CN114736950A - DNA next-generation sequencing library, and construction method and construction kit thereof - Google Patents
DNA next-generation sequencing library, and construction method and construction kit thereof Download PDFInfo
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- CN114736950A CN114736950A CN202210175829.XA CN202210175829A CN114736950A CN 114736950 A CN114736950 A CN 114736950A CN 202210175829 A CN202210175829 A CN 202210175829A CN 114736950 A CN114736950 A CN 114736950A
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- 238000007481 next generation sequencing Methods 0.000 title claims 8
- 238000010276 construction Methods 0.000 title claims 3
- 238000000034 method Methods 0.000 claims 9
- 230000005415 magnetization Effects 0.000 claims 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 4
- 229920001661 Chitosan Polymers 0.000 claims 3
- 235000015097 nutrients Nutrition 0.000 claims 3
- 238000001712 DNA sequencing Methods 0.000 claims 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims 2
- 238000006243 chemical reaction Methods 0.000 claims 2
- 239000011780 sodium chloride Substances 0.000 claims 2
- 238000003756 stirring Methods 0.000 claims 2
- 238000012408 PCR amplification Methods 0.000 claims 1
- 229920002565 Polyethylene Glycol 400 Polymers 0.000 claims 1
- 239000012295 chemical reaction liquid Substances 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000001976 enzyme digestion Methods 0.000 claims 1
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical group O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims 1
- 229910001629 magnesium chloride Inorganic materials 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 239000001103 potassium chloride Substances 0.000 claims 1
- 235000011164 potassium chloride Nutrition 0.000 claims 1
- 238000002360 preparation method Methods 0.000 claims 1
- 230000036632 reaction speed Effects 0.000 claims 1
- 238000012163 sequencing technique Methods 0.000 claims 1
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- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- 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
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
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Abstract
The invention provides a construction method of a DNA next generation sequencing library, which comprises the following steps: the method comprises the following steps: carrying out enzyme digestion on the DNA sample to obtain fragmented original template DNA; step two: preparation of reaction solution: and (2) sending 10-30 parts of chitosan solution into a magnetic stirrer, and then adding 5-10 parts of nutrient, 1-5 parts of DTT and 1-2 parts of PEG 4001. According to the invention, the DNA sample is subjected to enzyme cutting treatment, and through improvement of reaction liquid and a magnetizer, chitosan in the reaction liquid is matched with a nutrient, so that sufficient nutrition is provided for raw materials, and the power of raw materials of a product is improved; meanwhile, the magnetizing agent is used for further providing energy for ferroferric oxide, sodium chloride and sodium chloride, and the ferroferric oxide aggregation is matched, so that the construction efficiency of the product is improved.
Description
Technical Field
The invention relates to the technical field of sequencing libraries, in particular to a DNA second-generation sequencing library, a construction method and a construction kit thereof.
Background
Construction of sequencing libraries first prepares the genome (although sequencing companies require sample sizes of 200ng, the amount of sample required by the Gnome Analyzer system can be as low as 100ng, and can be used in many sample-limited experiments), then fragments the DNA randomly into small fragments of several hundred bases or less, and adds specific linkers (adaptors) at both ends. In the case of transcriptome sequencing, library construction is relatively cumbersome, with fragmentation of the RNA followed by inversion into cDNA and then linker addition, or with inversion of the RNA into cDNA followed by fragmentation and linker addition. The size of the fragments (Insert size) has an effect on the subsequent data analysis and can be selected as desired. For genome sequencing, several different insert sizes are usually chosen in order to obtain more information at the time of Assembly (Assembly).
2) An anchor bridge; reactions for Solexa sequencing were performed in glass tubes called flow cells, which were in turn subdivided into 8 Lanes, each of which had innumerable single linker immobilized on its inner surface. The DNA fragment with the joint obtained in the step is denatured into a single chain and then combined with the joint primer on the sequencing channel to form a bridge structure for subsequent pre-amplification.
The existing DNA next generation sequencing library construction method is simple and low in construction efficiency, and further improvement treatment is needed based on the method.
Disclosure of Invention
The invention aims to provide a DNA second generation sequencing library, a construction method and a construction kit thereof, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
the construction method of the DNA next generation sequencing library comprises the following steps:
the method comprises the following steps: carrying out enzyme digestion on a DNA sample to obtain fragmented original template DNA;
step two: preparation of reaction solution:
sending 10-30 parts of chitosan solution into a magnetic stirrer, and then adding 5-10 parts of nutrient, 1-5 parts of DTT and 1-2 parts of PEG 400;
step three: the original template DNA is treated by reaction liquid reaction, then multiple PCR amplification is carried out, the amplified DNA is sent into a magnetizer for reaction for 1-5min, and the reaction speed is 100-500 r/min;
step four: then, the single-stranded linker method was performed to construct DNA.
Preferably, the preparation method of the nutritional agent comprises the following steps: stirring 1-5 parts of magnesium chloride, 1-5 parts of sodium chloride and 1-2 parts of potassium chloride at the rotating speed of 100-500r/min for 10-20 min; then stirring for 15-25min at the rotating speed of 500-1000r/min to obtain the nutrient.
Preferably, the mass fraction of the chitosan solution is 10-20%.
Preferably, the mass fraction of the chitosan solution is 15%.
Preferably, the magnetizer is prepared by mixing ferroferric oxide and sodium chloride according to the weight ratio of 3:1 and then magnetizing in a magnetizing tank.
Preferably, the magnetization intensity is 1-5Bt, and the magnetization time is 10-20 min.
Preferably, the magnetization is 3Bt and the magnetization time is 15 min.
A sequencing library constructed by the construction method of a DNA next generation sequencing library.
A kit for constructing a DNA next generation sequencing library.
Compared with the prior art, the invention has the beneficial effects that:
the DNA sample is cut off by enzyme, and the chitosan in the reaction solution is matched with the nutrient by improving the reaction solution and the magnetizer, so that sufficient nutrition is provided for the raw material, and the power of the raw material of the product is improved; meanwhile, the magnetizing agent is used for further providing energy for ferroferric oxide, sodium chloride and sodium chloride, and the ferroferric oxide aggregation is matched, so that the construction efficiency of the product is improved.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The construction method of the DNA next generation sequencing library comprises the following steps:
the method comprises the following steps: carrying out enzyme digestion on a DNA sample to obtain fragmented original template DNA;
step two: preparation of reaction solution:
sending 10-30 parts of chitosan solution into a magnetic stirrer, and then adding 5-10 parts of nutrient, 1-5 parts of DTT and 1-2 parts of PEG 400;
step three: the original template DNA is treated by reaction liquid reaction, then multiple PCR amplification is carried out, the amplified DNA is sent into a magnetizer for reaction for 1-5min, and the reaction speed is 100-500 r/min;
step four: then, the single-chain linker method is performed to construct DNA.
The preparation method of the nutritional agent of the embodiment comprises the following steps: stirring 1-part of magnesium chloride, 1-5 parts of sodium chloride and 1-2 parts of potassium chloride at the rotating speed of 100-500r/min for 10-20 min; then stirring for 15-25min at the rotating speed of 500-1000r/min to obtain the nutrient.
The chitosan solution of this example was present in a mass fraction of 10-20%.
The chitosan solution of this example was 15% by mass.
The magnetizing agent in the embodiment is prepared by mixing ferroferric oxide and sodium chloride according to the weight ratio of 3:1 and then magnetizing in a magnetizing tank.
The magnetization intensity of the present example is 1-5Bt, and the magnetization time is 10-20 min.
The magnetization of this example was 3Bt and the magnetization time was 15 min.
The sequencing library constructed by the method for constructing the DNA next generation sequencing library.
This example is a DNA next generation sequencing library construction kit.
Example 1.
The construction method of the DNA next generation sequencing library comprises the following steps:
the method comprises the following steps: carrying out enzyme digestion on the DNA sample to obtain fragmented original template DNA;
step two: preparation of reaction solution:
sending 10 parts of chitosan solution into a magnetic stirrer, and then adding 5 parts of nutrient, 1 part of DTT and 1 part of PEG 4001;
step three: the original template DNA is subjected to reaction treatment by adopting reaction liquid, then multiple PCR amplification is carried out, the amplified DNA is sent into a magnetizer to react for 1min, and the reaction speed is 100 r/min;
step four: then, the single-stranded linker method was performed to construct DNA.
The preparation method of the nutritional agent of the embodiment comprises the following steps: stirring 1 part of magnesium chloride, 1 part of sodium chloride and 1 part of potassium chloride at the rotating speed of 100r/min for 10 min; then stirring for 15min at the rotating speed of 500r/min to obtain the nutritional agent.
The chitosan solution of this example was 10% by mass.
The magnetizing agent in the embodiment is prepared by mixing ferroferric oxide and sodium chloride according to the weight ratio of 3:1 and then magnetizing in a magnetizing tank.
The magnetization of this example was 1Bt and the magnetization time was 10 min.
The sequencing library constructed by the method for constructing the second generation DNA sequencing library of the embodiment.
This example is a DNA second generation sequencing library construction kit.
Example 2.
The construction method of the DNA next generation sequencing library comprises the following steps:
the method comprises the following steps: carrying out enzyme digestion on a DNA sample to obtain fragmented original template DNA;
step two: preparation of reaction solution:
sending 30 parts of chitosan solution into a magnetic stirrer, and then adding 10 parts of nutrient, 5 parts of DTT and 2 parts of PEG 4002;
step three: the original template DNA is subjected to reaction treatment by adopting reaction liquid, then multiple PCR amplification is carried out, the amplified DNA is sent into a magnetizer to react for 5min, and the reaction speed is 500 r/min;
step four: then, the single-chain linker method is performed to construct DNA.
The preparation method of the nutritional agent of the embodiment comprises the following steps: stirring 5 parts of magnesium chloride, 5 parts of sodium chloride and 2 parts of potassium chloride at the rotating speed of 500r/min for 20 min; then stirring at the rotating speed of 1000r/min for 25min to obtain the nutritional agent.
The chitosan solution of this example was 20% by mass.
The magnetizing agent in the embodiment is prepared by mixing ferroferric oxide and sodium chloride according to the weight ratio of 3:1 and then magnetizing in a magnetizing tank.
The magnetization of this example was 5Bt, and the magnetization time was 20 min.
The sequencing library constructed by the method for constructing the second generation DNA sequencing library of the embodiment.
This example is a DNA next generation sequencing library construction kit.
Example 3.
The construction method of the DNA next generation sequencing library comprises the following steps:
the method comprises the following steps: carrying out enzyme digestion on the DNA sample to obtain fragmented original template DNA;
step two: preparation of reaction solution:
feeding 20 parts of chitosan solution into a magnetic stirrer, and then adding 7.5 parts of nutrient, 3 parts of DTT and 4001.5 parts of PEG;
step three: the original template DNA is subjected to reaction treatment by adopting reaction liquid, then multiple PCR amplification is carried out, the amplified DNA is sent into a magnetizer to react for 3min, and the reaction rotating speed is 300 r/min;
step four: then, the single-stranded linker method was performed to construct DNA.
The preparation method of the nutritional agent of the embodiment comprises the following steps: stirring 3 parts of magnesium chloride, 3 parts of sodium chloride and 1.5 parts of potassium chloride at the rotating speed of 300r/min for 15 min; then stirring for 20min at the rotating speed of 750r/min to obtain the nutritional agent.
The chitosan solution of this example was 15% by mass.
The magnetizer in the embodiment is prepared by mixing ferroferric oxide and sodium chloride according to the weight ratio of 3:1 and then magnetizing in a magnetizing tank.
The magnetization of this example was 3Bt and the magnetization time was 15 min.
The sequencing library constructed by the method for constructing the second generation DNA sequencing library of the embodiment.
This example is a DNA next generation sequencing library construction kit.
Example 4.
The construction method of the DNA next generation sequencing library comprises the following steps:
the method comprises the following steps: carrying out enzyme digestion on the DNA sample to obtain fragmented original template DNA;
step two: preparation of reaction solution:
sending 15 parts of chitosan solution into a magnetic stirrer, and then adding 7.5 parts of nutrient, 2 parts of DTT and 4001.5 parts of PEG;
step three: the original template DNA is subjected to reaction treatment by adopting reaction liquid, then multiple PCR amplification is carried out, the amplified DNA is sent into a magnetizer to react for 2min, and the reaction speed is 200 r/min;
step four: then, the single-stranded linker method was performed to construct DNA.
The preparation method of the nutritional agent of the embodiment comprises the following steps: stirring 2 parts of magnesium chloride, 2 parts of sodium chloride and 1.3 parts of potassium chloride at the rotating speed of 200r/min for 13 min; then stirring for 17min at the rotating speed of 600r/min to obtain the nutritional agent.
The chitosan solution of this example was 13% by mass.
The magnetizing agent in the embodiment is prepared by mixing ferroferric oxide and sodium chloride according to the weight ratio of 3:1 and then magnetizing in a magnetizing tank.
The magnetization of this example is 2Bt and the magnetization time is 12 min.
The sequencing library constructed by the method for constructing the second generation DNA sequencing library of the embodiment.
This example is a DNA next generation sequencing library construction kit.
Example 5.
The construction method of the DNA next generation sequencing library comprises the following steps:
the method comprises the following steps: carrying out enzyme digestion on the DNA sample to obtain fragmented original template DNA;
step two: preparation of reaction solution:
sending 10-30 parts of chitosan solution into a magnetic stirrer, and then adding 7.5 parts of nutrient, 3 parts of DTT and 4001.8 parts of PEG;
step three: the original template DNA is subjected to reaction treatment by adopting reaction liquid, then multiple PCR amplification is carried out, the amplified DNA is sent into a magnetizer to react for 4min, and the reaction rotating speed is 300 r/min;
step four: then, the single-stranded linker method was performed to construct DNA.
The preparation method of the nutritional agent of the embodiment comprises the following steps: stirring 4 parts of magnesium chloride, 4 parts of sodium chloride and 1.6 parts of potassium chloride at the rotating speed of 400r/min for 15 min; then stirring for 22min at the rotating speed of 80r/min to obtain the nutritional agent.
The chitosan solution of this example was 18% by mass.
The magnetizing agent in the embodiment is prepared by mixing ferroferric oxide and sodium chloride according to the weight ratio of 3:1 and then magnetizing in a magnetizing tank.
The magnetization of this example was 3Bt, and the magnetization time was 18 min.
The sequencing library constructed by the method for constructing the second generation DNA sequencing library of the embodiment.
This example is a DNA next generation sequencing library construction kit.
The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (9)
- The construction method of the DNA next generation sequencing library is characterized by comprising the following steps:the method comprises the following steps: carrying out enzyme digestion on the DNA sample to obtain fragmented original template DNA;step two: preparation of reaction solution:sending 10-30 parts of chitosan solution into a magnetic stirrer, and then adding 5-10 parts of nutrient, 1-5 parts of DTT and 1-2 parts of PEG 400;step three: the original template DNA is subjected to reaction treatment by adopting reaction liquid, then multiple PCR amplification is carried out, the amplified DNA is sent into a magnetizer to react for 1-5min, and the reaction speed is 100-;step four: then, the single-stranded linker method was performed to construct DNA.
- 2. The method for constructing the DNA next-generation sequencing library according to claim 1, wherein the nutrient is prepared by the following steps: stirring 1-5 parts of magnesium chloride, 1-5 parts of sodium chloride and 1-2 parts of potassium chloride at the rotating speed of 100-500r/min for 10-20 min; then stirring the mixture for 15 to 25min at the rotating speed of 500-1000r/min to obtain the nutrient.
- 3. The method for constructing the DNA next-generation sequencing library according to claim 2, wherein the mass fraction of the chitosan solution is 10-20%.
- 4. The method for constructing a DNA next-generation sequencing library according to claim 3, wherein the mass fraction of the chitosan solution is 15%.
- 5. The method for constructing the DNA next-generation sequencing library according to claim 1, wherein the magnetizing agent is ferroferric oxide and sodium chloride which are mixed according to a weight ratio of 3:1, and then magnetization treatment is carried out in a magnetizing tank.
- 6. The method for constructing the DNA next-generation sequencing library according to claim 5, wherein the magnetization intensity is 1-5Bt, and the magnetization time is 10-20 min.
- 7. The method for constructing the DNA next-generation sequencing library according to claim 6, wherein the magnetization intensity is 3Bt, and the magnetization time is 15 min.
- 8. A sequencing library constructed by the method of constructing a secondary DNA sequencing library according to claim 7.
- 9. A kit for constructing the second generation DNA sequencing library of any of claims 1 to 8.
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| US20070254294A1 (en) * | 2004-09-07 | 2007-11-01 | Goodgene Inc. | Method for Storing Dna by Using Chitosan, and Products Using the Methods |
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| CN112251821A (en) * | 2020-10-20 | 2021-01-22 | 南京实践医学检验有限公司 | Kit for quickly and efficiently constructing second-generation sequencing library |
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2022
- 2022-02-24 CN CN202210175829.XA patent/CN114736950A/en active Pending
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| US20070254294A1 (en) * | 2004-09-07 | 2007-11-01 | Goodgene Inc. | Method for Storing Dna by Using Chitosan, and Products Using the Methods |
| US20160257984A1 (en) * | 2015-01-12 | 2016-09-08 | 10X Genomics, Inc. | Processes and Systems for Preparation of Nucleic Acid Sequencing Libraries and Libraries Prepared Using Same |
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