CN1986832A - Electromagnetic method and device for controlling single-chain nucleic acid perforating speed - Google Patents
Electromagnetic method and device for controlling single-chain nucleic acid perforating speed Download PDFInfo
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- CN1986832A CN1986832A CNA200610147233XA CN200610147233A CN1986832A CN 1986832 A CN1986832 A CN 1986832A CN A200610147233X A CNA200610147233X A CN A200610147233XA CN 200610147233 A CN200610147233 A CN 200610147233A CN 1986832 A CN1986832 A CN 1986832A
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- magnetic bead
- target molecule
- nanoporous
- dna
- electromagnetic force
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
- C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
Abstract
The present invention relates to bioengineering technology, and is electromagnetic method and equipment for controlling single-chain nucleic acid perforation. The method includes the following steps: 1. fixing the composition of one single-chain target DNA or RNA and one connected magnetic bead onto the negative pole of electrophoresis tank with two nanometer hole separated poles by using one electromagnet; and 2. turning on the power source to apply the free end of the negatively charged target molecule with one pull force towards the positive pole, trimming the electromagnetic force to release the magnetic bead while maintaining certain attraction of the electromagnet on the magnetic bead so as to make the target molecule to move slowly towards the positive pole and control the nanometer hole passing speed in 0.5-1 base/ms, recording the signal with the patch clamp and converting the signal into sequence information in the computer. The equipment includes electrophoresis tank, nanometer element, computer, etc.
Description
Technical field
What the present invention relates to is a kind of method of technical field of bioengineering, specifically is a kind of method and apparatus of electromagnetic force control single-chain nucleic acid perforating speed.
Background technology
Along with the arrival of genome times afterwards comprehensively, the demand of gene order-checking is increased severely day by day, but present sequence measurement speed is slow, expense is high, jagged.In fact, before the Human Genome Project starts, just the someone has begun the discussion of dna sequencing novel method, particularly the The National Institutes ofHealth of the U.S. in 2004 starts $1000 Genome (promptly about 10 years of future, about expense Jiang Didao $1000 with the Mammals order-checking, speed improves 3-4 the order of magnitude, error rate ten thousand/in, basic non-notch) since, there are many new order-checking thinkings to continue to bring out again, wherein, detect the electrical signal difference that single stranded DNA (abbreviation single stranded DNA) stays when molecule passes through nanoporous and realize order-checking with patch clamp, have very big potentiality, but under effective voltage, it is too fast that nucleic acid passes through the speed of nanoporous at present, head and shoulders above the discernible limit of patch clamp (1 incident/ms), so reduce the punching rate of single stranded DNA and RNA, become the key factor of nanoporous order-checking.
Through the prior art literature search is found, Fologea, people such as D 2005 " Slowing DNA translocation in a solid-statenanopore " (" receive a grade communication " of publishing an article at " Nano Letters " the 5th phase 1734-1737 page or leaf, " slow down DNA passing through in the solid nano hole "), provided their result of study in this article, main by add means such as glycerine, reduction voltage to damping fluid, though the punching rate of single stranded DNA has been reduced to 3nt/ μ s, but still has exceeded 3 orders of magnitude of instrumental resolution.So the nanoporous single-molecule sequencing still is in the key stage at present, can't be used for actual order-checking.
Summary of the invention
The present invention is directed to the deficiency and the defective that have now based on the nucleic acid sequencing technology of nanoporous, a kind of method and apparatus of electromagnetic force control single-chain nucleic acid perforating speed is provided, make it single stranded DNA punching rate can be controlled at every millisecond of 0.5-1 base, patch clamp record electrical signal, computer is a sequence information with electrical signal conversion, the needs of the realistic order-checking of energy.
The present invention is achieved by the following technical solutions, and the method for electromagnetic force control single-chain nucleic acid perforating speed of the present invention comprises the steps:
1. the mixture that a strand target DNA or RNA (abbreviation target molecule) are connected with a magnetic bead is fixed with electro-magnet at the electrophoresis chamber negative pole, and electromagnetic force is at 50-1000pN; Described electrophoresis chamber, its positive and negative polarities are separated by nanoporous.
2. opening power, give electronegative target molecule free end to an anodal pulling force (540-1000pN), because the end that target molecule is connected with magnetic bead is fixed by electro-magnet, such result is that target molecule is straightened, and by fine setting electromagnetic force is weakened again, discharge magnetic bead, but electro-magnet is still attractive to magnetic bead, and target molecule is slowly moved to positive pole, will pass through the speed control of nanoporous in every millisecond of 0.5-1 base, patch clamp record electrical signal, computer is a sequence information with electrical signal conversion.
Described strand target DNA or RNA are connected mixture with a magnetic bead, concrete making step is: magnetic bead is mixed with biotin labeled single stranded DNA (being called connector), make each magnetic bead only connect a single strand dna or do not have single strand dna, handle magnetic bead, single stranded DNA and magnetic bead mixed solution with the single stranded DNA fluorescence dye, fluorescent microscope is auxiliary to be confirmed, selects single magnetic bead and 1 connector mixture.This mixture is connected with strand target DNA or RNA under the dna ligase effect, also 3 ' free end of connector can be designed to poly T, catches mRNA by the mode of base pairing, obtains the mixture that target DNA or RNA are connected with a magnetic bead.
The present invention also provides a kind of device of electromagnetic force control single-chain nucleic acid perforating speed, this device comprises: electrophoresis chamber, nano-component, adjustable solenoid, patch clamp and computer, electrophoresis chamber the two poles of the earth are separated by nano-component, two interpolar ion-exchanges can only be undertaken, the transmitter of the resistance that each base pair ionic current produced when nanoporous passed through nanoporous as DNA or RNA molecule by the nanoporous of nano-component; Electrophoresis chamber the two poles of the earth connect patch clamp, by the nanoporous sensor detecting and put down in writing the size of each base pair ionic current resistance; The outer wall of electrophoresis chamber negative pole connects electromagnetic force at the adjustable electro-magnet of 50-1000pN, be used for fixing with discharging and be connected target molecule, and at target molecule when positive pole is mobile, give an opposite direction of target molecule gravitation, to control its translational speed; When target molecule passes through nanoporous, patch clamp record electrical signal; Patch clamp connects computer, and computer is a sequence information with electrical signal conversion.
The present invention is to provide and a kind ofly pass through the method and apparatus of nanoporous speed carrying out in the testing sequence of nucleic acid single molecule system control target molecule with nanoporous, catch gentle slow release by the adjustable electro-magnet of intensity and put magnetic bead, the punching rate of target molecule is controlled at 0.5-1 base/millisecond, has overcome the too fast shortcoming that causes patch clamp can't discern base perforation electrical signal of nucleic acid perforating speed in the past.
Description of drawings
Fig. 1 is apparatus of the present invention example structure synoptic diagram
Embodiment
Below embodiments of the invention are elaborated: present embodiment has provided detailed embodiment and process being to implement under the prerequisite with the technical solution of the present invention, but protection scope of the present invention is not limited to following embodiment.
As shown in Figure 1, be apparatus of the present invention example structure synoptic diagram, comprise: electrophoresis chamber 1, nano-component 2, adjustable solenoid 3, patch clamp 4 and computer 5, electrophoresis chamber 1 the two poles of the earth are separated by nano-component 2, two interpolar ion-exchanges can only be undertaken, the transmitter of the resistance that each base pair ionic current produced when nanoporous passed through nanoporous as DNA or RNA molecule by the nanoporous of nano-component 2; Electrophoresis chamber 1 the two poles of the earth connect patch clamp 4, by the nanoporous sensor detecting and put down in writing the size of each base pair ionic current resistance; The outer wall of electrophoresis chamber 1 negative pole connects electromagnetic force at 50-1000pN adjustable solenoid 3, be used for fixing with discharging and be connected target molecule, and at target molecule when positive pole is mobile, give an opposite direction of target molecule gravitation, to control its translational speed; When target molecule passes through nanoporous, patch clamp 4 record electrical signal; Patch clamp 4 connects computer 5, and computer 5 is a sequence information with electrical signal conversion.
The nanoporous of described nano-component 2, for diameter in 1-2nm, hole depth cylindrical at about 0.5nm, the material of nanoporous is solid insulating materials such as silicon chip, silica glass, diamond or mica.
The punching rate control of embodiment 1:BAC single stranded DNA
1. the mixture that a strand target DNA molecule is connected with a magnetic bead is fixed with electro-magnet at the electrophoresis chamber negative pole; Electrophoresis chamber the two poles of the earth are separated by nanoporous.
2. opening power, give electronegative target molecule free end to an anodal pulling force (540pN), because the end that target molecule is connected with magnetic bead is fixed (electromagnetic force is at 50pN) by electro-magnet, such result is that target molecule is straightened, by fine setting electromagnetic force is weakened again, discharge magnetic bead, but electro-magnet is still attractive to magnetic bead, target molecule is slowly moved to positive pole, to pass through the speed control of nanoporous every millisecond of 0.5 base, patch clamp record signal, computer is a sequence information with electrical signal conversion.
Effect: by single stranded DNA one end is connected magnetic bead, utilize the punching rate of electromagnetic attracting force magnetic bead control DNA when extra electric field draws, punching rate can be controlled at every millisecond of 0.5 base.
The punching rate control of embodiment 2:mRNA
1. the mixture that a strand target RNA molecule is connected with a magnetic bead is fixed with electro-magnet at the electrophoresis chamber negative pole; Electrophoresis chamber the two poles of the earth are separated by nanoporous.
2. opening power, give electronegative target molecule free end to an anodal pulling force (1000pN), because the end that target molecule is connected with magnetic bead is fixed (electromagnetic force is at 1000pN) by electro-magnet, such result is that target molecule is straightened, by fine setting electromagnetic force is weakened again, discharge magnetic bead, but electro-magnet is still attractive to magnetic bead, target molecule is slowly moved to positive pole, to pass through the speed control of nanoporous every millisecond of 1 base, patch clamp record signal, computer is a sequence information with electrical signal conversion.
Effect: by single stranded RNA one end is connected magnetic bead, utilize the punching rate of electromagnetic attracting force magnetic bead control RNA when extra electric field draws, punching rate can be controlled at every millisecond of 1 base.
Embodiment 3: the punching rate control of clay single stranded DNA
1. the mixture that a clay single stranded RNA molecule is connected with a magnetic bead is fixed with electro-magnet at the electrophoresis chamber negative pole; Electrophoresis chamber the two poles of the earth are separated by nanoporous.
2. opening power, give electronegative target molecule free end to an anodal pulling force (500pN), because the end that target molecule is connected with magnetic bead is fixed (electromagnetic force is at 500pN) by electro-magnet, such result is that target molecule is straightened, by fine setting electromagnetic force is weakened again, discharge magnetic bead, but electro-magnet is still attractive to magnetic bead, target molecule is slowly moved to positive pole, to pass through the speed control of nanoporous every millisecond of 1 base, patch clamp record signal, computer is a sequence information with electrical signal conversion.
Effect: by clay single stranded DNA one end is connected magnetic bead, utilize electromagnetic attracting force magnetic bead control DNA Punching rate when extra electric field draws can be controlled at punching rate every millisecond of 1 base.
Claims (10)
1, a kind of method of electromagnetic force control single-chain nucleic acid perforating speed is characterized in that, comprises the steps:
1. be target molecule with a strand target DNA or RNA, the mixture that is connected with a magnetic bead is fixed with adjustable solenoid at the electrophoresis chamber negative pole, and the positive and negative polarities of electrophoresis chamber are separated by nanoporous;
2. opening power, give electronegative target molecule free end to anodal pulling force, because the end that target molecule is connected with magnetic bead is fixed by adjustable solenoid, such result is that target molecule is straightened, and by fine setting electromagnetic force is weakened again, discharge magnetic bead, but electro-magnet is still attractive to magnetic bead, and target molecule is slowly moved to positive pole, will pass through the speed control of nanoporous in every millisecond of 0.5-1 base, patch clamp record electrical signal, computer is a sequence information with electrical signal conversion.
2, the method for electromagnetic force control single-chain nucleic acid perforating speed according to claim 1 is characterized in that, described adjustable solenoid, and its electromagnetic force is adjustable in the 50-1000pN scope.
3, the method for electromagnetic force according to claim 1 control single-chain nucleic acid perforating speed is characterized in that, describedly gives electronegative target molecule free end to anodal pulling force, and weight range is 540-1000pN.
4, device according to claim 1 is characterized in that, the nanoporous of described nano-component is for diameter is that 1-2nm, hole depth are the cylindrical of 0.5nm.
5, the method of electromagnetic force control single-chain nucleic acid perforating speed according to claim 1, it is characterized in that, described strand target DNA or RNA are connected mixture with a magnetic bead, concrete making step is: with magnetic bead and biotin labeled single stranded DNA is that connector mixes, make each magnetic bead only connect a single strand dna or do not have single strand dna, handle magnetic bead with the single stranded DNA fluorescence dye, single stranded DNA and magnetic bead mixed solution, fluorescent microscope is auxiliary to be confirmed, select single magnetic bead and 1 connector mixture, this mixture is under the dna ligase effect, be connected with strand target DNA or RNA, or 3 ' free end of connector is designed to poly T, mode by base pairing is caught mRNA, obtains the mixture that target DNA or RNA are connected with a magnetic bead.
6, a kind of device of electromagnetic force control single-chain nucleic acid perforating speed, comprise: electrophoresis chamber, nano-component, adjustable solenoid, patch clamp and computer, it is characterized in that, electrophoresis chamber the two poles of the earth are separated by nano-component, two interpolar ion-exchanges can only be undertaken by the nanoporous of nano-component, electrophoresis chamber the two poles of the earth connect patch clamp, the outer wall of electrophoresis chamber negative pole connects electro-magnet, patch clamp connects computer, electrical signal when patch clamp record target molecule passes through nanoporous, computer is a sequence information with electrical signal conversion.
7, device according to claim 6 is characterized in that, the nanoporous of described nano-component, and the transmitter of the resistance that each base pair ionic current produces when passing through nanoporous as DNA or RNA molecule detects and puts down in writing the size of each base pair ionic current resistance.
8, according to claim 6 or 7 described devices, it is characterized in that, the nanoporous of described nano-component, for diameter is that 1-2nm, hole depth are the cylindrical of 0.5nm, the material of nanoporous is a solid insulating material.
9, device according to claim 6 is characterized in that, described adjustable solenoid is used for fixing with discharging and is connected target molecule, and at target molecule when positive pole is mobile, give an opposite direction of target molecule gravitation, to control its translational speed.
10, according to claim 6 or 9 described devices, it is characterized in that, described adjustable solenoid, its electromagnetic force is 50-1000pN.
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CN102590314A (en) * | 2012-02-20 | 2012-07-18 | 北京大学 | Method for decelerating nucleic acid molecules in solid nanopore |
CN102621214A (en) * | 2012-03-13 | 2012-08-01 | 北京大学 | Method for carrying out deceleration and monomolecular capture on nucleic acid molecule based on solid-state nano hole |
CN103328973A (en) * | 2011-07-20 | 2013-09-25 | 加利福尼亚大学董事会 | Dual-pore device |
CN103509852A (en) * | 2012-06-18 | 2014-01-15 | 北京大学 | Detection method for biomolecular probe-calibrated specific sites of DNA based on nanopore device |
CN104076138A (en) * | 2011-07-20 | 2014-10-01 | 加利福尼亚大学董事会 | Compensated patch-clamp amplifier for nanopore polynucleotide sequencing and other applications |
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Family Cites Families (3)
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US6936433B2 (en) * | 2000-11-27 | 2005-08-30 | The Regents Of The University Of California | Methods and devices for characterizing duplex nucleic acid molecules |
US8278055B2 (en) * | 2002-05-01 | 2012-10-02 | Intel Corporation | Methods and device for analyte characterization |
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CN103328973A (en) * | 2011-07-20 | 2013-09-25 | 加利福尼亚大学董事会 | Dual-pore device |
CN103328973B (en) * | 2011-07-20 | 2015-04-01 | 加利福尼亚大学董事会 | Dual-pore device |
CN102590314B (en) * | 2012-02-20 | 2014-03-12 | 哈佛大学 | Method for decelerating nucleic acid molecules in solid nanopore |
CN102590314A (en) * | 2012-02-20 | 2012-07-18 | 北京大学 | Method for decelerating nucleic acid molecules in solid nanopore |
CN102621214A (en) * | 2012-03-13 | 2012-08-01 | 北京大学 | Method for carrying out deceleration and monomolecular capture on nucleic acid molecule based on solid-state nano hole |
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CN103509852B (en) * | 2012-06-18 | 2014-11-19 | 北京大学 | Detection method for biomolecular probe-calibrated specific sites of DNA based on nanopore device |
WO2020005994A1 (en) * | 2018-06-26 | 2020-01-02 | Electronic Biosciences, Inc. | Controlled nanopore translocation utilizing extremophilic replication proteins |
CN111122525A (en) * | 2019-12-11 | 2020-05-08 | 浙江大学 | Fluorescence-patch clamp-micro suction tube detection device |
CN111455034A (en) * | 2020-04-09 | 2020-07-28 | 南京罗岛纳米科技有限公司 | Single molecule detection method and system based on solid-state nanopore mechanism |
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