CN109852678A - DNA sequencing method based on carbon nanotube and single stranded DNA winding arrangement - Google Patents
DNA sequencing method based on carbon nanotube and single stranded DNA winding arrangement Download PDFInfo
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- CN109852678A CN109852678A CN201910158843.7A CN201910158843A CN109852678A CN 109852678 A CN109852678 A CN 109852678A CN 201910158843 A CN201910158843 A CN 201910158843A CN 109852678 A CN109852678 A CN 109852678A
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- 108020004414 DNA Proteins 0.000 title claims abstract description 55
- 102000053602 DNA Human genes 0.000 title claims abstract description 42
- 108020004682 Single-Stranded DNA Proteins 0.000 title claims abstract description 42
- 239000002041 carbon nanotube Substances 0.000 title claims abstract description 41
- 229910021393 carbon nanotube Inorganic materials 0.000 title claims abstract description 41
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 20
- 238000001712 DNA sequencing Methods 0.000 title claims abstract description 11
- 238000004804 winding Methods 0.000 title claims abstract description 11
- 239000002131 composite material Substances 0.000 claims abstract description 28
- 238000010276 construction Methods 0.000 claims abstract description 26
- 238000007672 fourth generation sequencing Methods 0.000 claims abstract description 18
- 238000012163 sequencing technique Methods 0.000 claims abstract description 13
- 239000011148 porous material Substances 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 18
- 239000012266 salt solution Substances 0.000 claims description 11
- 238000005259 measurement Methods 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000002604 ultrasonography Methods 0.000 claims description 6
- 239000010410 layer Substances 0.000 claims description 5
- 239000002356 single layer Substances 0.000 claims description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 239000005457 ice water Substances 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 2
- 239000013049 sediment Substances 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 239000003115 supporting electrolyte Substances 0.000 claims 2
- 238000004458 analytical method Methods 0.000 claims 1
- 239000012895 dilution Substances 0.000 claims 1
- 238000010790 dilution Methods 0.000 claims 1
- 238000001962 electrophoresis Methods 0.000 claims 1
- 239000011259 mixed solution Substances 0.000 claims 1
- 230000035772 mutation Effects 0.000 claims 1
- 239000011435 rock Substances 0.000 claims 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims 1
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 2
- 230000009471 action Effects 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 description 8
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 229910052710 silicon Inorganic materials 0.000 description 4
- 239000010703 silicon Substances 0.000 description 4
- 102100033072 DNA replication ATP-dependent helicase DNA2 Human genes 0.000 description 3
- 101000927313 Homo sapiens DNA replication ATP-dependent helicase DNA2 Proteins 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 229910052581 Si3N4 Inorganic materials 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
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- 239000007788 liquid Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- HQVNEWCFYHHQES-UHFFFAOYSA-N silicon nitride Chemical compound N12[Si]34N5[Si]62N3[Si]51N64 HQVNEWCFYHHQES-UHFFFAOYSA-N 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000010409 thin film Substances 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000010884 ion-beam technique Methods 0.000 description 1
- 238000001755 magnetron sputter deposition Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
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- 238000001338 self-assembly Methods 0.000 description 1
- 239000002109 single walled nanotube Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
Abstract
The DNA sequencing method based on carbon nanotube and single stranded DNA winding arrangement that the invention discloses a kind of, step are carbon nanotube and single stranded DNA to be mixed to form a kind of composite construction first, and the composite construction is placed in containing Na+And Mg2+In the reactive tank of equal solution, nano-pore sequencing device is passed through by the driving force of electrophoretic action, realizes the capture of signal, and then determines the DNA sequence dna of winding on the carbon nanotubes.The composite construction perforates generated signal greater than signal caused by single stranded DNA perforation, is easier to read, the efficiency and accuracy rate of sequencing greatly improved.
Description
Technical field
The present invention relates to gene sequencing technology fields, and in particular to a kind of detection of the type and sequence of single-chain DNA base
Method further relates to the DNA sequencing system based on carbon nanotube and single stranded DNA composite construction, and be related to based on carbon nanotube and
The preparation method of single stranded DNA composite construction.
Background technique
Gene sequencing technology refers to by analyzing specific DNA fragments, determines a kind of technology of base sequence.It is in several species
Gene sequencing, disorder in screening and diagnosis, personalized treatment, forensic identification, biological information reading etc. are of great significance.
DNA sequencing is developed so far from the first generation technique, and sequencing cost constantly reduces, and sequencing speed greatly improves, and is achieved
It is a series of to be in progress breakthroughly.Different bases institute when nano-pore sequencing technology of new generation passes through nano-pore by detecting single stranded DNA
The electrical signal of generation and determine DNA sequence dna, mainly have current type and condenser type two types.The main base of nano-pore sequencing at present
In biological nano hole and solid nano hole, the former can be primarily used for nano-pore and being self-assembly of because its noise is small
Sequencing, but not can avoid the biomolecule defect sensitive to environmental changes such as pH value and temperature.Stability is good as a result, persistence
High and easily commercial production solid nano hole is come into being, however since electrical signal is excessively faint, it is dry vulnerable to environment
It disturbs, need to often be aided with complicated material modification technique reduces noise, it is also necessary to by limitation perforation rate to improve resolution ratio, not only
Reading rate is sacrificed, Detection accuracy is also affected.Therefore, need to develop a kind of new technology to realize fast and accurately gene
Sequencing.
Summary of the invention
(1) technical problems to be solved
In view of this, the purpose of the present invention is to provide a kind of DNA sequencing sides wound based on carbon nanotube and single stranded DNA
Method, sequencing system and preparation method, at least partly to overcome the signal that single strand dna perforation generates in the prior art excessively micro-
It is small, it is not easy to read, and vulnerable to noise jamming；Single strand dna is flexible structure, foldable, and the low technology of Detection accuracy is asked
Topic.
(2) technical solution
The DNA sequencing method based on carbon nanotube and single stranded DNA winding arrangement that the invention discloses a kind of: by carbon nanotube
Ratio mixing with single stranded DNA to set forms an one compound winding form of packet, places it in conductive salt solution, lead to
It crosses electrode drive and passes through the nanopore-channel for being placed in conductive salt solution, perforation when institute can be measured using ion blocking effect
The electrical response of generation, and then determine the type of base and put in order.
(3) beneficial effect
It can be seen from the above technical proposal that the present invention is based on the DNA sequencing sides of carbon nanotube and single stranded DNA winding arrangement
Method or system at least have the advantages that
(1) carbon nanotube and single stranded DNA of specific caliber and length is equal with the ratio mixing of setting under solution state
It is even, an one compound winding form of packet is formed, autofolding phenomenon of the single stranded DNA separately through nano-pore when can be greatly avoided,
Realize the sequencing of single stranded DNA；
(2) carbon nanotube and single stranded DNA composite construction are enhanced into the response intensity of punch signal, greatly by nano-pore
Width improves the accuracy of sequencing；
(3) composite construction for passing through carbon nanotube and single stranded DNA, increases the scale of test sample, realizes usable
Larger solid nano hole is sequenced.
Detailed description of the invention
Fig. 1 is the DNA nano-pore sequencing based on carbon nanotube and single stranded DNA composite construction that the embodiment of the present invention proposes
Implementation steps figure.
Fig. 2 is the making step figure of the single layer that the embodiment of the present invention proposes or plural layers nano-pore sequencing device.
Fig. 3 is the schematic diagram that the carbon nanotube that the embodiment of the present invention proposes and single stranded DNA one wrap a composite wound structure.
Fig. 4 is that a kind of of proposition of the embodiment of the present invention is surveyed based on the DNA nano-pore of carbon nanotube and single stranded DNA composite construction
The schematic diagram of sequence device.
Fig. 5 is a kind of preparation process based on carbon nanotube and single stranded DNA composite construction that the embodiment of the present invention proposes.
[component symbol illustrates in attached drawing]
1- carbon nanotube；2- single stranded DNA；The composite construction of 3- carbon nanotube and single stranded DNA；4- nano-pore sequencing device；
5- reactive tank；6- electrode (conducting wire)；7- conductivity measurement.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, and reference
Attached drawing, the present invention is described in more detail.
The present invention provides a kind of DNA sequencing technologies wound based on carbon nanotube and single stranded DNA, and wherein Fig. 1 is this hair
Bright implementation steps, as shown in Figure 1, mixing single stranded DNA to be detected and carbon nanotube under solution state in step A
It closes；Existing single layer or plural layers nano-pore sequencing device are placed in conductive salt solution in step B, conductive salt is molten
Liquid can be Na+And Mg2+Solution；Electrophoretic action flows conductive salt in solution by electrode energization in step C
Dynamic, drive mixture flows through nano-pore；Conductivity measurement test nano-pore sequencing device both ends are used in step D
Conductivity variations, to release the type and sequence of surveyed single-chain DNA base.
Fig. 2 is the making step of single layer proposed by the present invention or plural layers nano-pore sequencing device, as shown in Fig. 2,
To nitrogenize silicon substrate as target, the silicon nitride that thickness is about 30nm is prepared on the silicon substrate of 100nm by magnetron sputtering technique
Then film etches the cavity that diameter is hundreds of nanometers by lithographic technique, finally by focused ion beam on silicon thin film
Technology gets nano-pore, in one embodiment of the invention, nano-pore by support construction of silicon thin film on silicon nitride film
Aperture be 10nm.
Fig. 3 is the schematic diagram that carbon nanotube 1 proposed by the present invention and single stranded DNA 2 one wrap a composite wound structure, a packet
One compound winding form can greatly avoid autofolding phenomenon of the single stranded DNA 2 separately through nano-pore when.Fig. 5 is carbon nanometer
The preparation step of 2 composite construction of pipe 1 and single stranded DNA, as shown in figure 5, the conductive salt solution of configuration setting concentration first, with this
Solution is that solvent configures DNA2 solution to be measured, is in one embodiment in the present invention, conductive salt solution is NaCl solution, concentration
For 0.1M, DNA2 solution concentration to be measured is 10 μM；It takes a certain amount of above-mentioned solution to be placed in centrifuge tube, and single-walled carbon nanotube is added
1, it is mixed through vortex oscillation, in one embodiment of the invention, DNA2 solution volume is 2mL, and 1 mass of carbon nanotube is 2mg；
Centrifuge tube is placed in Ultrasound Instrument, ultrasound setting duration, in one embodiment of the invention, ultrasonic power are carried out in ice-water bath
For 100W, Shi Changwei 1 hour；Above-mentioned solution is continued to carry out centrifugally operated setting duration in setting centrifugal force, in the present invention
One embodiment in, centrifugal force 12000g, Shi Changwei 0.5 hour；Above-mentioned processed mixture is subjected to supernatant point
From, wherein separation after supernatant be 2 composite construction of required carbon nanotube 1 and single stranded DNA, lower sediment be not with it is single-stranded
The carbon nanotube that DNA is combined.
Fig. 4 be a kind of DNA nano-pore sequencing based on carbon nanotube and single stranded DNA composite construction proposed by the present invention
The schematic diagram of device, comprising: carbon nanotube and single stranded DNA composite construction 3, nano-pore sequencing device 4, conduction built in reactive tank 5
Property salting liquid, electrode (conducting wire) 6, conductivity measurement 7.It will be by the carbon nanotube and single stranded DNA composite junction of preceding method preparation
Structure 3 is placed in the conductive salt solution of reactive tank 5, and electrode 6 is powered, and moves the electron ion in conductive salt solution, band
Dynamic composite construction 3 moves and passes through the nano-pore in nano-pore sequencing device 4, and conductivity measurement 7 can detecte nano-pore survey
The corresponding conductivity variations in 4 both ends of sequence device, to realize the sequencing of DNA to be measured.
Particular embodiments described above has carried out further in detail the purpose of the disclosure, technical scheme and beneficial effects
Describe in detail it is bright, it is all it should be understood that be not limited to the disclosure the foregoing is merely the specific embodiment of the disclosure
Within the spirit and principle of the disclosure, any modification, equivalent substitution, improvement and etc. done should be included in the guarantor of the disclosure
Within the scope of shield.
Claims (9)
1. a kind of DNA sequencing method based on carbon nanotube and single stranded DNA winding arrangement, comprising:
Single stranded DNA to be detected and carbon nanotube are mixed to form to one composite construction of packet of carbon nanotube winding DNA；
So that the composite construction is passed through the nano-pore of nano-pore sequencing device, is received when being measured by conductivity measurement across nano-pore
The variation of device two sides conductivity is sequenced in metre hole, according to the mutation analysis go out across base type and sequence, described in realization
The sequencing of DNA.
2. according to the method described in claim 1, wherein, the nano-pore sequencing device is to run through in single layer or plural layers
Nano-pore.
3. according to the method described in claim 1, wherein, the mixing include: by single stranded DNA to be detected and carbon nanotube with
Setting ratio mixes under solution state.
4. according to the method described in claim 2, wherein, the mode which passes through nano-pore sequencing device is electrophoresis side
Formula, electrophoretic specifically include:
The nano-pore sequencing device is inserted into conductive salt solution, and the composite construction of carbon nanotube and single stranded DNA is also put
In the conductive salt solution；
Flow conductive salt in solution by electrode energization, drive composite construction flows through nano-pore.
5. according to the method described in claim 1, wherein, in the nano-pore sequencing device aperture of nano-pore be 2 nanometers extremely
20 nanometers.
6. a kind of DNA sequencing system based on carbon nanotube and single stranded DNA composite construction, characterized by comprising:
Reactive tank is internally configured as being packed into the composite construction of supporting electrolyte and carbon nanotube and single stranded DNA；
Nano-pore sequencing device, including single-layer or multi-layer film have an at least nano-pore, are configured to be inserted into supporting electrolyte
In；
Conductivity measurement includes positive and negative electrode, and wherein positive and negative electrode is configured to be inserted into conducting solution and is located at support
The two sides of film, the conductivity measurement are configured to conductivity variations when measurement composite construction passes through nano-pore.
7. a kind of preparation method based on carbon nanotube and single stranded DNA composite construction, comprising:
Single stranded DNA to be detected and carbon nanotube are blended in conductive salt solution by setting ratio；Said mixture is set
In centrifuge tube, mixed through vortex oscillation；
Centrifuge tube is placed in Ultrasound Instrument, carries out ultrasound to set power in ice-water bath；
It is centrifuged with setting centrifugal force to the solution after carrying out ultrasound；
Mixture after being centrifuged carries out supernatant dilution separation, wherein the supernatant after separation is required carbon nanotube
With single stranded DNA composite construction, lower sediment is the carbon nanotube not in conjunction with single stranded DNA.
8. according to the method described in claim 7, wherein the conductive salt solution is NaCl solution.
9. according to the method described in claim 7, wherein the ice-water bath refers on the rocks in the water of Ultrasound Instrument or ice bag
Guarantee the temperature added with carbon nanotube and the centrifuge tube of DNA mixed solution.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910158843.7A CN109852678A (en) | 2019-03-01 | 2019-03-01 | DNA sequencing method based on carbon nanotube and single stranded DNA winding arrangement |
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| CN201910158843.7A CN109852678A (en) | 2019-03-01 | 2019-03-01 | DNA sequencing method based on carbon nanotube and single stranded DNA winding arrangement |
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| CN209816171U (en) * | 2019-03-01 | 2019-12-20 | 天津大学 | DNA sequencing system based on carbon nanotube and single-stranded DNA composite structure |
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2019
- 2019-03-01 CN CN201910158843.7A patent/CN109852678A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050009039A1 (en) * | 2002-11-21 | 2005-01-13 | Anand Jagota | Dispersion of carbon nanotubes by nucleic acids |
| CN101203740A (en) * | 2005-04-06 | 2008-06-18 | 哈佛大学校长及研究员协会 | Molecular identification with carbon nanotube control |
| CN1986791A (en) * | 2005-12-20 | 2007-06-27 | 国际商业机器公司 | Helical wrapping method and structure of single-walled carbon nanotubes |
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