EP2635708A1 - Analyse d'adn génomique fragmenté dans des gouttelettes - Google Patents

Analyse d'adn génomique fragmenté dans des gouttelettes

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Publication number
EP2635708A1
EP2635708A1 EP11838712.5A EP11838712A EP2635708A1 EP 2635708 A1 EP2635708 A1 EP 2635708A1 EP 11838712 A EP11838712 A EP 11838712A EP 2635708 A1 EP2635708 A1 EP 2635708A1
Authority
EP
European Patent Office
Prior art keywords
droplets
dna
genomic dna
per
droplet
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP11838712.5A
Other languages
German (de)
English (en)
Other versions
EP2635708A4 (fr
Inventor
Kevin D. Ness
Amy L. Hiddessen
Paul W. Wyatt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bio Rad Laboratories Inc
Original Assignee
Bio Rad Laboratories Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US12/976,827 external-priority patent/US9598725B2/en
Application filed by Bio Rad Laboratories Inc filed Critical Bio Rad Laboratories Inc
Publication of EP2635708A1 publication Critical patent/EP2635708A1/fr
Publication of EP2635708A4 publication Critical patent/EP2635708A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • Emulsions hold substantial promise for revolutionizing high-throughput assays for targets.
  • Emulsification techniques can create large numbers of aqueous droplets that function as independent reaction chambers for biochemical reactions. For example, an aqueous sample (e.g., 20 microliters) can be partitioned into droplets (e.g., 20,000 droplets of one nanoliter each) to allow an individual test for the target to be performed with each of the droplets.
  • Aqueous droplets can be suspended in oil to create a water-in-oil emulsion (W/O).
  • W/O water-in-oil emulsion
  • the emulsion can be stabilized with a surfactant to reduce coalescence of droplets during heating, cooling, and transport, thereby enabling thermal cycling to be performed.
  • emulsions have been used to perform single-copy amplification of nucleic acid target molecules in droplets using the polymerase chain reaction (PCR).
  • Digital assays are enabled by the ability to detect the presence of individual molecules of a target in droplets.
  • a sample is partitioned into a set of droplets at a limiting dilution of a target (i.e., some of the droplets contain no molecules of the target). If molecules of the target are distributed randomly among the droplets, the probability of finding exactly 0, 1 , 2, 3, or more target molecules in a droplet, based on a given average concentration of the target in the droplets, is described by a Poisson distribution. Conversely, the concentration of target molecules in the droplets (and thus in the sample) may be calculated from the probability of finding a given number of molecules in a droplet.
  • each droplet can be tested to determine whether the droplet is positive and contains at least one molecule of the target, or is negative and contains no molecules of the target.
  • the probability of finding no molecules of the target in a droplet can be approximated by the fraction of droplets tested that are negative (the "negative fraction"), and the probability of finding at least one target molecule by the fraction of droplets tested that are positive (the "positive fraction").
  • the value of the positive fraction or the negative fraction then may be utilized in a Poisson algorithm to calculate the concentration of the target in the droplets.
  • the digital assay may generate data that is greater than binary.
  • the assay may measure how many molecules of the target are present in each droplet with a resolution greater than negative (0) or positive (>0) (e.g., 0, 1 , or >1 molecules; 0, 1 , 2, or >2 molecules; or the like).
  • negative (0) or positive >0
  • the assay may measure how many molecules of the target are present in each droplet with a resolution greater than negative (0) or positive (>0) (e.g., 0, 1 , or >1 molecules; 0, 1 , 2, or >2 molecules; or the like).
  • droplets should be generated rapidly and with a uniform size (i.e., monodisperse droplets).
  • sample components can interfere with the ability of droplets to separate from the bulk sample phase, particularly as the frequency of droplet generation is increased.
  • the size of droplets formed, or even the ability to form droplets at all can vary from sample to sample, diminishing the reliability of the assays.
  • New approaches are needed to provide reliable and consistent generation of droplets at a higher generation frequency.
  • Genomic DNA including a target may be obtained.
  • the genomic DNA may be fragmented volitionally to produce fragmented DNA.
  • the fragmented DNA may be passed through a droplet generator to generate aqueous droplets containing the fragmented DNA.
  • a digital assay may be performed on the droplets to determine a level of the target.
  • the droplets may contain the genomic DNA at a concentration of at least about five nanograms per microliter, the droplets may be generated at a droplet generation frequency of at least about 50 droplets per second, the droplets may have an average volume of less than about 10 nanoliters per droplet, the droplets may generated at a sample flow rate of greater than about 50 nanoliters per second, or any combination thereof.
  • Figure 1 is a flowchart illustrating an exemplary method of analyzing genomic DNA, in accordance with aspects of the present disclosure.
  • Figure 2 is a matrix of drawings made from photographs of a droplet generator processing three different samples at each of four different driving pressures.
  • Figure 3 is a graph of droplet volume plotted as a function of droplet generation frequency for samples containing no genomic DNA or genomic DNA (Raji or Coriell) that is digested (EcoRI) or undigested.
  • Figure 4 is a graph of droplet volume plotted as a function of sample flow rate for the samples of Figure 3.
  • Figure 5 is a graph of maximum extension plotted as a function of droplet generation frequency for the samples of Figure 3.
  • Figure 6 is a graph of maximum extension plotted as a function of sample flow rate for the samples of Figure 3.
  • Genomic DNA including a target may be obtained.
  • the genomic DNA may be fragmented volitionally to produce fragmented DNA.
  • the fragmented DNA may be passed through a droplet generator to generate aqueous droplets containing the fragmented DNA.
  • An assay may be performed on the droplets to determine a level of the target.
  • the droplets may contain the genomic DNA at a concentration of at least about five nanograms per microliter, the droplets may be generated at a droplet generation frequency of at least about 50 droplets per second, the droplets may have an average volume of less than about 10 nanoliters per droplet, the droplets may generated at a flow rate of greater than about 50 nanoliters per second, or any combination thereof.
  • the method of analyzing genomic DNA in droplets has substantial advantages over other droplet-based approaches.
  • the advantages may include generating droplets at a higher frequency, with greater monodispersity, with a higher load of DNA, and/or with substantially less interference from genomic DNA.
  • Figure 1 shows a flowchart illustrating an exemplary method 20 of analyzing genomic DNA. The steps presented may be performed in any suitable order and in any suitable combination.
  • Genomic DNA may be obtained, indicated at 22.
  • the DNA may be obtained from any suitable organism, such as a mammal (e.g., human, mouse, rat, monkey, etc.), a non-mammalian vertebrate, an invertebrate, a yeast or fungus, a plant, a protozoan, a bacterium, or the like.
  • the DNA may be obtained by any suitable process, such as purchased commercially, received as a gift, acquired by extraction from cells or fluid, received as a clinical sample, or the like.
  • the DNA may be obtained in a relatively high molecular weight form, such as having a molecular weight of at least about 10 4 , 10 5 or 10 6 kilodaltons, among others (e.g., having an average length of at least about 25, 50, 100, 200, 500, or 1 ,000 kilobases).
  • the genomic DNA may be fragmented, indicated at 24, before droplet generation. Fragmentation may be a volitional act, that is, performed deliberately. Fragmentation generally involves any procedure that substantially reduces the molecular weight of the genomic DNA, such as by cutting or breaking DNA strands. The fragmentation may reduce the average molecular weight and/or length by any suitable amount, such as at least about 5, 10, 20, 50, or 100-fold, among others.
  • An exemplary approach to fragmenting genomic DNA includes digestion with a restriction enzyme (e.g., an enzyme having a 4, 5, 6 or 8 nucleotide recognition site, among others).
  • the target may contain no recognition sites for the restriction enzyme, to avoid any cleavage of target molecules.
  • the restriction enzyme digestion may be performed to completion or may be a partial digestion.
  • an aqueous sample of the genomic DNA may be heated to fragment the DNA.
  • Exemplary heating that fragments the DNA may be performed at a temperature of at least 95 °C, for at least about 10, 15, 20, or 30 minutes, among others.
  • the DNA may be fragmented by shearing, sonicating, nebulizing, irradiating, or the like.
  • the genomic DNA may include a target, generally a sequence of interest to be tested. Fragmentation of the genomic DNA may be performed without substantially disrupting the target, meaning that less than one-half of target sequences in the genomic DNA are disrupted (e.g., broken or cut) by the fragmentation process.
  • Droplets containing the fragmented DNA may be generated, indicated at 26.
  • the droplets may be generated serially with each of one or more droplet generators.
  • the fragmented DNA may be passed through at least one droplet generator to generate droplets.
  • the fragmented DNA is disposed in an aqueous sample, and the aqueous sample and an immiscible continuous phase are passed through the droplet generator to form aqueous droplets containing the fragmented DNA and disposed in the continuous phase.
  • Further aspects of droplet generators and emulsion phases that may be suitable are described in the documents listed above under Cross- References, which are incorporated herein by reference, particularly U.S. Patent Application Publication No. 2010/0173394 A1 , published July 8, 2010; and PCT Patent Application No. WO 201 1/120024, published September 29, 201 1 .
  • the droplets may have any suitable size.
  • the droplets may have an average volume of less than about 1 ⁇ _, 100 nl_, 10 nl_, 1 nl_, 100 pL, 10 pL, or 1 pL, among others.
  • the droplets may have an average volume of greater than about 10 fl_, 100 fl_, 1 pL, 10 pL, or 100 pL, among others.
  • the droplets may have an average volume of about 1 pL to 100 nl_, 1 pL to 10 nl_, or 0.1 to 10 nl_, among others.
  • the droplets may be monodisperse.
  • the droplets may contain any suitable concentration of fragmented DNA.
  • the fragmented DNA may be disposed in the droplets at a concentration of at least about 0.1 , 0.2, 0.5, 1 , 2, 5, 10, 20, 50 ng/ L, among others. In some cases, the concentration may be about 0.1 -50 or 0.2-20 ng/ L. Fragmenting the DNA allows a higher DNA load to be incorporated into droplets.
  • the fragmented DNA may be present at an average of less than about two genome-equivalents per droplet.
  • the target may be present at an average of less than about two molecules per droplet.
  • the droplets may be formed at any suitable droplet generation frequency, such as at least about 10, 20, 50, 100, 200, 500, or 1 ,000 Hz (droplets/second), among others.
  • the droplet generation frequency is inversely related to the size of droplets being generated, with smaller droplets allowing a higher droplet generation frequency.
  • An aqueous sample used to form the droplets (and containing the fragmented DNA) may be passed through the droplet generator and/or converted into droplets at any suitable flow rate.
  • Exemplary flow rates that may be suitable include at least about 1 , 5, 10, 20, 50, 100, 200, 500, 1 ,000, 5,000, or 10,000 nL/second, among others.
  • the sample flow rate is directly related to the size of droplets being generated, with larger droplets permitting a higher flow rate.
  • the assay may be performed on the droplets, indicated at 28.
  • the assay may be a digital assay that detects individual target molecules in the droplets.
  • the digital assay may involve amplifying target molecules, such as by PCR or a ligase chain reaction, among others.
  • the digital assay also may involve detecting fluorescence from the droplets.
  • the assay further may involve determining a level (e.g., a concentration) of the target in the droplets with a Poisson algorithm.
  • Droplet generation in a microfluidic device may depend on the flow rate at which the sample travels to a droplet generator, and on the frequency with which droplets are generated. At high flow rates, a sample stream may jet into the immiscible continuous phase, no longer generating droplets. At generation rates close to the jetting limit, the sample starts to extend deeper into the outlet channel before droplets are generated. This extension length can be used to see how close a set of generation conditions is to the jetting limit.
  • Figure 2 shows a matrix of drawings made from photographs of a droplet generator 30 processing three different aqueous samples 32-36 at each of four different driving pressures and thus flow rates.
  • the three samples are (a) a control sample 32 containing no DNA (PCR buffer with no template), (b) an aqueous sample 34 of human genomic DNA (Raji, 18.75 ng/ L) that is undigested and has a high molecular weight (MW), and (c) an aqueous sample 36 of human genomic DNA (Raji, 18.75 ng/ L) that has been digested with a restriction enzyme and has a reduced molecular weight.
  • the four driving pressures (1 , 2, 3, and 4) are negative (vacuum) pressures applied downstream of the droplet generator and are expressed in pounds per square inch (psi), with 1 psi equal to about 6.9 kilopascals.
  • the vacuum level may control the sample flow rate, the total flow rate, and the droplet generation frequency.
  • Droplet generator 30 may be formed by a channel network composed of a sample inlet channel 38, at least one or a pair of oil inlet channels 40, 42, and an outlet channel 44.
  • Inlet channel 38 carries a bulk aqueous phase 46 of aqueous sample 32, 34, or 36 to the droplet generator.
  • Inlet channels 40, 42 carry a continuous phase 48 (e.g., oil with a surfactant) to the droplet generator.
  • Outlet channel 44 carries droplets 50 in continuous phase 48 away from a channel intersection 52.
  • the top row shows droplet generation of control sample 32, which contains no genomic DNA.
  • Droplets 50 are approximately 1 nl_ and do not vary much in size with the different vacuum levels.
  • the middle row shows droplet generation with sample 34 containing undigested genomic DNA.
  • the genomic DNA strongly impairs droplet generation, which occurs only at the lowest vacuum level tested (1 psi). Even at this lowest level, there is a considerable extension of bulk aqueous phase 46 past channel intersection 52, indicated by an arrow at 54, and droplets 50 are larger.
  • higher vacuum levels e.g., compare 1 psi with 2-4 psi
  • flow rates no droplets are generated, because the sample stream jets into outlet channel 44, indicated by an arrow at 56, without breaking up into droplets. Accordingly, the presence of human genomic DNA can strongly interfere with droplet generation, and may require use of lower DNA concentrations, flow rates, and droplet generation frequencies.
  • sample processing may be slowed considerably.
  • the frequency of target-positive droplets in the emulsion may be reduced substantially (due to the lower DNA concentration), which would require more droplets to be analyzed to achieve to the same confidence for the target level determined.
  • the bottom row shows droplet generation with sample 36 containing the same concentration (mass per unit volume) of genomic DNA as sample 34, but after the DNA has been digested with a restriction enzyme into shorter fragments. At the pressures (and flow rates) shown here, the genomic DNA in fragmented form does not detectably impair droplet generation.
  • the sample produces droplets 50 that are similar to control sample 32 lacking DNA.
  • aqueous samples used were Spectral Dye Buffer (the same control sample as in Figure 2, but without DNA polymerase), Raji human genomic DNA (Loftstrand Laboratories) ("Raji"), and 19205 human DNA (Coriell Institute) ("Corell”).
  • DNA samples were either undigested or digested with a restriction enzyme, EcoRI. DNA digestion was performed with a 20 ⁇ / ⁇ _ concentration of EcoRI (New England Biolabs) in NEB #4 buffer, with the genomic DNA at a final concentration of 200 ng/ L. The mixture was incubated at 37°C for one hour, and then was diluted to various final concentrations.
  • the graphs of Figures 3-6 show the results of droplet generation experiments performed with samples of Master Mix (no DNA), EcoRI-digested Raji DNA at 18.75 ng/pL, EcoRI-digested Coriell 19205 DNA at 18.75 ng/pL, undigested Raji DNA at 18.75 ng/ L, and undigested Coriell 19205 DNA at 18.75 ng/ L.
  • Figure 3 shows a graph of droplet volume plotted as a function of droplet generation frequency for the samples.
  • Figure 4 shows a graph of droplet volume plotted as a function of sample flow rate for the samples.
  • Figure 5 shows a graph of maximum extension plotted as a function of droplet generation frequency for the samples.
  • Figure 6 shows a graph of maximum extension plotted as a function of sample flow rate for the samples of Figure 3.
  • the graphs also show that for digested DNA at the same concentration, the effects of DNA on droplet generation are not detectable. No jetting or long sample extensions were observed at any of the flow rates or generation frequencies that were tested, and the droplet volumes are the same as with the sample with no DNA present.
  • a method of analyzing genomic DNA comprising: (i) obtaining genomic DNA including a target; (ii) fragmenting the genomic DNA volitionally to produce fragmented DNA; (iii) passing the fragmented DNA through at least one droplet generator to generate aqueous droplets containing the fragmented DNA; and (iv) performing a digital assay on the droplets to determine a level of the target.
  • the droplets have an average volume of less than about 10 nanoliters, wherein the genomic DNA is disposed in an aqueous sample, and wherein the droplets are generated at a flow rate of greater than about 50 nanoliters per second of the aqueous sample through the droplet generator.
  • the droplets contain the genomic DNA at a concentration of at least about 5 nanograms per microliter, wherein the genomic DNA is disposed in an aqueous sample, and wherein the droplets are generated at a flow rate of greater than about 50 nanoliters per second of the aqueous sample through the droplet generator.
  • a method of partitioning an aqueous sample comprising DNA into droplets comprising: (i) obtaining a sample comprising DNA at a concentration of at least about 5 ng per microliter; (ii) fragmenting the DNA volitionally to produce fragmented DNA; and (iii) passing the sample through a droplet generator, to generate aqueous droplets containing the fragmented DNA, the droplets being generated at a droplet generation frequency of at least about 50 droplets per second and having an average volume of less than about 10 nanoliters.
  • a method of partitioning an aqueous sample comprising DNA into droplets comprising: (i) obtaining a sample comprising genomic DNA; (ii) fragmenting the DNA volitionally to produce fragmented DNA; and (iii) passing the sample through a droplet generator, to generate aqueous droplets containing the fragmented DNA, the droplets being generated at a droplet generation frequency of at least about 50 droplets per second and having an average volume of less than about 10 nanoliters, wherein the genomic DNA is at a concentration that interferes with droplet generation if the step of passing is performed with the genomic DNA under the same conditions without fragmenting the DNA.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé d'analyse d'ADN génomique. De l'ADN génomique comprenant une cible peut être obtenu. L'ADN génomique peut être fragmenté volontairement pour produire de l'ADN fragmenté. L'ADN fragmenté peut être passé à travers un générateur de gouttelettes pour générer des gouttelettes aqueuses contenant l'ADN fragmenté. Un dosage peut être réalisé sur les gouttelettes pour déterminer un niveau de la cible. Dans certains modes de réalisation, les gouttelettes peuvent contenir de l'ADN génomique à une concentration d'au moins environ cinq nanogrammes par microlitre, les gouttelettes pouvant être générées à une fréquence de génération de gouttelettes d'au moins environ 50 gouttelettes par seconde, les gouttelettes pouvant avoir un volume moyen inférieur à environ 10 nanolitres par gouttelette, les gouttelettes pouvant être générées à un débit supérieur à environ 50 nanolitres par seconde, ou toute combinaison de ceci.
EP11838712.5A 2010-11-01 2011-11-01 Analyse d'adn génomique fragmenté dans des gouttelettes Withdrawn EP2635708A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US40910610P 2010-11-01 2010-11-01
US12/976,827 US9598725B2 (en) 2010-03-02 2010-12-22 Emulsion chemistry for encapsulated droplets
PCT/US2011/058854 WO2012061442A1 (fr) 2010-11-01 2011-11-01 Analyse d'adn génomique fragmenté dans des gouttelettes

Publications (2)

Publication Number Publication Date
EP2635708A1 true EP2635708A1 (fr) 2013-09-11
EP2635708A4 EP2635708A4 (fr) 2014-10-29

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EP11838712.5A Withdrawn EP2635708A4 (fr) 2010-11-01 2011-11-01 Analyse d'adn génomique fragmenté dans des gouttelettes

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EP (1) EP2635708A4 (fr)
JP (1) JP2013541347A (fr)
CN (1) CN103443289A (fr)
AU (1) AU2011323507A1 (fr)
CA (1) CA2816702A1 (fr)
SG (1) SG190076A1 (fr)
WO (1) WO2012061442A1 (fr)

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WO2017038682A1 (fr) 2015-08-28 2017-03-09 国立研究開発法人理化学研究所 Procédé d'analyse d'acide nucléique utilisé comme gabarit, procédé d'analyse de substance cible, kit d'analyse d'acide nucléique utilisé comme gabarit ou de substance cible, et dispositif d'analyse d'acide nucléique utilisé comme gabarit ou de substance cible
WO2018098438A1 (fr) * 2016-11-28 2018-05-31 Arizona Board Of Regents On Behalf Of Arizona State University Systèmes et procédés liés à une réaction de gouttelettes à écoulement continu

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WO2005043154A2 (fr) * 2003-10-27 2005-05-12 Massachusetts Institute Of Technology Chambres de réaction haute densité et procédés d'utilisation
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US8383345B2 (en) * 2008-09-12 2013-02-26 University Of Washington Sequence tag directed subassembly of short sequencing reads into long sequencing reads
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BENJAMIN J. HINDSON ET AL: "High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number", ANALYTICAL CHEMISTRY, vol. 83, no. 22, 28 October 2011 (2011-10-28), pages 8604-8610, XP055047554, ISSN: 0003-2700, DOI: 10.1021/ac202028g *
RYAN TEWHEY ET AL: "Microdroplet-based PCR enrichment for large-scale targeted sequencing", NATURE BIOTECHNOLOGY, vol. 27, no. 11, 1 November 2009 (2009-11-01), pages 1025-1031, XP055103848, ISSN: 1087-0156, DOI: 10.1038/nbt.1583 *
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Also Published As

Publication number Publication date
SG190076A1 (en) 2013-06-28
JP2013541347A (ja) 2013-11-14
WO2012061442A1 (fr) 2012-05-10
AU2011323507A1 (en) 2013-06-13
CA2816702A1 (fr) 2012-05-10
CN103443289A (zh) 2013-12-11
EP2635708A4 (fr) 2014-10-29

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