CN108411374A - A kind of efficient denier TE banking process based on Wafergen Smartchip systems - Google Patents
A kind of efficient denier TE banking process based on Wafergen Smartchip systems Download PDFInfo
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- CN108411374A CN108411374A CN201810066751.1A CN201810066751A CN108411374A CN 108411374 A CN108411374 A CN 108411374A CN 201810066751 A CN201810066751 A CN 201810066751A CN 108411374 A CN108411374 A CN 108411374A
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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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- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The invention discloses a kind of efficient denier TE banking process based on Wafergen Smartchip systems, concrete operation step is as follows：Step 1：MSND liquid separation equipment has been debugged, and has carried software using MSND liquid separation equipment and liquid separation program is set；Step 2：Reaction reagent is configured according to demand；Step 3：The correspondence enzymatic reagent mixed and sample DNA are added into 384 plates；Step 4：Chip is placed into MSND, 384 orifice plates configured with sample and enzymatic reagent are placed into MSND liquid separation equipment, executes liquid separation program, sample in 384 orifice plates is sprayed into chip；Step 5：The chip for having sprayed into sample reagent reaction solution is placed into SmartChipTMIn TE PCR Cycler, the program of PCR (PCR) is executed；Step 6：After the completion of PCR (PCR).The present invention can greatly save reagent cost, improve reaction flux, simultaneously, moreover it is possible to reduce error or failure caused by artificial problem.
Description
Technical field
The present invention relates to Target Enrichment (TE) technical fields, more particularly to a kind of to be based on Wafergen
The efficient denier TE banking process of Smartchip systems.
Background technology
Target area is enriched with database technology can be to realize two generation PCR (polymerase chain reactions of batch samples in the short time
Answer) library construction.Its principle is the primer sequence amplification target area for designing one section for specific site, then uses to carry and survey
The universal primer of sequence joint sequence carries out secondary amplification to extension increasing sequence, by secondary amplification is sequenced to required connector sequence two generations
Row are added to target area, to complete the purpose of library construction.At present the technology can apply to 16S 18S ITS etc. be based on
In the two generation library constructions of PCR (PCR) technology.But normal PCR (PCR) region is enriched with skill
Amount of reagent generally is 10ul or more needed for the reaction of each of art, and dosage is all artificially to control, and is easy to cause mistake in this way
Difference or failure, increase the cost of reagent, while decreasing reaction flux.
Therefore, it invents and a kind of is solved based on the efficient denier TE banking process of Wafergen Smartchip systems
It is necessary to state problem.
Invention content
The purpose of the present invention is to provide a kind of efficient denier TE based on Wafergen Smartchip systems to build library
Method, to solve the problems mentioned in the above background technology.
To achieve the above object, the present invention provides the following technical solutions：It is a kind of based on WafergenSmartchip systems
Efficient denier TE banking process, concrete operation step are as follows：
Step 1：MSND (WaferGen Biosystems SmartChip Multisample are debugged
Nanodispenser) liquid separation equipment, and utilize MSND (WaferGen Biosystems SmartChip Multisample
Nanodispenser) liquid separation equipment carries software setting liquid separation program；
Step 2：Reaction reagent is configured according to demand；
Step 3：The correspondence enzymatic reagent mixed and sample DNA are added into 384 plates；
Step 4：Chip is placed into MSND (WaferGen Biosystems SmartChip Multisample
Nanodispenser) in liquid separation equipment, 384 orifice plates configured with sample and enzymatic reagent are placed into MSND (WaferGen
Biosystems SmartChip Multisample Nanodispenser) in liquid separation equipment, liquid separation program is executed, by 384
Sample sprays into chip in orifice plate；
Step 5：The chip for having sprayed into sample reagent reaction solution is placed into SmartChipTMIn TE PCR Cycler,
Execute the program of PCR (PCR)；
Step 6：After the completion of PCR (PCR), reaction solution in chip is collected into using centrifugation retracting device
In clean centrifuge tube；
Step 7：Using the product of PCR (PCR) in magnetic beads for purifying centrifuge tube, can make after Quality Control qualification
It is sequenced for machine on two Valsartan libraries
Preferably, the reaction reagent in the step 2 includes enzymatic reagent and primer.
Preferably, 10~20ul enzymatic reagents, 5~15ul sample DNAs is added in each hole in the step 3 on 384 plates.
The technique effect and advantage of the present invention：
1, each amount of reagent needed for reaction generally is 10ul or more to normal PCR region beneficiation technologies, and Wafergen
Each reaction reagent dosage can be reduced to 100nl by Smartchip systems, and reagent cost is compared to tradition needed for each reaction
Method can save 90%, and reagent cost is greatly saved；
2, reaction reagent is added in MSND liquid separation equipment, and carries the liquid separation of software setting using MSND liquid separation equipment
Program executes liquid separation operation, the accuracy of reaction reagent input amount is substantially increased, to reduce error caused by artificial problem
Or failure；
3, using the product of PCR (PCR) in magnetic beads for purifying centrifuge tube, two be can be used as after Quality Control qualification
Machine is sequenced on Valsartan library, substantially increases reaction flux.
Specific implementation mode
Below in conjunction with the embodiment of the present invention, technical solution in the embodiment of the present invention is clearly and completely retouched
It states, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the present invention
In embodiment, every other implementation obtained by those of ordinary skill in the art without making creative efforts
Example, shall fall within the protection scope of the present invention.
Embodiment 1：
A kind of efficient denier TE banking process based on Wafergen Smartchip systems, concrete operation step is such as
Under：
Step 1：MSND (WaferGen Biosystems SmartChip Multisample are debugged
Nanodispenser) liquid separation equipment, and utilize MSND (WaferGen Biosystems SmartChip Multisample
Nanodispenser) liquid separation equipment carries software setting liquid separation program；
Step 2：Reaction reagent, including enzymatic reagent and primer are configured according to demand；
Step 3：The correspondence enzymatic reagent mixed and sample DNA are added into 384 plates, 10ul enzymatic reagents are added per hole,
5ul sample DNAs；
Step 4：Chip is placed into MSND (WaferGen Biosystems SmartChip Multisample
Nanodispenser) in liquid separation equipment, 384 orifice plates configured with sample and enzymatic reagent are placed into MSND (WaferGen
Biosystems SmartChip Multisample Nanodispenser) in liquid separation equipment, liquid separation program is executed, by 384
Sample sprays into chip in orifice plate；
Step 5：The chip for having sprayed into sample reagent reaction solution is placed into SmartChipTMIn TE PCR Cycler,
Execute the program of PCR (PCR)；
Step 6：After the completion of PCR (PCR), reaction solution in chip is collected into using centrifugation retracting device
In clean centrifuge tube；
Step 7：Using the product of PCR (PCR) in magnetic beads for purifying centrifuge tube, can make after Quality Control qualification
It is sequenced for machine on two Valsartan libraries.
Embodiment 2：
A kind of efficient denier TE banking process based on Wafergen Smartchip systems, concrete operation step is such as
Under：
Step 1：MSND (WaferGen Biosystems SmartChip Multisample are debugged
Nanodispenser) liquid separation equipment, and utilize MSND (WaferGen Biosystems SmartChip Multisample
Nanodispenser) liquid separation equipment carries software setting liquid separation program；
Step 2：Reaction reagent, including enzymatic reagent and primer are configured according to demand；
Step 3：The correspondence enzymatic reagent mixed and sample DNA are added into 384 plates, 15ul enzymatic reagents are added per hole,
10ul sample DNAs；
Step 4：Chip is placed into MSND (WaferGen Biosystems SmartChip Multisample
Nanodispenser) in liquid separation equipment, 384 orifice plates configured with sample and enzymatic reagent are placed into MSND (WaferGen
Biosystems SmartChip Multisample Nanodispenser) in liquid separation equipment, liquid separation program is executed, by 384
Sample sprays into chip in orifice plate；
Step 5：The chip for having sprayed into sample reagent reaction solution is placed into SmartChipTMIn TE PCR Cycler,
Execute the program of PCR (PCR)；
Step 6：After the completion of PCR (PCR), reaction solution in chip is collected into using centrifugation retracting device
In clean centrifuge tube；
Step 7：Using the product of PCR (PCR) in magnetic beads for purifying centrifuge tube, can make after Quality Control qualification
It is sequenced for machine on two Valsartan libraries.
Embodiment 3：
A kind of efficient denier TE banking process based on Wafergen Smartchip systems, concrete operation step is such as
Under：
Step 1：MSND (WaferGen Biosystems SmartChip Multisample are debugged
Nanodispenser) liquid separation equipment, and utilize MSND (WaferGen Biosystems SmartChip Multisample
Nanodispenser) liquid separation equipment carries software setting liquid separation program；
Step 2：Reaction reagent, including enzymatic reagent and primer are configured according to demand；
Step 3：The correspondence enzymatic reagent mixed and sample DNA are added into 384 plates, 20ul enzymatic reagents are added per hole,
15ul sample DNAs；
Step 4：Chip is placed into MSND (WaferGen Biosystems SmartChip Multisample
Nanodispenser) in liquid separation equipment, 384 orifice plates configured with sample and enzymatic reagent are placed into MSND (WaferGen
Biosystems SmartChip Multisample Nanodispenser) in liquid separation equipment, liquid separation program is executed, by 384
Sample sprays into chip in orifice plate；
Step 5：The chip for having sprayed into sample reagent reaction solution is placed into SmartChipTMIn TE PCR Cycler,
Execute the program of PCR (PCR)；
Step 6：After the completion of PCR (PCR), reaction solution in chip is collected into using centrifugation retracting device
In clean centrifuge tube；
Step 7：Using the product of PCR (PCR) in magnetic beads for purifying centrifuge tube, can make after Quality Control qualification
It is sequenced for machine on two Valsartan libraries.
By the comparison of different embodiments as can be seen that when the enzymatic reagent amount being added in each hole on 384 orifice plates is
For 15ul when sample DNA amount is 10ul, reacts flux highest, and reagent cost is compared to conventional method needed for each reaction
And it substantially reduces.
Operation principle of the present invention：First, it designs one section of primer sequence for specific site and expands target area, then,
Secondary amplification is carried out to extension increasing sequence with the universal primer with sequence measuring joints sequence, finally, is expanded two generations by secondary
Joint sequence needed for sequencing is added to target area, to complete the purpose of library construction.
Finally it should be noted that：The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
With technical scheme described in the above embodiments is modified or equivalent replacement of some of the technical features,
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in the present invention's
Within protection domain.
Claims (3)
1. a kind of efficient denier TE banking process based on Wafergen Smartchip systems, which is characterized in that it is specific
Operating procedure is as follows：
Step 1：MSND liquid separation equipment has been debugged, and has carried software using MSND liquid separation equipment and liquid separation program is set；
Step 2：Reaction reagent is configured according to demand；
Step 3：The correspondence enzymatic reagent mixed and sample DNA are added into 384 plates；
Step 4：Chip is placed into MSND, 384 orifice plates configured with sample and enzymatic reagent are placed into MSND liquid separation equipment
On, liquid separation program is executed, sample in 384 orifice plates is sprayed into chip；
Step 5：The chip for having sprayed into sample reagent reaction solution is placed into SmartChipTMIn TE PCR Cycler, execute
The program of PCR (PCR)；
Step 6：After the completion of PCR (PCR), reaction solution in chip is collected into cleaning using centrifugation retracting device
In centrifuge tube；
Step 7：Using the product of PCR (PCR) in magnetic beads for purifying centrifuge tube, two are can be used as after Quality Control qualification
Machine is sequenced on Valsartan library.
2. a kind of efficient denier TE based on Wafergen Smartchip systems according to claim 1 builds library side
Method, it is characterised in that：Reaction reagent in the step 2 includes enzymatic reagent and primer.
3. a kind of efficient denier TE based on Wafergen Smartchip systems according to claim 1 builds library side
Method, it is characterised in that：10~20ul enzymatic reagents, 5~15ul sample DNAs is added in each hole in the step 3 on 384 plates.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810066751.1A CN108411374A (en) | 2018-01-24 | 2018-01-24 | A kind of efficient denier TE banking process based on Wafergen Smartchip systems |
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| CN201810066751.1A CN108411374A (en) | 2018-01-24 | 2018-01-24 | A kind of efficient denier TE banking process based on Wafergen Smartchip systems |
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| CN108411374A true CN108411374A (en) | 2018-08-17 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114181996A (en) * | 2021-12-14 | 2022-03-15 | 中国科学院城市环境研究所 | Technical method for high-throughput construction of 16S rDNA full-length library based on Pacbio sequencing |
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| WO2017084023A1 (en) * | 2015-11-17 | 2017-05-26 | 深圳华大基因研究院 | Library creating method for single-cell transcriptome profile having high throughput |
| CN106834275A (en) * | 2017-02-22 | 2017-06-13 | 天津诺禾医学检验所有限公司 | The analysis method of the construction method, kit and library detection data in ctDNA ultralow frequency abrupt climatic changes library |
| CN107250447A (en) * | 2015-04-20 | 2017-10-13 | 深圳华大基因研究院 | A kind of DNA long fragment library constructing method |
| CN107400705A (en) * | 2016-05-20 | 2017-11-28 | 深圳华大基因研究院 | A kind of high-throughout unicellular whole genome amplification method |
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2018
- 2018-01-24 CN CN201810066751.1A patent/CN108411374A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107250447A (en) * | 2015-04-20 | 2017-10-13 | 深圳华大基因研究院 | A kind of DNA long fragment library constructing method |
| WO2017084023A1 (en) * | 2015-11-17 | 2017-05-26 | 深圳华大基因研究院 | Library creating method for single-cell transcriptome profile having high throughput |
| CN107400705A (en) * | 2016-05-20 | 2017-11-28 | 深圳华大基因研究院 | A kind of high-throughout unicellular whole genome amplification method |
| CN106834275A (en) * | 2017-02-22 | 2017-06-13 | 天津诺禾医学检验所有限公司 | The analysis method of the construction method, kit and library detection data in ctDNA ultralow frequency abrupt climatic changes library |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114181996A (en) * | 2021-12-14 | 2022-03-15 | 中国科学院城市环境研究所 | Technical method for high-throughput construction of 16S rDNA full-length library based on Pacbio sequencing |
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Application publication date: 20180817 |
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