( ⁇ )2, such as 0.2-9 ⁇ /( ⁇ )2, such as 0.3-8 ⁇ /( ⁇ )2, such as 0.4-7 ⁇ /( ⁇ )2, such as 0.5-6 ⁇ /( ⁇ )2, such as 0.6-5 ⁇ /( ⁇ )2, such as 0.7-4 ⁇ /( ⁇ )2, such as 0.8- 3 ⁇ /( ⁇ )2, such as 0.9-2 ⁇ /( ⁇ )2, such as 1-2 ⁇ /( ⁇ )2, and/or with an illumination time of 10-1000 seconds, such as in the range of 10-900 seconds, such as in the range of 10-800 seconds, such as in the range of 10-700 seconds, such as in the range of 10-600 seconds, such as in the range of 10-500 seconds, such as in the range of 10-400 seconds, such as in the range of 10-300 seconds, such as in the range of 20-100 seconds, such as in the range of 20-200 seconds, such as in the range of 20-300 seconds,
  • a cell from a cancer cell line is trapped in a disposable polymer lab-on-a-chip device. Its cytoplasm is removed by a detergent while the nucleus remains in the trap. Here its DNA is stained with an
  • FIG. 2 shows an overview of the device.
  • the device comprises a cell trap, in which a single cell is trapped and lysed, and a nanochannel array to stretch and visualize the DNA extracted from the cell.
  • Cells introduced in the feeding channel are aligned against the walls of the microchannel. A narrow constriction connects it to another microchannel, thus constituting a cell trap.
  • a random, single cell is typically captured in the trap after a few minutes. Cells that escape the trap are directed to the waste outlet.
  • the cell can be imaged with fluorescence imaging (Fig. 3A).
  • the trapping force is sufficient to allow the exchange of solution up to a flow rate of 50 nL/min in the feeding channel although over time cells can slowly deform, release parts of the cytoplasm, or escape.
  • the trap dimension allows us to keep the nucleus in the trap, so the chromatin can be further investigated.
  • YOYO-1 is only fluorescent when attached to DNA
  • the cell's entire genome should be fragmented into molecules larger than
  • Photonicking cannot produce a monodispersed length distribution but is still an efficiently tool for fragmentation. Accordingly, in this present invention photonicking is used to obtain a predicted length distribution. Photonicking and photobleaching are unwanted effects when working with genomic DNA in nanofluidics. They are suppressed by adding BME and/or an oxygen scavenging system to the buffer [Perkins 1994], but from an engineering point of view this also provides a way to turn photonicking on and off. First we fragment the DNA by photonicking in a buffer flow without added BME. Then we simultaneously add BME to the buffer and reduce the excitation light intensity to arrest photonicking.
  • the DNA plug contains less than 10 nl_ of solution. It can be stored temporarily in the channel while the device temperature is lowered from the temperature at which proteolysis is made. The oil immersion objective of the microscope is moved into place, which improves the cooling of the device.
  • a heat cycle is introduced after the proteolysis while keeping the DNA in the microchannel (see 'Materials and Methods' for details) [Reisner 2010] .
  • the temperature is raised to a level between the melting temperature of the AT- and the GC-bonds.
  • the double strand opens up, and the intercalating dye YOYO-1 is released and washed away.
  • the double strands closes, leaving only YOYO-1 in GC-rich regions.
  • the stability of the polymer device and the generation of air bubbles in the microchanels are a concern.
  • the staining ratio during the cell lysis is adjusted so the temperature range at which the DNA double-strand starts to melt can be tolerated by the polymer device.
  • the characteristic length scale varies between 70 ⁇ 10 kb for an illumination time of 300 s to at 140 ⁇ 10 kb for 150 s.
  • the size distribution is exponential, less than 1% of the total amount of genomic DNA falls below the lower cut-off.
  • FIG. 8a-b In a different device we replace the nanochannel array by a cross-shaped nanoslit device ( Figure 8a-b).
  • the DNA is barcoded with denaturation-renaturation patterns by heating and cooling the DNA while it travels through the meandering channel.
  • mega-base pair-long fragments barcoded with denaturation-renaturation patterns can be introduced using electrophoresis and stretched by the double elongation flow in the cross-shaped nanoslit (see Marie 2013).
  • Figure 9 shows the result of the optical mapping of such a fragment resulting in the mapping of the DNA fragment to its location in the genome.
  • Device fabrication The device architecture for optical mapping is summarized in Fig. 2.
  • Microchannels are designed to accommodate cells from a human cancer cell line with a typical diameter of 15 ⁇ , so the microfluidic network has a minimal depths and widths of 33 ⁇ .
  • the device comprises a cell and a buffer inlet (Fig. 2), which merge into a single channel to feed the single-cell trap (blue box in Fig. 2). At the intersection between the cell and buffer inlets, cells get aligned along the side wall of the feeding channel where the trap is located.
  • the trap is a simple constriction dimensioned to capture cells from a human cancer cell line. The constriction for cell trapping is 4.3 ⁇ .
  • the cell trap connects the feeding channel with the outlet channels, which consist of two meandering channels: one for waste and the other (green box in Fig. 2) leading to the outlet and the DNA-stretch device, in this case a nanochannel array device (red box in Fig. 2).
  • the nanochannel array device is replaced by a flow-stretch device shaped as a cross (figure 8b).
  • the flow-stretch device consists of a lOOnm-deep cross-shaped nanoslit that connects the outlet channel to three other microchannels.
  • a DNA molecule is introduced to the nanoslit by electrophoresis thus it extends across the nanoslit then it is stretched by a double-elongation flow and imaged (see Marie 2013).
  • the device is fabricated by replicating a nickel shim using injection molding of TOPAS 5013 (TOPAS) as previously reported (Utko 2011, Ostergaard 2015). Briefly, a silicon master is produced by UV lithography and reactive ion etching. A 100 nm NiV seeding layer is deposited and nickel is electroplated to a final thickness of 330 ⁇ . The Si master is chemically etched away in KOH. Injection molding is performed using a melt temperature of 250 ° C, a mold temperature of 120 ° C, a maximum holding pressure of 1500 bar for 2 seconds, and an injection rate varying between 20cm3/s and 45 cm3/s.
  • TOPAS 5013 TOPAS 5013
  • a 150 ⁇ TOPAS foil is used to seal the device by a combined UV and thermal treatment under a maximum pressure of 196 Pa.
  • the device is mounted on an inverted fluorescence microscope (Nikon T2000) equipped with an air objective (50x/0.8), an oil objective (60x/1.40) and an EMCCD camera (Photometries cascade II 512). Liquids are driven through the device using a pressure controller (MFCS, Fluigent).
  • MFCS Fluigent
  • the device is primed with ethanol, then degassed FACSFlow (BD Biosciences) is loaded in all microchannels but the microchannel connecting the flow-stretch device. Instead, a buffer suitable for single molecule imaging and electrophoresis
  • DMEM fetal bovine serum
  • FBS fetal bovine serum
  • penicillin/streptomycin Lico-streptomycin
  • cell suspension is mixed 1 : 1 with FACSFlow buffer, centrifuged at 400 rpm for 5 minutes and resuspended in FACSFlow. Finally, the cells are stained with 1 ⁇ Calcein AM (Invitrogen) and loaded in the chip at 0.35 106 cells/mL.
  • the protocol for operating the device is sketched in figure 6 C-F.
  • cells and buffer are introduced simultaneously aligning the cells along the side wall of the micro channel where the trap is located.
  • a cell is captured and kept in the trap for a buffer flow through the trap up to 30 nL/min.
  • the lysis buffer composed of 0.5xTBE+0.5% v/v triton- XlOO+0.1 ⁇ YOYO-1 (Invitrogen) is loaded in one of the inlets and injected at 10 nL/min through the trap for 10 minutes. Then the solution is exchanged to a buffer without YOYO-1 in all wells in order to stop the staining.
  • the cell nucleus is exposed to blue excitation light at a dose of 1 ⁇ /( ⁇ )2 for up to 300 seconds causing a partial photonicking of the DNA.
  • the buffer is changed to a solution containing BME (0.5xTBE+0.5 % v/v triton- X100+l% v/v BME) and the excitation of the fluorescence lamp is lowered to the minimum intensity allowed for fluorescence imaging.
  • temperature is raised to 60 ° C and the proteolysis solution (0.5xTBE+0.5 % v/v triton-X100+l% v/v BME- ⁇ -200 ⁇ g/mL) is introduced, pushing the lysate through the trap.
  • the DNA staining is modified thus the lysis buffer contains 0.04 ⁇ YOYO-1 instead of 0.1 ⁇ .
  • the device is heated further after the proteolysis for 10 minutes at 80 ° C before rapid cooling to room temperature by moving the oil immersion objective in place.
  • the temperature at which the DR-pattern is created is highly depending on the dye loading and the ionic strength of the buffer and should be adjusted by steps of 2 degrees if necessary.
  • DNA travels through the meandering channel as the temperature is lowered and an oil immersion objective is moved into place for single molecule imaging (60x/1.4).
  • DNA fragments are introduced from the micro channel to the nanoslit of the flow-stretch device using electrophoresis by applying a voltage of 5-10 V across the nanoslit.
  • Barcode imaging and mapping The 450 ⁇ portion of the molecule stretched in the nanoslit is imaged 50ms exposure in 5 overlapping field-of-views.
  • the DR-pattern along the DNA is extracted by simply averaging across the DNA and over twenty frames to produce a barcode for each field-of-view.

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Abstract

La présente invention concerne des procédés pour la cartographie optique et l'analyse ultérieure, tels que le séquençage de polynucléotides, par exemple l'ADN génomique, dans des dispositifs à micro-canaux.
PCT/EP2017/065200 2016-06-21 2017-06-21 Procédés de cartographie optique de polynucléotides WO2017220637A1 (fr)

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WO2021247394A1 (fr) * 2020-06-01 2021-12-09 Dimensiongen Dispositifs et procédés d'analyse génomique
EP3984640A1 (fr) * 2020-10-16 2022-04-20 Universität Hamburg Dispositif d'analyse nanofluidique autonome et procédé d'analyse de molécules d'adn
EP4121560B1 (fr) * 2020-08-10 2024-07-10 Dimensiongen Dispositifs et procédés d'analyse de génome multidimensionnelle

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WO2021247394A1 (fr) * 2020-06-01 2021-12-09 Dimensiongen Dispositifs et procédés d'analyse génomique
CN111889082A (zh) * 2020-08-07 2020-11-06 南昌航空大学 一种接枝巯基的介孔有机聚合物的制备方法
EP4121560B1 (fr) * 2020-08-10 2024-07-10 Dimensiongen Dispositifs et procédés d'analyse de génome multidimensionnelle
EP3984640A1 (fr) * 2020-10-16 2022-04-20 Universität Hamburg Dispositif d'analyse nanofluidique autonome et procédé d'analyse de molécules d'adn
WO2022078690A1 (fr) * 2020-10-16 2022-04-21 Universität Hamburg Dispositif d'analyse nanofluidique autonome et procédé d'analyse de molécules d'adn

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