CN104630202A - Amplification method capable of decreasing bias generation during trace nucleic acid substance entire amplification - Google Patents
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Abstract
The invention relates to an amplification method capable of decreasing bias generation during trace nucleic acid substance entire amplification. The amplification method is characterized in that the nucleic acid substances are randomly dispersed into several individual reaction systems, which don't communicate with one another; and primer sequences for amplification include 3-20 random basic groups, which are two, three or four groups randomly selected from A, G, C, and T. Compared the amplification method in the invention with an amplification method of trace nucleic acid substances in a large system, the amplification bias is reduced one order of magnitude or more; an amplification result can accurately reflect quantification information of original nucleic acid substances; and more amplification cycles are allowed, so that more products are obtained.
Description
Technical field
The present invention relates to a kind of nucleic acid substances amplification method, particularly a kind of amplification method that can reduce generation bias when trace dna material entirety increases.
Background technology
Along with deepening continuously of life science, the analysis of individual cell level is more and more important.On the one hand, due to the heterogeneity between cell, the change that the average data of some a large amount of cells can not authentic representative cell levels occur, and often cover the signal of individual cell level, be such as only present in the sudden change etc. in a few cell (as early cancer cell); On the other hand, in the experiment that some starting material are very rare, the sample etc. that the early stage cell of such as embryo, circulating tumor cell and puncture obtain, a large amount of cells checked order or other are analyzed enough are carried out in very difficult acquisition.
In order to carry out the analyses such as order-checking to individual cells, the overall amplification technique comprising PCR, MDA, RCA must be first utilized to generate a large amount of DNA copies.But the shortcoming that these technology exist is: when carrying out entirety amplification, some part genomic compares other can generate more substantial copy, and this can cause the region generating copy number less in genome to be submerged, thus cannot be detected.This problem is referred to as amplification bias.Amplification bias extensively exists in existing whole genome amplification method, and become important weak point, reason is that amplification bias can cause final product cannot represent the initial relative content of fragment, the karyomit(e) times type made the mistake and gene copy number are estimated, and can cause some fragments due to amplification times very few and cannot detect in the product, single cell analysis cannot accurately be carried out.In addition, in unit molecule amplification, because the content of initial nucleic acid is very few, the nucleic acid molecule that the nucleic acid molecule in the surrounding environment of trace or operator bring into is all easy to be mixed in amplification system pollute.
Summary of the invention
For the problems referred to above, the invention provides a kind of amplification method that can reduce to produce when trace dna material entirety increases bias, it is characterized in that, described nucleic acid substances is dispersed randomly to after in some mutual disconnected independent reaction systems and increases again, comprise 3-20 randomized bases in the primer sequence that amplification uses, randomized bases is selected from two, three or four in A, G, C, T at random.
(1 more than μ L is often referred in traditional large system, can direct liquid-transfering gun carry out the system that operates) in unicellular amplification, in unicellular, each nucleic acid molecule or fragment are equal to monomolecular amplification at whole genome amplification, and the whether startup of unit molecule amplified reaction is a stochastic process with when starting.Once the amplification of a molecule is by random " startup ", this molecule just can obtain the copy not starting molecule several times higher than other at short notice, advantage on this number then further increases it and continues by the probability of " startup ", and cause the amplification raw material that should belong to other molecules to be copied shared by the more macromolecular amplified reaction of number, the copy number of some molecule in final product is made to be far longer than other molecules, the ratio of product nucleic acid molecule copy number can not the copy number ratio of same molecular in actual response raw material, and this just causes amplification bias.And in the method for the invention, molecule to be amplified is dispersed randomly in some independent reaction systems, make the molecule number in each reaction system meet Poisson's distribution, only have 0 to 50 molecule, even 0-20, or 0-10 following molecule.Even if certain molecule takes the lead in " startup " obtain more copy, what affect by it is also only other molecules being in same reaction system, and the molecule of other reaction systems still can obtain amplification chance.In addition, after although " startup " time of the unit molecule amplification in each reaction system has and first has, but owing to being limited for the raw material increased in each reaction system, the amplification rate of " startup " and the amplification rate molecule faster that takes the lead in can tend to be steady over time.When proliferation time sufficiently long, stop the reaction in all reaction systems when the reaction of reaction system is all tended to be steady, the multiple of now all in theory molecular clonings should be identical simultaneously, thus reaches the object of evenly amplification.Meanwhile, due to by molecular dispersion to be amplified in several reaction systems, the impact of pollutent on W-response just consequently reduces.
Further, increasing the primer used in the present invention can for the sequence be made up of 3-20 randomized bases, and randomized bases is selected from two, three or four in A, G, C, T at random.The sequence of nucleic acid substances of the present invention can be unknown.Require in traditional PCR method to be amplified fragment at least there is a part of known array, then according to this section of known array design can be combined thereon one or more particular sequence as primer.And method of the present invention uses the sequence be made up of randomized bases as " random primer ", because its randomness can have a large amount of combinations, therefore this primer can carry out base pairing with the unknown fragment of the unknown nucleotide sequence of corresponding length, thus the arbitrary portion of unknown nucleotide sequence is gone out as primer amplification, reach the overall expanding effect of unknown nucleotide sequence, thus allow amplification, the order-checking of unknown nucleotide sequence being carried out to full-length genome.Experimentally need, the base of non-natural or modified in primer, can be comprised, and with other materials of base effect, such as lock nucleic acid, 5 '-nitroindoline, phosphorothioate oligonucleotide etc.
Further, the volume of independent reaction system of the present invention is 0.5fL-100nL, preferred 1fL-10nL, more preferably 10fL-1nL.What be dispersed in some independent reaction systems is some, refers to more than 2, preferred 2-10
20individual, more preferably 2-10
8individual.Independent reaction system can be disperseed to form by immiscible two-phase any in oil, water, hydrogel, polydimethylsiloxane, glass, plastics, silicon chip, air.A phase in two-phase can dissolve the reactant of amplified reaction, is called disperse phase, and another phase is not dissolved each other with this phase, is called external phase or separates phase.The method of dispersion can be ultrasonic disperse or use fluid-operating system to disperse.Fluid-operating system can be micro-fluidic chip.The method using micro flow chip to produce mutual disconnected disperse phase microlayer model has polyphasic flow method, Electrowetting, thermo-capillary method, dielectrophoresis method etc.Polyphasic flow ratio juris is, by to the unique design of fluid Micro Channel Architecture and the control of fluid flow rate, utilize the shearing force between liquid stream, viscous force and capillary interaction, dispersed phase fluid is made to produce velocity slope in local, microchannel, thus split point generates microlayer model, the microlayer model produced is evenly distributed in disperse phase in immiscible external phase, forms mono-disperse system.In order to reduce surface tension, generating stable microlayer model, tensio-active agent can also be added in liquid stream.The pipeline of micro flow chip can be T-shaped, Y type, or cross type.By the stream that adjusts the flow velocity of external phase and external phase and disperse phase than the formation speed that can control microlayer model and the size of microlayer model formed, thus the size of control independent reaction system and the quantity of each reaction system nucleic acid molecule.The content of each reaction system amplifying nucleic acid material is 0 to 50 molecule, is preferably 0-20 molecule, is more preferably 0-10 molecule.Can produce more product when system is larger, nucleic acid molecule quantity is fewer, and influencing each other in single reaction chamber will be less, can reach evenly amplification
Amplification of the present invention can be MDA (multiple displacement amplification), PCR (polymerase chain reaction), RCA (rolling circle amplification) or MALBAC (ring-type cyclic amplification of repeatedly annealing).
Proliferation time can be 30 minutes to 40 hours, is preferably 1 hour to 20 hours.In the present invention, raw material for increasing in each reaction system is limited, the take the lead in amplification rate of " startup " and amplification rate molecule faster can tend to be steady over time, therefore need sufficiently long proliferation time that the reaction of reaction system is all tended to be steady, just can reach the object of evenly amplification.Different amplification methods (as MDA, PCR, RCA, MALBAC) is different with the minimum proliferation time needed for different reaction systems, and proliferation time is selected by normal experiment means.In traditional amplification method, the prolongation of proliferation time can aggravate the bias that increases, and therefore usually can control proliferation time in experiment.Method of the present invention can effectively avoid this problem, allows the means by extending proliferation time to obtain more substantial molecule and is convenient to downstream analysis.
Nucleic acid substances of the present invention comprises single stranded DNA; Double-stranded DNA; Single stranded RNA; Double-stranded RNA; Double-stranded RNA/DNA hybridization body; DNA and RNA of partial hybridization; Through DNA or RNA of zymetology, chemistry, biological method process, as: the DNA after the DNA extracted after immunoprecipitation, sulfiting, DNA, RNA cDNA etc. after reverse transcription process after Methyl transporters ferment treatment.Nucleic acid substances can not come from nature, can be the nucleic acid substances of synthetic or certain chemical treatment product of nature nucleic acid substances, can from one or more in cell, subcellular unit, karyomit(e), nucleic acid molecule, can from unicellular, monosome, single core acid molecule, one or more in trace nucleic acid.The quality of trace dna material can be less than 10ng, is preferably less than 1ng.
According to subsequent experimental procedure needs, after amplification terminates, product recovery can be carried out.The method that product reclaims can be carried out for respectively product being taken out rear mixing or do not mix from each separation reaction system.Also the method for two-phase mixtures can be carried out by adding the emulsion splitter such as ethanol, isopropylcarbinol.
Present invention also offers a kind of method of trace dna material being carried out to full-length genome or transcript profile order-checking, first use and produce the amplification method of bias when can reduce the amplification of unknown trace dna material entirety and increase, then carry out product recovery, build storehouse and order-checking.
Use amplification method of the present invention relative to the amplification method of trace dna material in large system, amplification bias reduces more than 1 order of magnitude, amplification can quantitative information in accurate response parent acid substance, can allow more amplification cycles, thus obtain more product.
Accompanying drawing explanation
Below in conjunction with the drawings and the specific embodiments, the present invention is described in further detail.
Fig. 1 is the microfluidic chip structure forming independent reaction room in embodiment 1.
The light field picture of independent reaction room of Fig. 2 for being formed in embodiment 1, reaction chamber is dispersed in continuous print mineral oil phase.
Fig. 3 is the chromosome segment copy number distribution plan after the single human cell's amplification obtained in embodiment 1 and comparative example 1.
Embodiment
Embodiment 1
1. single celled separation:
Cell is blown and beaten into single dispersing, is placed in PBS damping fluid and puts and carry out under the microscope observing and operating, kapillary is blocked into the capillary needle of cross-sectional diameter 20-30 μm, utilize wicking action to draw individual cells, and cell is transferred in new PCR pipe.
2. single celled cracking:
Be formulated as follows lysate:
| Content | Volume (μ l) | Final concentration |
| 1M Tris-HCl pH=8.0 | 0.12 | 30mM |
| 500mM NaCl | 0.08 | 10mM |
| 10mg／mL Proteinase | 1 | 1mg／mL |
| 20mM EDTA | 1 | 5mM |
| 5％Triton(v／v) | 0.4 | 0.5％ |
| Water | 1.4 | - |
| Volume altogether | 4 |
Wherein Proteinase (Qiagen, #19155) needs to use 50% glycerol to become the storage liquid of 10mg/mL in advance.By 4 μ L lysates mixing after join containing in single celled PCR pipe, in PCR instrument, carry out following program: 50 DEG C 3 hours for scission reaction, 70 DEG C 30 minutes for inactivating protein enzyme.The genomic dna obtained) to be placed in-80 DEG C of refrigerators stand-by.
3. the preparation of single celled amplified reaction solution:
Use MDA method to increase, be formulated as follows single celled amplified reaction solution:
| Content | Volume (μ l) | Final concentration |
| 10x Phi29buffer(NEB#B0269) | 1 | 1x |
| 500μM N6primer | 1 | 50μM |
| 10mM dNTP(NEB#N0447) | 1 | 1mM each dNTP |
| 2mg／mL BSA(NEB#M0269) | 1 | 0.2mg／mL |
| Phi29polymerase(NEB#M0269) | 0.8 | 0.8U |
| Water | 1.2 | - |
| 1 cell Lysis (previous step product) | 4 | - |
| Volume altogether | 10 |
Wherein BSA is subsidiary in phi29 polymerase.
Wherein N6primer is the sequence be made up of 6 randomized bases, and randomized bases is selected from any one in A, G, C, T at random.
4. the foundation of reaction chamber
In micro-fluidic chip, utilize polyphasic flow method to produce diameter be about the drop of 50 μm as the reaction chamber separated.The pipeline configuration of micro flow chip as shown in Figure 1.Pipeline is cruciform shape, upper and lower two pipelines are connected sample inlet with left side pipeline, mineral oil is passed into from upper and lower two pipelines, the unicellular amplified reaction solution prepared is passed into the 3rd step from left side pipeline, the pressure of 0.5 standard atmospheric pressure is applied to sample inlet and sentences promotion mineral oil phase and reaction soln phase Flows to the right, reaction soln is separated to form and is dispersed in volume in oil phase by oil phase is the some independent reaction rooms of 50fL, and in each reaction chamber, molecule number is 0-5.The volume of reaction chamber regulates by the sample introduction speed of adjustment two-phase and the cross-section of pipeline of chip.The process that reaction chamber is formed as shown in Figure 1.After the foundation of reaction chamber completes as shown in Figure 2.
5. amplified reaction
MDA method is used to increase.The reaction chamber pipettor separated in oil phase is placed in new PCR pipe, PCR instrument carries out following program: 30 DEG C 6 hours for carrying out amplified reaction, 65 DEG C 20 minutes for inactivation use Phi29 polysaccharases.
6. the purifying of amplified production
Add 700 μ L isopropylcarbinols after amplified reaction terminates, the water react room be separated by oil is merged mutually, and carry out purifying by DNA Clean & Concentrator kit (Zymo, #D4033), step comprises:
A) add the Binding Buffer of 70 μ L, 15000rpm rotates 3 minutes, removes the isopropylcarbinol of the superiors.
B) solution of lower floor is all transferred to the pillar of pellosil, 15000prm rotates one minute, removes liquid phase.
C) add 350 μ l Wash Buffer, 15000rpm rotates one minute, removes liquid phase.
D) repeating step c twice.
E) silicagel column is transferred in new 1.5mL centrifuge tube (Eppendorf#0030120.086), add the TE buffer of 20 μ L, leave standstill after 5 minutes, 15000rpm rotates one minute, the DNA be attached on pellosil by wash-out, will be placed in-80 DEG C of refrigerators stand-by.
The fragmentation of 7.DNA
Because the length of reading of high-flux sequence limits, need the fragment that the broken DNA generated is extremely shorter.
Take out the product 100ng that step 6 obtains, dilute with the Low TE damping fluid of 130 μ l, carry out fragmentation by Covaris ultrasonic apparatus, service routine is the DNA-200 carried.With DNA Clean & Concentrator kit (Zymo, #D4033) purifying, key step comprises:
A) add the Binding Buffer of 260 μ L, the solution after mixing all transfers to the pillar of pellosil, and 15000prm rotates one minute, removes liquid phase.
B) add 350 μ l Wash Buffer, 15000rpm rotates one minute, removes liquid phase.
C) repeating step b twice.
D) silicagel column is transferred in new 1.5mL centrifuge tube (Eppendorf#0030120.086), add the distilled water of 20 μ L, leave standstill after 5 minutes, 15000rpm rotates one minute, the DNA be attached on pellosil by wash-out, will be stored in-20 DEG C of refrigerators stand-by.
8. the foundation of sequencing library:
Use
ultraTM DNA Library Prep Kit for Illumina (NEB, #N7370) carries out the foundation of sequencing library.Key step comprises:
A) polishing of DNA.Because 5 ' and 3 ' holds the region having some mutually not match when DNA fragmentation, carry out polishing or excision in this step, make all DNA all become normal double chain DNA molecule.
Be formulated as follows reaction system:
| Content | Volume (μ L) |
| DNA fragmentation | 20 |
| End Prep Enzyme Mix | 3 |
| End Repair Reaction Buffer(10X) | 6.5 |
| Water | 35.5 |
| Volume altogether | 65 |
In PCR instrument, following program is performed after mixing:
20 DEG C 30 minutes for reaction, 65 DEG C 30 minutes for inactivation end repair enzymes
B) sequence measuring joints is connected
Joint is carried out diluting according to 1:10 and makes following reaction system:
| Content | Volume (uL) |
| Blunt／TA Ligase Master Mix | 15 |
| The joint of dilution | 2.5 |
| The DNA molecular repaired | 65 |
| Volume altogether | 82.5 |
In PCR instrument, following program is performed after mixing:
20 DEG C 30 minutes for reaction.
C) experimental program identical (experimental program version number is Version1.213 version in February) that the Piece Selection after provides to this product with amplification step with NEB company, namely the DNA library established can check order.
9. order-checking and bioinformatic analysis
We use the miSeq sequenator of Illumine company to check order, and the reading of generation is the fragment of 50 bases longs.For being not less than 1,000,000, the CNV of 000 base number detects, and each sample needs 0.01 times to the order-checking degree of depth of its species gene group size, obtains about 600 at this each sample, and 000 reading is approximately the order-checking degree of depth of 0.03G.After obtaining original order-checking image data, convert thereof into the base sequence that can read, then after deleting the too low data of sequence quality by sequence by the comparison of comparison software to reference on genome, genome is divided into size identical 1,000,000, the fragment of 000 bases longs, calculate each fragment altogether by the number of times recorded, divided by a homogenization value: (clip size * check order the total bases obtained)/genome total bases certificate, is the copy number of the homogenization of each fragment.To be checked order again the fragment different for human genome that obtain and the corresponding copies data mapping obtained by unicellular amplification, as shown in Fig. 3 upper part.Some of them are the fragment of 0 value is the part of not including with reference to genome, and be generally positioned near centrosome or chromosomal two ends, tumor-necrosis factor glycoproteins is more herein, cannot know real sequence, therefore be got rid of.In figure, the value of each corresponding ordinate zou is the copy number after homogenization, the fragment of 1,000,000,000 base of corresponding chromosome numbers that what X-coordinate was corresponding is.Difference between the copy number of the different fragments of embodiment 1 is less, and euchromosome is average out to 2 copy all, and X chromosome and Y chromosome are a copy, and this is consistent with known knowledge.
Embodiment 2
When setting up reaction chamber, the unicellular amplified reaction solution that 10 μ L are prepared and 100 μ L mineral oil, 3 minutes are shaken in 500HZ frequency ultrasound, form the water react room be dispersed in oil phase, reaction chamber system is 10fL, and the volume of reaction chamber can be changed by control ultrasonic time and frequency.Except the establishment step of reaction chamber, other steps are all identical with embodiment 1.
Comparative example 1
The reaction soln that step 3 prepares directly carries out amplified reaction in 10 μ L systems, and amplified reaction terminates the purifying carrying out DNA, and purification step is that given experimental program version number 1.0 of Zymo company is consistent, eliminates the step setting up reaction chamber.The step of the fragmentation of the preparation of single celled separation, cracking, reaction soln, amplified reaction, DNA, the foundation of sequencing library, comparison is all identical with embodiment 1.
According to the copies data mapping of comparison result for the different fragment of human genome and the corresponding homogenization obtained, as shown in Fig. 3 lower part.From the figure of comparative example 1, widely different between the copy number of different fragments, autosomal copy number has distribution between 1-4, the chromosomal copy number of every bar can not be found out intuitively completely from this figure, illustrate that the result after amplification cannot represent the front single celled state of amplification, there is more amplification bias.The variation coefficient (mean value of the standard deviation/euchromosome fragment homogenization copy number of euchromosome fragment homogenization copy number) of the chromosome segment copy number of embodiment 1 is 0.04, compared with the variation coefficient 0.4 of comparative example 1, amplification bias reduces by 10 times.
Claims (18)
1. one kind can reduce the amplification method producing bias when trace dna material entirety increases, it is characterized in that, described nucleic acid substances is dispersed randomly to after in some mutual disconnected independent reaction systems and increases again, comprise 3-20 randomized bases in the primer sequence that amplification uses, randomized bases is selected from two, three or four in A, G, C, T at random.
2. method according to claim 1, the primer that amplification uses is the sequence be made up of 3-20 randomized bases, and randomized bases is selected from two, three or four in A, G, C, T at random.
3. method according to claim 1, can comprise the base of non-natural or modified in described primer, and with other materials of base effect, as lock nucleic acid, 5 '-nitroindoline, phosphorothioate oligonucleotide.
4. method according to claim 1, the volume of described independent reaction system is 0.5fL-100nL, preferred 1fL-10nL, more preferably 10fL-1nL.
5. method according to claim 1, described independent reaction system can be disperseed to form by immiscible two-phase any in oil, water, hydrogel, polydimethylsiloxane, plastics, silicon chip, glass, air.
6. method according to claim 1, the method for described dispersion can be ultrasonic disperse or use fluid-operating system to disperse.
7. method according to claim 6, described fluid-operating system can be micro-fluidic chip.
8. method according to claim 1, described amplification can be MDA (multiple displacement amplification), PCR (polymerase chain reaction), RCA (rolling circle amplification), MALBAC (ring-type cyclic amplification of repeatedly annealing).
9. method according to claim 1, the sequence of described nucleic acid substances is unknown.
10. method according to claim 1, the quality of described trace dna material is less than 10ng, is preferably less than 1ng.
11. methods according to claim 1, the content of each reaction system amplifying nucleic acid material is 0 to 50 molecule, is preferably 0-20 molecule, is more preferably 0-10 molecule.
12. methods according to claim 1, described to be dispersed in some independent reaction systems some, refer to more than 2, preferred 2-10
20individual, more preferably 2-10
8individual.
13. methods according to claim 1, described nucleic acid substances comprises single stranded DNA; Double-stranded DNA; Single stranded RNA; Double-stranded RNA; Double-stranded RNA/DNA hybridization body; DNA and RNA of partial hybridization; Through DNA or RNA of zymetology, chemistry, biological method process, as: the DNA after the DNA extracted after immunoprecipitation, sulfiting, DNA, RNA cDNA etc. after reverse transcription process after Methyl transporters ferment treatment.
14. methods according to claim 1, described nucleic acid substances can not come from nature, can be the nucleic acid substances of synthetic or certain chemical treatment product of nature nucleic acid substances, can from one or more in cell, subcellular unit, karyomit(e), nucleic acid molecule, can from unicellular, monosome, single core acid molecule, one or more in trace nucleic acid.
15. methods according to claim 1, can carry out product recovery after amplification terminates.
16. methods according to claim 15, the method that product reclaims can make independently reaction system be connected for adding the emulsion splitter such as ethanol, isopropylcarbinol.
17. 1 kinds are carried out the method for genome sequencing to trace dna material, it is characterized in that, use the amplification method in claim 1-16 to increase, then carry out product recovery, build storehouse and order-checking.
18. 1 kinds are carried out the method for transcript profile order-checking to trace dna material, it is characterized in that, use the amplification method in claim 1-16 to increase, then carry out product recovery, build storehouse and order-checking.
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| CN109439734A (en) * | 2018-10-17 | 2019-03-08 | 东南大学 | A kind of whole genome amplification method based on Ago-Gel medium |
| CN109439734B (en) * | 2018-10-17 | 2022-01-28 | 东南大学 | Whole genome amplification method based on agarose gel medium |
| CN112251500A (en) * | 2020-10-21 | 2021-01-22 | 东南大学 | Method capable of improving amplification efficiency of trace nucleic acid |
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