CN103443289A - Analysis of fragmented genomic DNA in droplets - Google Patents
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- CN103443289A CN103443289A CN2011800528318A CN201180052831A CN103443289A CN 103443289 A CN103443289 A CN 103443289A CN 2011800528318 A CN2011800528318 A CN 2011800528318A CN 201180052831 A CN201180052831 A CN 201180052831A CN 103443289 A CN103443289 A CN 103443289A
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- 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]
Abstract
Method of analyzing genomic DNA. 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. In some embodiments, 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.
Description
the cross reference of priority application
The application requires to enjoy the following right of priority in first to file: the U.S. Provisional Patent Application series number 61/409,106 that on November 1st, 2010 submits to; U.S. Provisional Patent Application series number 12/976,827 with on December 22nd, 2010 submits to, came forth for No. 2011/0217712A1 with U.S. Patent Application Publication on September 8th, 2011.Two patent applications are incorporated to this paper based on all purposes by reference of text at this.
the cross reference of extra data
The application is incorporated to following data based on all purposes by reference of text at this: the United States Patent (USP) the 7th that on May 9th, 2006 authorizes, 041, No. 481; The U.S. Patent Application Publication No. 2010/0173394A1 that on July 8th, 2010 announces; On September 29th, 2011 disclosed PCT number of patent application WO2011/120024; And JosephR.Lakowicz, PRINCIPLES OF FLUORESCENCE SPECTROSCOPY (second edition, 1999).
foreword
Many biomedical applications depend on the high throughput assay of nucleic acid target sample.For example, in studies and clinical application, use target-specific reagent high-throughput heredity test can for drug discovery, biomarker discovery and clinical diagnosis and other aspects provide nucleic acid target accurately and accurate the quantification.
Emulsion has been brought sizable hope for the high throughput assay of innovation target.Emulsifying technology can produce the water-containing drop of a large amount of independently reaction chambers as biochemical reaction.For example, aqueous specimen (as, 20 microlitres) can be allocated to drop (as, 20,000 drops, each receive liters), with permission, each drop carried out to the independent test of target.
Water-containing drop can be suspended in oil to produce water-in-oil emulsion (W/O).Can with the tensio-active agent stable emulsion with reduce heating, cooling and carry during drop coalescent, make it possible to thus carry out thermal cycling.Therefore, emulsion has been used to be used the single copy amplification of polymerase chain reaction (PCR) to the nucleic acid target molecule in drop.Due to the ability that the target that has individual molecule detected in drop, thereby make it possible to carry out number mensuration (digital assay).
In the exemplary number based on drop is measured, sample is allocated to one group of drop (that is, some drops do not comprise target molecule) of the target of limiting dilution.If target molecule is by random distribution in drop, Poisson's distribution has been described given average target concentration based in drop and has accurately been found 0 in drop, 1,2,3 or the probability of more target molecules so.On the contrary, can calculate from the probability of the molecule of discovery given number drop the concentration of the target molecule of (and thereby in sample) in drop.
Do not find the probability of target molecule and find that the estimated value of the probability of one or more target molecules can be measured in number is measured.In binary approach, each drop can be tested to determine that whether drop is positive and comprises at least one target molecule, or negative and do not comprise target molecule.It is that negative ratio (" negative ratio ") be similar to and draws that the probability of not finding target molecule in drop can pass through tested drop, and the probability of finding at least one target molecule to pass through tested drop be that positive ratio (" positive ratio ") quilt is similar to and draws.Then can in the Poisson algorithm, utilize the value of positive ratio or negative ratio to calculate the concentration that drop hits.In other situations, digital mensuration can produce and be greater than binary data.For example, mensuration can be measured in each drop and have how many target molecules, and resolving power be greater than feminine gender (0) or positive (>0) (as, 0,1 or>1 molecule; 0,1,2 or>2 molecules; Or similarly).
In order in the DNA mensuration based on drop of different samples, to have high-throughput and accuracy concurrently, drop should be produced and be had uniform size (that is, the drop of monodispersity) fast.Yet the ability that sample component may disturb the sample of drop and main body to be separated, especially when having improved the frequency of drop generation.Therefore, the size of formed drop, or the ability that even is completed into drop may change from the sample to the sample, thus weakened the reliability of measuring.Need new method to provide with higher generation frequency and reliably and as one man produce drop.
general introduction
Present disclosure provides the method for analyzing gene group DNA a kind of.Can obtain the genomic dna that comprises target.Genomic dna wittingly (volitionally) by fragmentation to produce the DNA of fragmentation.The water-containing drop of the DNA that the DNA of fragmentation can contain fragmentation with generation through droplet generator.Can carry out digital mensuration to determine the level of target to drop.In some embodiments, drop can comprise the genomic dna of every microlitre at least about 5 nanogram concentration, drop can the drop generation frequency at least about 50 drops be produced with per second, drop can have every and be less than approximately 10 and receive the average-volume risen, drop can be greater than approximately 50 sample flow rates of receiving liter with per second and produce, or its any combination.
the accompanying drawing summary
Fig. 1 is the schema of having explained according to the illustrative methods of the analyzing gene group DNA of the each side of present disclosure.
Fig. 2 is the matrix at each figure that photo of three kinds of different samples of processing obtains down of four kinds of different driving pressure by droplet generator.
Fig. 3 does not comprise genomic dna or comprises digested (EcoRI) or the droplet size of the sample of not digested genomic dna (Raji or Coriell) produces the function of frequency as drop and the figure that draws.
Fig. 4 is that the droplet size of sample of Fig. 3 is as the function of sample flow rate and the figure drawn.
Fig. 5 is that the maximum of the sample of Fig. 3 is extended as drop and produced the function of frequency and the figure that draws.
Fig. 6 is that the maximum of the sample of Fig. 3 is extended the figure drawn as the function of sample flow rate.
describe in detail
Present disclosure provides the method for analyzing gene group DNA a kind of.Can obtain the genomic dna that comprises target.Genomic dna is the DNA with the generation fragmentation by fragmentation wittingly.The water-containing drop of the DNA that the DNA of fragmentation can contain fragmentation with generation through droplet generator.Can be measured to determine to drop the level of target.In some embodiments, drop can comprise the genomic dna of every microlitre at least about 5 nanogram concentration, drop can the drop generation frequency at least about 50 drops be produced with per second, drop can have every and be less than approximately 10 and receive the average-volume risen, drop can be greater than approximately 50 flow velocitys of receiving liter with per second and produce, or its any combination.
As disclosed herein, the method for the genomic dna in the analysis drop has than the sizable advantage of the additive method based on drop.Advantage can comprise with higher frequency generation drop, have higher monodispersity, has larger DNA charge capacity and/or have the remarkable more weak interference from genomic dna.
These and other aspects of present disclosure have been described: the general introduction of the illustrative methods that (I) genomic dna is analyzed, (II) example data of drop generation test and (III) preferred embodiment in part below.
I.
the general introduction of the illustrative methods that genomic dna is analyzed
Fig. 1 shows the schema of the illustrative methods 20 of having explained analyzing gene group DNA.The step presented can and be carried out according to any suitable combination according to any suitable order.
Can obtain genomic dna, with 22 signs.Can obtain DNA, vertebrates, invertebrates, yeast or fungi, plant, protozoon, bacterium or the analogue of organism such as Mammals (as the mankind, mouse, rat, monkey etc.), nonmammalian from any suitable organism.Can obtain DNA by any suitable method, method such as commercial, with present accept, by from cell or fluid extraction, obtaining, accept or similar approach with clinical sample.DNA can obtain with the form of relative high molecular, such as having at least about 10
4, 10
5or 10
6kilodalton and other (as, there is the mean length at least about 25,50,100,200,500 or 1000 kilobase) molecular weight.
Before producing drop, can the fragmentation genomic dna, with 24 signs.Fragmentation can be act of will, carries out wittingly.Fragmentation generally includes any program of the molecular weight that significantly reduces genomic dna, such as passing through cutting or DNA breakage chain.Fragmentation can reduce any appropriate vol by molecular-weight average and/or length, such as at least about 5,10,20,50 or 100 times and other.The illustrative methods of fragmentation genomic dna comprise with restriction enzyme (as, there are the enzyme of 4,5,6 or 8 Nucleotide recognition sites and other) digestion.Target can not comprise the recognition site of restriction enzyme to avoid any cracking of target molecule.It can be fully maybe that part digests that restriction enzyme digestion can be carried out.Selectively or in addition, the aqueous specimen of genomic dna can be heated with fragmentation DNA.The exemplary heating of fragmentation DNA can be carried out at least about 10,15,20 or 30 minutes and other times at the temperature of at least 95 ℃.In other situations, DNA can be by shearing, supersound process, atomization, radiation or similar approach by fragmentation.
Genomic dna can comprise target, interested sequence normally to be tested.Can carry out the fragmentation of genomic dna and basically can not destroy target, this means by the fragmentation method, the target sequence less than half in genomic dna destroyed (as fracture or cutting).
Can produce the drop of the DNA that comprises fragmentation, with 26 signs.Drop can be in one or more droplet generators each produce in order.The DNA of fragmentation can be through at least one droplet generator to produce drop.Usually, the DNA of fragmentation is disposed in aqueous specimen, and aqueous specimen and immiscible external phase are through the DNA that droplet generator comprises fragmentation with formation and are disposed in the water-containing drop in external phase.The further aspect of the emulsion phase that droplet generator and possibility are suitable is described in document listed under cross reference, and these documents are incorporated at this by reference, the U.S. Patent Application Publication No. 2010/0173394A1 especially announced on July 8th, 2010; The PCT number of patent application WO2011/120024 that on September 29th, 2011 announces.
Drop can have any suitable size.For example, drop can have and is less than approximately 1 μ L, 100nL, 10nL, 1nL, 100pL, 10pL or 1pL and other average-volume.Selectively or in addition, drop can have and is greater than about 10fL, 100fL, 1pL, 10pL or 100pL and other average-volume.In some cases, drop can have about 1pL to 100nL, 1pL to 10nL or 0.1 to 10nL and other average-volume.Drop can be monodispersity.
Drop can comprise the DNA of the fragmentation of any suitable concn.For example, the DNA of fragmentation can with at least about 0.1,0.2,0.5,1,2,5,10,20,50ng/ μ L and other concentration is disposed in drop.In some cases, concentration can be about 0.1-50 or 0.2-20ng/ μ L.Fragmentation DNA allows higher DNA charge capacity to be incorporated in drop.The DNA of fragmentation can be less than approximately 2 genome Equivalents with average every drop and exist.Target can be less than approximately 2 molecules with average every drop and exist.
Drop can produce frequency with any suitable drop and form, such as at least about 10,20,50,100,200,500 or 1,000Hz (liquid drops/sec) and other.Usually, drop produces the size negative correlation of frequency and the drop be produced, and less drop allows higher drop to produce frequency.
The aqueous specimen that is used to form drop (and the DNA that comprises fragmentation) can pass droplet generator and/or be converted to drop with any suitable flow velocity.May suitable exemplary flow velocity comprise at least about 1,5,10,20,50,100,200,500,1,000,5,000 or 10,000nL/ second and other.Usually, the size positive correlation of sample flow rate and the drop that is produced, and larger drop allows higher flow velocity.
The example values (or scope) that the droplet size gone out listed above, DNA concentration, drop produce frequency and flow velocity can be combined with any suitable combination.
Can be measured drop, with 28 signs.Mensuration can be that the number that detects the independent target molecule in drop is measured.Digital mensuration can comprise the amplified target molecule, such as by PCR or ligase chain reaction (LCR) and other.Digital mensuration can also comprise the fluorescence detected from drop.Mensuration can also comprise with the Poisson algorithm determines the level (as concentration) that drop hits.
II.
drop produces the example data of test
The drop that this part has presented by the genomic dna that adopts or do not adopt fragmentation produces the example data of test; Referring to Fig. 2-6.
Drop in microfluidic device produces and can depend on that sample marches to the frequency of flow velocity and the generation drop of droplet generator.When high flow rate, sample flow can be spurted in immiscible external phase, no longer produces drop.When producing the approaching injection of speed limit value, before producing drop, sample starts to extend to such an extent that more go deep in exit passageway.This development length can produce condition and the degree of approach of spraying limit value for understanding one group.
Fig. 2 shown by lower in each of four kinds of different driving pressures and thereby different in flow rate under process the matrix of the picture that the photo of the droplet generator 30 of three kinds of different aqueous specimen 32-36 obtains.Three kinds of samples are the not digested and human genome DNA's that have high molecular (MW) of (a) control sample 32 (without the PCR damping fluid of template), (b) of not comprising DNA aqueous specimen 34 (Raji, 18.75ng/ μ L) and (c) with restriction enzyme digestion and there is human genome DNA's the aqueous specimen 36 (Raji, 18.75ng/ μ L) of the molecular weight of reduction.Four kinds of driving pressures (1,2,3 and 4) be applied to droplet generator downstream negative (vacuum) pressure and with PSI (psi), mean, and 1psi equals approximately 6.9 kPas.Vacuum level can the Quality control flow velocity, overall flow rate and drop produce frequency.
Upper row has shown that the drop of the control sample 32 that does not comprise genomic dna produces.Drop 50 be about 1nL and under different vacuum levels dimensional change little.
Middle row has shown that the drop of the sample 34 that comprises indigested genomic dna produces.Only at the lower genomic dna that occurs of tested minimum vacuum level (1psi), greatly damage the drop generation.Even at this minimum level, exist main body water 46 to extend through in a large number passage joint 52, by arrow 54, indicated, and drop 50 is larger.Higher vacuum level (as, 1psi compares with 2-4psi) and flow velocity under, dripless produces, reason is that sample flow is sprayed and entered in exit passageway 44, by arrow 56, is indicated, and can not be broken into drop.Therefore, human genome DNA's existence can disturb drop to produce consumingly, and may require to use lower DNA concentration, flow velocity and drop to produce frequency.Therefore, sample preparation may be quite slow.In addition, the frequency of the positive drop of the target in emulsion may significantly be reduced (due to lower DNA concentration), and this will need to analyze more drop to obtain the same degree of confidence of determined target level.
Lower row shown by the drop contained with the sample 36 of the genomic dna of sample 34 same concentrations (quality of per unit volume) and produced, but after DNA is digested shorter fragment with restriction enzyme.The shown pressure (under flow velocity) at this paper, the genomic dna that is the fragmentation form is not detected the infringement drop and produces.Sample produces drop 50, and this is similar to the control sample 32 that lacks DNA.
Carry out other research and carried out the relation between the maximum sample extension during quantitative measurment produces speed, drop size and the drop generation in vacuum, sample flow rate, drop generation frequency, exit passageway.The aqueous specimen used is Spectral Dye Buffer (with control sample identical in Fig. 2, but there is no archaeal dna polymerase), Raji human genome DNA (Loftstrand Laboratories) (" Raji ") and 19205 human DNAs (Coriell Institute) (" Corell ").The DNA sample is indigested or digested with limiting enzyme EcoRI.The EcoRI (New England biolabs) that is used in the 20U/ μ L concentration in the NEB# 4 damping fluid carries out DNA digestion, and the ultimate density of genomic dna is 200ng/ μ L.Mixture, 37 ℃ of lower incubations 1 hour, then is diluted to different ultimate densities.
The figure of Fig. 3-6 has shown that the drop that the sample by the indigested Coriell 19205DNA of the indigested Raji DNA of Coriell 19205 DNA, the 18.75ng/ μ L of the EcoRI-digestion of Raji DNA, the 18.75ng/ μ L of the EcoRI-of Master Mix (without DNA), 18.75ng/ μ L digestion and 18.75ng/ μ L carries out produces the result of test.The droplet size that Fig. 3 has shown sample produces the function of frequency as drop and the figure that draws.The droplet size that Fig. 4 has shown sample is as the function of sample flow rate and the figure drawn.Fig. 5 has shown that the maximum of sample is extended as drop and has produced the function of frequency and the figure that draws.Fig. 6 has shown that the maximum of the sample of Fig. 3 extends the figure drawn as the function of sample flow rate.
For indigested human genome DNA, before spraying, only low-down drop generation frequency or sample flow rate are possible.For example, for Coriell 19205 DNA, maximum value is 60Hz and 83nL/ second.For Raji DNA, maximum value is 120Hz and 162nL/ second.Yet even, lower than these limit values, the drop produced has volume high when not having DNA, and produce and extend in output channel with much longer sample.
Figure has also shown the DNA for the digestion of same concentrations, and the impact that DNA produces drop can not detect.All do not observe and spray or the extension of long sample under tested any flow velocity or generation frequency, and droplet size is identical with the volume of the sample that does not have DNA.
These results have shown that drop is created in while having indigested human DNA and have been subject to strong damage, but with not being damaged after restriction enzyme digestion.
III.
preferred embodiment
This part has been described the preferred embodiment of present disclosure with the paragraph of a series of index.These embodiments should in no way limit the gamut of present disclosure.
A. the method for an analyzing gene group DNA, comprising: (i) obtain the genomic dna that comprises target; (ii) wittingly the described genomic dna of fragmentation to produce the DNA of fragmentation; (iii) water-containing drop of the DNA that the DNA that makes described fragmentation contains described fragmentation through at least one droplet generator with generation; And (iv) described drop is carried out to number and measure to determine the level of described target.
B. the method for paragraph A, wherein said drop has and is less than approximately 10 and receives the average-volume risen.
C. the method for paragraph A, wherein said drop comprises the described genomic dna of every microlitre at least about 5 nanogram concentration.
D. the method for paragraph A, wherein said genomic dna is disposed in aqueous specimen, and wherein said drop is greater than approximately 50 flow velocitys of receiving the described aqueous specimen that rises by described droplet generator with per second and is produced.
E. the method for arbitrary section in paragraph A to D, wherein said drop produces frequency with per second at least about the drop of 50 drops and is produced.
F. the method for paragraph A, wherein said drop has and is less than approximately 10 and receives the average-volume that rises and comprise the described genomic dna of every microlitre at least about 5 nanogram concentration.
G. the method for paragraph A, wherein said drop has and is less than approximately 10 and receives the average-volume risen, and wherein said genomic dna is disposed in aqueous specimen, and wherein said drop is greater than approximately 50 flow velocitys of receiving the described aqueous specimen that rises by described droplet generator with per second and is produced.
H. the method for paragraph A, wherein said drop comprises the described genomic dna of every microlitre at least about 5 nanogram concentration, and wherein said genomic dna is disposed in aqueous specimen, and wherein said drop is greater than approximately 50 flow velocitys of receiving the described aqueous specimen that rises by described droplet generator with per second and is produced.
I. the method for paragraph F, wherein said genomic dna is disposed in aqueous specimen, and wherein said drop is greater than approximately 50 flow velocitys of receiving the described aqueous specimen that rises by described droplet generator with per second and is produced.
J. the method for paragraph F, wherein said drop produces frequency with per second at least about the drop of 50 drops and is produced.
K. the method for paragraph G, wherein said drop produces frequency with per second at least about the drop of 50 drops and is produced.
L. the method for paragraph H, wherein said drop produces frequency with per second at least about the drop of 50 drops and is produced.
M. the method for paragraph L, wherein said drop has and is less than approximately 10 and receives the average-volume risen.
N. the method for arbitrary section in paragraph A to M, the step of wherein said fragmentation comprises the step that digests described genomic dna with restriction enzyme.
O. the method for paragraph N, wherein said restriction enzyme is less than and cuts described genomic dna approximately once with average every kilobase.
P. the method for arbitrary section in paragraph A to M, the step of wherein said fragmentation comprises the step of shearing described genomic dna.
Q. the method for arbitrary section in paragraph A to M, the step of wherein said fragmentation comprises the step of the described genomic dna of supersound process.
R. the method for arbitrary section in paragraph A to Q, wherein said drop comprises average every drop and is less than the approximately described target of 2 copies.
S. the method for arbitrary section in paragraph A to R, wherein said drop comprises average every drop and is less than the approximately genome Equivalent of 2 described genomic dnas.
T. the method for arbitrary section in paragraph A to S, the step of wherein said fragmentation is not destroyed described target basically.
U. the method for arbitrary section in paragraph A to T, wherein saidly carry out the step that the digital step of measuring comprises the described target in the described drop of amplification.
V. the method for paragraph U, wherein said target is amplified by PCR.
W. the method for arbitrary section in paragraph A to V, wherein saidly carry out the digital step of measuring and comprise the step detected from the fluorescence of described drop.
X. the method for arbitrary section in paragraph A to W, wherein saidly carry out the digital step of measuring and comprise the step of determining the level of described target with the Poisson algorithm.
Y. the method for arbitrary section in paragraph A to X, wherein said drop has approximately 0.1 to 10 and receives the average-volume risen.
Z. the aqueous specimen that will comprise DNA is distributed into the method for drop, and described method comprises: (i) obtain and comprise the sample of every microlitre at least about the DNA of 5ng concentration; (ii) wittingly the described DNA of fragmentation to produce the DNA of fragmentation; And the water-containing drop of the DNA that (iii) makes described sample comprise described fragmentation with generation through droplet generator, described drop produces frequency with per second at least about the drop of 50 drops and is produced and has and be less than approximately 10 and receive the average-volume risen.
A1. the aqueous specimen that will comprise DNA is distributed into the method for drop, and described method comprises: (i) obtain the sample that comprises genomic dna; (ii) wittingly the described DNA of fragmentation to produce the DNA of fragmentation; And the water-containing drop of the DNA that (iii) makes described sample comprise described fragmentation through droplet generator with generation, described drop produces frequency with per second at least about the drop of 50 drops and is produced and has and be less than approximately 10 and receive the average-volume risen, the concentration of wherein said genomic dna is, if the step of passing described adopts described genomic dna disturbs drop to produce under the same terms of the described DNA of fragmentation not.
Disclosure presented above can comprise the multinomial different invention with independent function.Although each in the present invention is disclosed with its preferred form, its specific embodiment disclosed herein and explaination is not considered to restrictive meaning, and reason is that many versions are possible.Theme of the present invention comprise different key element, feature, function and/or characteristic disclosed herein all novelties and non-obvious combination and sub-portfolio.Below claim particularly point out and be considered to novel and non-obvious some combination and sub-portfolio.The invention embodied in other combinations of feature, function, key element and/or characteristic and sub-portfolio can be protected in the application of the right of priority that requires this application or related application.No matter such claim, relate to different inventions or identical invention, and, no matter than the wide ranges of initial claim, narrow, equal or different, also all be believed to comprise in the subject matter of present disclosure.In addition, for the ordinal number sign of confirmed key element, such as first, second or the 3rd for distinguishing key element, and do not show specific position or the order of such key element, unless otherwise specific statement.
Claims (27)
1. the method for an analyzing gene group DNA comprises:
The genomic dna that acquisition comprises target;
The described genomic dna of fragmentation is to produce the DNA of fragmentation wittingly;
The water-containing drop of the DNA that the DNA that makes described fragmentation contains described fragmentation through at least one droplet generator with generation; And
Described drop is carried out to digital mensuration to determine the level of described target.
2. the method for claim 1, wherein said drop has and is less than approximately 10 and receives the average-volume risen.
3. the method for claim 1, wherein said drop comprises the described genomic dna of every microlitre at least about 5 nanogram concentration.
4. the method for claim 1, wherein said genomic dna is disposed in aqueous specimen, and wherein said drop is greater than approximately 50 flow velocitys of receiving the described aqueous specimen that rises by described droplet generator with per second and is produced.
5. method as described as any one in claim 1 to 4, wherein said drop produces frequency with per second at least about the drop of 50 drops and is produced.
6. the method for claim 1, wherein said drop has and is less than approximately 10 and receives the average-volume that rises and comprise the described genomic dna of every microlitre at least about 5 nanogram concentration.
7. the method for claim 1, wherein said drop has and is less than approximately 10 and receives the average-volume risen, and wherein said genomic dna is disposed in aqueous specimen, and wherein said drop is greater than approximately 50 flow velocitys of receiving the described aqueous specimen that rises by described droplet generator with per second and is produced.
8. the method for claim 1, wherein said drop comprises the described genomic dna of every microlitre at least about 5 nanogram concentration, and wherein said genomic dna is disposed in aqueous specimen, and wherein said drop is greater than approximately 50 flow velocitys of receiving the described aqueous specimen that rises by described droplet generator with per second and is produced.
9. method as claimed in claim 6, wherein said genomic dna is disposed in aqueous specimen, and wherein said drop is greater than approximately 50 flow velocitys of receiving the described aqueous specimen that rises by described droplet generator with per second and is produced.
10. method as claimed in claim 6, wherein said drop produces frequency with per second at least about the drop of 50 drops and is produced.
11. method as claimed in claim 7, with per second, the drop generation frequency at least about 50 drops is produced wherein said drop.
12. method as claimed in claim 8, with per second, the drop generation frequency at least about 50 drops is produced wherein said drop.
13. method as claimed in claim 12, wherein said drop has and is less than approximately 10 and receives the average-volume risen.
14. the method for claim 1, the step of wherein said fragmentation comprises the step that digests described genomic dna with restriction enzyme.
15. method as claimed in claim 14, wherein said restriction enzyme is less than and cuts described genomic dna approximately once with average every kilobase.
16. the method for claim 1, the step of wherein said fragmentation comprises the step of shearing described genomic dna.
17. the method for claim 1, the step of wherein said fragmentation comprises the step of the described genomic dna of supersound process.
18. the method for claim 1, wherein said drop comprises average every drop and is less than the approximately described target of 2 copies.
19. the method for claim 1, wherein said drop comprises average every drop and is less than the approximately genome Equivalent of 2 described genomic dnas.
20. the method for claim 1, the step of wherein said fragmentation is not destroyed described target basically.
21. the method for claim 1, wherein said step of carrying out digital mensuration comprises the step of the described target in the described drop of amplification.
22. method as claimed in claim 21, wherein said target is amplified by PCR.
23. method as claimed in claim 21, wherein said step of carrying out digital mensuration comprises the step detected from the fluorescence of described drop.
24. method as described as any one in claim 21 to 23, wherein said step of carrying out digital mensuration comprises the step of determining the level of described target with the Poisson algorithm.
25. the method for claim 1, wherein said drop has approximately 0.1 to 10 and receives the average-volume risen.
26. the aqueous specimen that will comprise DNA is distributed into the method for drop, described method comprises:
Acquisition comprises the sample of every microlitre at least about the DNA of 5ng concentration;
The described DNA of fragmentation is to produce the DNA of fragmentation wittingly; And
The water-containing drop of the DNA that makes described sample comprise described fragmentation with generation through droplet generator, described drop produces frequency with per second at least about the drop of 50 drops and is produced and has and be less than approximately 10 and receive the average-volume risen.
27. the aqueous specimen that will comprise DNA is distributed into the method for drop, described method comprises:
The sample that acquisition comprises genomic dna;
The described DNA of fragmentation is to produce the DNA of fragmentation wittingly; And
The water-containing drop of the DNA that makes described sample comprise described fragmentation with generation through droplet generator, described drop produces frequency with per second at least about the drop of 50 drops and is produced and has and be less than approximately 10 and receive the average-volume risen,
If wherein said genomic dna be with described step of passing under the same terms of the described DNA of fragmentation not the concentration of disturbing drop to produce.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US40910610P | 2010-11-01 | 2010-11-01 | |
| US61/409,106 | 2010-11-01 | ||
| US12/976,827 | 2010-12-22 | ||
| US12/976,827 US9598725B2 (en) | 2010-03-02 | 2010-12-22 | Emulsion chemistry for encapsulated droplets |
| PCT/US2011/058854 WO2012061442A1 (en) | 2010-11-01 | 2011-11-01 | Analysis of fragmented genomic dna in droplets |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103443289A true CN103443289A (en) | 2013-12-11 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011800528318A Pending CN103443289A (en) | 2010-11-01 | 2011-11-01 | Analysis of fragmented genomic DNA in droplets |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP2635708A4 (en) |
| JP (1) | JP2013541347A (en) |
| CN (1) | CN103443289A (en) |
| AU (1) | AU2011323507A1 (en) |
| CA (1) | CA2816702A1 (en) |
| SG (1) | SG190076A1 (en) |
| WO (1) | WO2012061442A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| ES2821650T3 (en) | 2015-08-28 | 2021-04-27 | Dnaform Kk | Template nucleic acid analysis method, target substance analysis method, template nucleic acid or target substance assay kit and template nucleic acid or target substance analyzer |
| WO2018098438A1 (en) * | 2016-11-28 | 2018-05-31 | Arizona Board Of Regents On Behalf Of Arizona State University | Systems and methods related to continuous flow droplet reaction |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5555191A (en) * | 1994-10-12 | 1996-09-10 | Trustees Of Columbia University In The City Of New York | Automated statistical tracker |
| US20050277125A1 (en) * | 2003-10-27 | 2005-12-15 | Massachusetts Institute Of Technology | High-density reaction chambers and methods of use |
| US20100069263A1 (en) * | 2008-09-12 | 2010-03-18 | Washington, University Of | Sequence tag directed subassembly of short sequencing reads into long sequencing reads |
| US20100137163A1 (en) * | 2006-01-11 | 2010-06-03 | Link Darren R | Microfluidic Devices and Methods of Use in The Formation and Control of Nanoreactors |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US9156010B2 (en) * | 2008-09-23 | 2015-10-13 | Bio-Rad Laboratories, Inc. | Droplet-based assay system |
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2011
- 2011-11-01 SG SG2013033238A patent/SG190076A1/en unknown
- 2011-11-01 WO PCT/US2011/058854 patent/WO2012061442A1/en active Application Filing
- 2011-11-01 JP JP2013537769A patent/JP2013541347A/en active Pending
- 2011-11-01 CN CN2011800528318A patent/CN103443289A/en active Pending
- 2011-11-01 EP EP11838712.5A patent/EP2635708A4/en not_active Withdrawn
- 2011-11-01 CA CA2816702A patent/CA2816702A1/en not_active Abandoned
- 2011-11-01 AU AU2011323507A patent/AU2011323507A1/en not_active Abandoned
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5555191A (en) * | 1994-10-12 | 1996-09-10 | Trustees Of Columbia University In The City Of New York | Automated statistical tracker |
| US20050277125A1 (en) * | 2003-10-27 | 2005-12-15 | Massachusetts Institute Of Technology | High-density reaction chambers and methods of use |
| US20100137163A1 (en) * | 2006-01-11 | 2010-06-03 | Link Darren R | Microfluidic Devices and Methods of Use in The Formation and Control of Nanoreactors |
| US20100069263A1 (en) * | 2008-09-12 | 2010-03-18 | Washington, University Of | Sequence tag directed subassembly of short sequencing reads into long sequencing reads |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2816702A1 (en) | 2012-05-10 |
| WO2012061442A1 (en) | 2012-05-10 |
| AU2011323507A1 (en) | 2013-06-13 |
| JP2013541347A (en) | 2013-11-14 |
| EP2635708A4 (en) | 2014-10-29 |
| EP2635708A1 (en) | 2013-09-11 |
| SG190076A1 (en) | 2013-06-28 |
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