CN106282179A - A kind of multiple PCR primer and method building Mus TCRA library based on high-flux sequence - Google Patents
A kind of multiple PCR primer and method building Mus TCRA library based on high-flux sequence Download PDFInfo
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- CN106282179A CN106282179A CN201610821985.3A CN201610821985A CN106282179A CN 106282179 A CN106282179 A CN 106282179A CN 201610821985 A CN201610821985 A CN 201610821985A CN 106282179 A CN106282179 A CN 106282179A
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- tcra
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- pcr primer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1068—Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
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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
Abstract
The invention discloses a set of multiple PCR primer based on high throughput sequencing technologies and method thereof, Mus T lymphocyte populations TCRA gene mRNA is expanded by the method, amplicon library checks order to produce the immunne response spectrum in φt cell receptor storehouse, understands immune state and disclose pathogenesis.It is specially the amplification T lymphocyte TCR A gene C DR3 region covered for masterplate height with mRNA, obtains the combined information of VDJC fragment simultaneously.The primer building TCRA gene amplification sublibrary includes: forward primer one group totally 39, covers 23 V fragments by one_to_one corresponding mode；Reverse primer one group totally 32, is composed in series by sequence measuring joints, key sequence, barcode coding and TRAC specificity reverse complementary sequence, and wherein barcode sequence is divided into 32 kinds, and can check order in once sequencing is tested 32 samples.By this kind of method, can effectively expand 98 TRAV fragments having function, TRAJ fragment realizes zero deflection amplification simultaneously, basis and clinical research for immunohistochemistry provide help.
Description
Technical field
The invention belongs to biological technical field, specifically, relate to one and utilize multiple PCR method amplification Mus T lymph thin
The primer sets in born of the same parents' TCRA gene C DR3 region, builds the method in TCRA amplicon library and determines the life of TCRA immune group planting modes on sink characteristic
Thing Informatics Method.
Background technology
Having benefited from fast development and the extensively application of high throughput sequencing technologies of future generation, DNA and RNA order-checking platform is at gene
The various aspects that group is learned play prominent impetus.Meanwhile, this emerging technical field be applied to T cell and
The immune group storehouse order-checking of B-cell receptor.In acquired immunity, T cell and B cell pass through φt cell receptor and the B cell on surface
The identification antigenic peptides of receptor-specific-MHC (pMHC) and then startup immune system activation.By surveying
The CDR3 immune group storehouse of sequence lymphocyte receptor, determines the composition of acceptor molecule in T, bone-marrow-derived lymphocyte, can be in order to evaluate immunity
The characteristic parameters such as the multiformity in group storehouse, and study response generating process and the mechanism thereof of acquired immune system further.T
Cell receptor (TCR) is made up of two peptide chains of alpha and beta, respectively by TCRA and TCRB gene code, and the embryo of TCRA gene
It is that gene is arranged in order by multiple open reading frame and forms, can be divided into TRAV, TRAJ, TRAC tri-pack section, wherein, TRAV district
About 98 kinds of fragments are contained in territory, and TRAJ contains in region 60 kinds of fragments, and TRAC region then only has a kind of fragment.In T lymphocyte development mistake
Cheng Zhong, three pack sections realize the random restructuring between different fragments by Somatic Rearrangement, produce ripe TCRA molecule, this mistake
Journey is inserted along with TRAV and TRAJ interregional non-template base or disappearance, thus the multiformity for TCR provides molecular genetic
Basis.During TCR identifies with antigenic peptides-MHC (pMHC), CDR1 and CDR2 is main and many
The MHC fragment combination that sample is relatively low, the antigenic peptides combination that the complementary determining region 3 (CDR3) of TCRA is then main and different,
Its multiformity is the abundantest.TCRA gene C DR3 region overlay TRBV-Junction-TRBJ this multiformity derived region,
Its sequence composition is carried out sequencing analysis and can well reflect composition and the response situation in TCR immune group storehouse.
Research method for T lymphocyte TCR A gene C DR3 region still haves much room for improvement and simplifies at present.
Summary of the invention
For solving above technical problem, an object of the present invention is to provide based on high-flux sequence structure Mus TCRA literary composition
The multiple PCR primer in storehouse.
The two of the object of the invention are to provide a kind of method utilizing described multiple PCR primer to build TCRA library.
A kind of multiple PCR primer building Mus TCRA library based on high-flux sequence, it is characterised in that: described multiplex PCR
Primer is SED ID NO.1-SED ID NO.79.
A kind of multiple PCR primer builds the method in Mus TCRA library, carries out as follows:
First extract normal mucosa tissues, stomach organization or peripheral blood total serum IgE, then reversion synthesis cDNA, with SEQ ID
Sequence shown in NO.72 be reverse transcription primer synthesize cDNA, then with synthesis cDNA as template, SEQ ID NO.1-SEQ ID
Sequence shown in NO.72 is that primer carries out multiplex PCR, reclaims PCR primer, PCR primer is carried out microemulsion and drips PCR, then carry out PGM
Order-checking, obtains Mus TCRA library.
The amplification condition of above-mentioned multiplex PCR is: 95 DEG C of denaturations 10min, 95 DEG C of degeneration 30s, 59 DEG C of annealing 90s, 72 DEG C
Extend 90s, circulate 35 times；Last 72 DEG C extend 10min.
Beneficial effect:
The invention discloses the multiple PCR primer building Mus TCRA library, this primer can expand TCRA gene C DR3 district
Territory multiformity, builds Mus TCRA library, and the present invention also constructs the method for Mus TCRA gene C DR3 district sequencing library, it is achieved T is thin
The stable detection in born of the same parents' recipient immune group storehouse, to further appreciating that immune group storehouse Changing Pattern, announcement disease incidence mechanism have important
Meaning.
Detailed description of the invention
Embodiment:
Below in conjunction with shown sequence, the preferred embodiments of the present invention are described in detail.In embodiment unreceipted
The experimental technique of actual conditions, generally according to normal condition, such as Molecular Cloning: A Laboratory guide (third edition, J. Pehanorm Brooker
Deng writing) described in condition, or according to the condition proposed by manufacturer of manufacturer.
1, total serum IgE in rat tissue is extracted
In rat tissue, the extracting method of total serum IgE, comprises the steps:
A. taking Mus pancreatic tissue and peripheral blood adds piping and druming after Trizol, room temperature stands 5min, extracting directly RNA or puts
Enter-80 DEG C of refrigerators frozen；
B. adding 200ul chloroform by every milliliter of Trizol, reverse mixing, room temperature places 3Min, then in 4 DEG C, 12000g
Centrifugal 15Min.
C. drawing upper strata aqueous phase, add 0.5ml isopropanol, reverse mixing by every milliliter of Trizol, room temperature places 10Min.
In 4 DEG C, 12000g be centrifuged 10Min.
D. precipitation adds 1ml 75% ethanol, gentle concussion by every milliliter of Trizol, and suspend precipitation, then in 4 DEG C,
8000g is centrifuged 5Min.Suck supernatant.
E. room temperature dries 5-10Min, with the RNA of water dissolution without RNase of suitable volumes.
The RNA extracted utilize Biotek EPOCH measure concentration and quality.Testing result shows, OD260/280 exists
1.8-2.0 left and right.
The RNA extracted is utilized agarose gel gel electrophoresis test strip integrity.Testing result shows, 28S and 18S
Band is obvious, and 5S band is inconspicuous, and 28S/18S is about 2.0.
Above-mentioned testing result shows that the RNA mass that step 1 is extracted meets requirement for construction data base, it is possible to continue storehouse after being used for.
2, reverse transcription and multiplex PCR
Total serum IgE sample step 1 obtained carries out reverse transcription respectively, and reverse transcription uses ReverAid First Strand
CDNA Synthesis kit, concrete operations are carried out according to test kit description, and reaction system is: total serum IgE 0.1-5ug, concentration
For 20pmol reverse transcription primer (SEQ ID NO.72) 1ul, add DEPC water and be settled to 12ul, then by mixed liquor in PCR instrument
In 65 DEG C reaction 5min, reaction terminate after put into rapidly ice bath 5min on ice, add 5x reaction buffer 4ul,
RibolockTM inhibitor 1ul, 1mM dNTP Mix 2ul, revertaidTMM-MLV reverse transcription 200u/ul 1ul, mixing
Rear centrifugal, then react 1h at 42 DEG C, obtain the first chain cDNA.
According to t lymphocyte receptor Alpha chain (TCRA gene) Somatic Rearrangement, the rule of non-template radom insertion disappearance
Rule, designs multiple PCR primer, for convenience of sequencing reaction, adds sequence measuring joints sequence in the upstream of forward primer and reverse primer,
Concrete primer is as shown in sequence table:
Then with obtain the first chain cDNA as template, with sequence shown in SEQ ID NO.1-SEQ ID NO.71 for drawing
Thing, carries out PCR amplification, and the reaction system of PCR amplification is as follows: multiplex-PCR premixed liquid 25ul, forward primer 5ul, reversely
Primer 5ul, cDNA 5ul, water 10ul, altogether 50ul；PCR amplification condition is: 95 DEG C of denaturations 10min, 95 DEG C of degeneration 30s, 59
DEG C annealing 90s, 72 DEG C extend 90s, circulate 35 times；Last 72 DEG C extend 10min.The pcr amplification product quality obtained is divided
Number is the agarose gel (low melting-point agarose gel) of 3%, electrophoresis 3h under 50V voltage, will contain under ultraviolet after electrophoresis again
The gel of purpose band cuts, and puts in 1.5ml EP pipe, is subsequently adding 1ml QG Stablization buffer, at 45 DEG C
Under the conditions of water-bath 5-10min until blob of viscose is completely dissolved, water-bath 1-1min；Waste liquid in collecting pipe is outwelled, by adsorption column after Li Xin
Being placed in collecting pipe, add 300ul QG Stablization buffer to adsorption column, 18000g is centrifuged 1min, outwells collection
Waste liquid in pipe, is placed on adsorption column in collecting pipe, and 18000g is centrifuged 2min, then adsorption column is placed on new 1.5ml EP pipe
In, with hair-dryer, the ethanol remained on adsorption column is thoroughly dried up, in adsorption column, add the ultra-pure water of 60 DEG C of preheatings, stand
2min, last 18000g are centrifuged 2min, collect this eluent, obtain building storehouse sample.
In order to determine that building storehouse sample quality meets the requirements, to build storehouse sample mass fraction be 1.5% agarose gel,
Electrophoresis 30min under 100V voltage, then by gel imaging testing goal band.Above-mentioned testing result shows that obtain builds storehouse sample
This meets builds storehouse demand, it is possible to check order for next step.
3, em-PCR (microemulsion drips PCR) and PGM order-checking
Utilize what Qubit measured the different samples that obtain to build storehouse concentration of specimens, then mix by equal amount.Sample Dilution
Method is, it is assumed that the concentration that Qubit records is N ng/ul, and the amplified library a length of M kb of son, then the extension rate in library is N*
1.515*1000/26*M。
Then with the library after dilution as template, carry out emPCR, emPCR and use Ion OneTouchTM sequencing system to carry
For reagent, operating procedure is carried out according to test kit description, then carries out PGM order-checking, and sequencing result is the TCRA library of Mus.
Then sequencing result is carried out statistical analysis.Result shows, the Mus TCRA library utilizing the method for the present invention to build can cover
The diversity information of TCRA gene.
Finally illustrate, preferred embodiment above only in order to the bright technical scheme of we to be described and unrestricted, although logical
Cross above preferred embodiment the present invention to be described in detail, but art technology Mus person should be appreciated that can be
In form and it is made various change, without departing from the scope of the claims in the present invention in details.
Claims (3)
1. the multiple PCR primer building Mus TCRA library based on high-flux sequence, it is characterised in that: described multiplex PCR draws
Thing is SED ID NO.1-SED ID NO.79.
2. the method that multiple PCR primer as claimed in claim 1 builds Mus TCRA library, is carried out as follows:
First extract normal mucosa tissues, stomach organization or peripheral blood total serum IgE, then reversion synthesis cDNA, with SEQ ID
Sequence shown in NO.72 be reverse transcription primer synthesize cDNA, then with synthesis cDNA as template, SEQ ID NO.1-SEQ ID
Sequence shown in NO.72 is that primer carries out multiplex PCR, reclaims PCR primer, PCR primer is carried out microemulsion and drips PCR, then carry out PGM
Order-checking, obtains Mus TCRA library.
Weigh the method that PCR primer builds Mus TCRA library the most according to claim 2, it is characterised in that: described multiplex PCR
Amplification condition is: 95 DEG C of denaturations 10min, 95 DEG C of degeneration 30s, and 59 DEG C of annealing 90s, 72 DEG C extend 90s, circulate 35 times；Finally
72 DEG C extend 10min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111575343A (en) * | 2019-02-18 | 2020-08-25 | 北京全谱医学检验实验室有限公司 | Construction method and kit of immune repertoire sequencing library |
Citations (4)
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| WO2013188831A1 (en) * | 2012-06-15 | 2013-12-19 | Adaptive Biotechnologies Corporation | Uniquely tagged rearranged adaptive immune receptor genes in a complex gene set |
| CN104531713A (en) * | 2015-01-20 | 2015-04-22 | 中国人民解放军第三军医大学 | Multiple PCR primers and method for constructing human TCBR library based on high-throughput sequencing |
| CN104531698A (en) * | 2015-01-20 | 2015-04-22 | 中国人民解放军第三军医大学 | Multi-PCR (Polymerase Chain Reaction) primer and method for constructing mouse TCRB (T-Cell Receptor Beta) library based on high-throughput sequencing |
| CN104560979A (en) * | 2015-01-20 | 2015-04-29 | 中国人民解放军第三军医大学 | Multiplex-polymerase chain reaction (PCR) primer and method for constructing mouse B cell receptor (BCR) heavy-chain library based on high-throughput sequencing |
-
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- 2016-09-13 CN CN201610821985.3A patent/CN106282179A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013188831A1 (en) * | 2012-06-15 | 2013-12-19 | Adaptive Biotechnologies Corporation | Uniquely tagged rearranged adaptive immune receptor genes in a complex gene set |
| CN104531713A (en) * | 2015-01-20 | 2015-04-22 | 中国人民解放军第三军医大学 | Multiple PCR primers and method for constructing human TCBR library based on high-throughput sequencing |
| CN104531698A (en) * | 2015-01-20 | 2015-04-22 | 中国人民解放军第三军医大学 | Multi-PCR (Polymerase Chain Reaction) primer and method for constructing mouse TCRB (T-Cell Receptor Beta) library based on high-throughput sequencing |
| CN104560979A (en) * | 2015-01-20 | 2015-04-29 | 中国人民解放军第三军医大学 | Multiplex-polymerase chain reaction (PCR) primer and method for constructing mouse B cell receptor (BCR) heavy-chain library based on high-throughput sequencing |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111575343A (en) * | 2019-02-18 | 2020-08-25 | 北京全谱医学检验实验室有限公司 | Construction method and kit of immune repertoire sequencing library |
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Effective date of registration: 20180919 Address after: 400000 5, 10-2, Chui Hui Road, Chunhui Road, Dadukou District, Chongqing, 5 Applicant after: Chongqing kozia Biotechnology Co. Ltd. Address before: Room 332, Building 1, No. 1, No. 1, No. 8, Life Park Road, Zhongguancun Life Science Park, Huilongba Town, Changping District, Beijing Applicant before: Beijing kozia Biotechnology Co. Ltd. |
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