CN106591293A - Method for separating known-sequence flanking sequences from unknown genomes based on enzyme cutting and connection - Google Patents
Method for separating known-sequence flanking sequences from unknown genomes based on enzyme cutting and connection Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
Abstract
The invention provides a method for separating known-sequence flanking sequences from unknown genomes based on enzyme cutting and connection. Synthesized linker primers can be complemented and have the cohesive ends the same as the cohesive ends of some enzyme cutting sites, and linkers for the corresponding enzyme cutting sites are formed through a denaturation annealing reaction. In addition, the invention discloses a method for constructing a target-gene flanking sequence library in the mode of conducting connection while enzyme cutting. The flanking sequences of target genes are accurately separated through 2-3 times of nested PCR, and the problems that when genomes for reference do not exist, obtaining of the information of the genomes of animals and plants is difficult and consumes time, and accuracy is poor can be solved; meanwhile, the method does not have the selectivity of species of to-be-separated genes, preparing of core-technology link linkers has multiple changing modes, the false positive result can be rapidly and simply identified with the electrophoresis method and the like, the sequence information of target genes can be efficiently, rapidly and cheaply separated from the unknown genomes.
Description
Technical field
The present invention relates to plant genetic engineering field, especially according to one section of known sequence point from unknown gene group
From the method for its flanking sequence.
Background technology
With the development of modern biotechnology, animals and plants breeding research enters molecular breeding via traditional experience selection-breeding
Epoch, molecular marker is widely used in spore analysis, and Variety fingerprinting builds and variation analyses and molecular breeding etc.
Many aspects, but either traditional SSR marker functional molecular marker or the height for doing GWAS analyses
Density SNP marker is required for the sequence information of correspondence species.
Earliest or sequence is that same Sanger methods are obtained, and high cost is sequenced, and the time is long, and the secondary sequencing after technological innovation
Technology has high flux, high accuracy, the advantage of low cost, and for animals and plants molecular breeding and genomics research new grinding is provided
Study carefully method and solution.And be successfully established using polymerase synthetic technology Illumina and 454 platforms and
Using the SOLiD platforms of ligase synthesis sequencing technologies.But no matter using which kind of microarray dataset, its relative cost is higher, and
Analysis needs to use professional very strong computer software such as SHRiMP, MAQ, BOWTIE etc., higher to technical requirements, needs the time
It is very long, also only it is widely used in large animals and plants research at present.
But for little ancestor crops, genome sequence determination is carried out without enough funds, can be believed with known sequence
Breath is less, and is concentrated mainly on gene coding region.In research and production, we can obtain some by methods such as homologous clones
The encoding gene of important character, but gene coding region encoding function albumen, conservative is higher, relative to the gene of coding region flank
Between area, its pleomorphism site is less, is unfavorable for the exploitation of molecular marker；Simultaneously gene transcription start site on have sequence often
It is the promoter region of gene, the research to gene function also has great importance.In order to obtain certain or some genes
Flanking sequence and carry out gene order-checking or sequence of resurveying, time and Financial cost are higher.
Conventional research based on degenerate primer it has been reported that enter the TAIL-PCR technologies of performing PCR amplification, it is in complicated base
Targeting locating effect is poor because in, and false positive is higher, and isolated fragment length is shorter.Therefore a kind of targeting precision is developed higher
The method of inexpensive Flanking sequence isolation be even more important in little ancestor's crop investigations.
The content of the invention
A kind of method that known array flanking sequence is separated from unknown gene group based on enzyme action connection is provided, its targeting is determined
Position effect is good, accuracy is high, and detached fragment length is preferable.
What the present invention was realized in：The side of known array flanking sequence is separated from unknown gene group based on enzyme action connection
Method：Comprise the steps：
1) using base pair complementarity principle synthetic primer, according to the target gene of flanking sequence to be obtained design 3
Bar PCR amplification reverse primers, wherein the binding site of 3 primers all near known 5 ' hold, according to from 5 ' from close to
R1, R2 and R3 are named as successively；One forward primer AF of engineer, it has SEQ ID NO：Nucleotide sequence shown in 1,
According to SEQ ID NO：Nucleotide sequence shown in 1, designs two forward primer sequences, according to from 5 ' close to far naming successively
To be named as AD1 and AD2；AD1 and R1 carries out the amplification of first round PCR, and AD2 and R2 carries out the second wheel PCR amplifications, and AD2 enters with R3
The wheel PCR amplifications of row the 3rd, the 1st wheel PCR primer fragment is most long, and the 3rd wheel is most short；The binding site of the 2nd wheel PCR primer is in the 1st wheel
In PCR primer, but overlap inside the binding site of the 1st wheel primer or with the 1st wheel primer binding site；Equally, the 3rd
To primer binding site in the 2nd wheel PCR primer, but primer bound site is taken turns inside the binding site of the 2nd wheel primer or with the 2nd
Point overlaps, and the 3 wheel PCR primer differs 50-100bp between often taking turns；
2) engineer's primer sequence AER, AHR, ABR, AXR, APR, they have respectively such as SEQ ID NO：2, SEQ
ID NO：3, SEQ ID NO：4, SEQ ID NO：5, SEQ ID NO：Nucleotide sequence shown in 6, by above-mentioned primer respectively with SEQ
ID NO：Primer sequence shown in 1 by degeneration annealing reaction sequentially generate with EcoR I, I/Bgl of BamH II, Hind III, Xho I,
I/EcoR of Sma V have the joint of identical sticky end, and the joint can be complementary with the cohesive end of corresponding digestion products, in ligase
Coupled reaction can be completed under effect, enzyme action position original after the completion of coupled reaction is made by the base outside artificial design cohesive end
Point disappears；
3) using step 2) described in one or more restricted enzyme digest the genome of target species respectively, respectively
Correspondence adds step 2) the ready joint of institute and ligase, in the following enzyme action side connection of 2-16 DEG C of condition, product conduct
Flanking sequence isolation the 1st takes turns the template of PCR；
4) by step 3) in obtain template dilute 5-10 times carry out the 1st wheel PCR amplification, with the 1st wheel amplification product it is dilute
Release 10-200 times carries out the 2nd wheel amplification for template, and by template of the 2nd 10-100 times of product dilution of wheel the 3rd wheel amplification is carried out, and point
Electrophoresis detection is not carried out, the visible two articles bands for differing 50-100bp between the 2nd wheel and the 3rd wheel；
5) by cutting glue reclaim purpose band, and the flanking sequence information that sequencing analysis obtain genes of interest is carried out.
Joint is prepared and carried out in PCR instrument, is prepared using denaturing gradient method for annealing, and system is as follows：AF 10μmol 10μ
The μ l of 10 μm of ol of l, A*R 10；A*R refers in 5 reverse primers, and mix homogeneously carries out joint and prepares reaction, response procedures
It is as follows：94 DEG C of 3min, 70 DEG C of 10min, 60 DEG C of 10min, 50 DEG C of 10min, 30 DEG C of 10min, 25 DEG C of 10min, terminate.
As a result of above-mentioned technical proposal, compared with prior art, the present invention has following innovative point：
1st, the high difficult problem of little ancestor's crop gene group sequencing analysis fund, time cost is solved.
The difficult problem such as the 2nd, solve targeting difference when TAIL-PCR degeneracys are expanded amplified fragments be shorter.
3rd, treating the species belonging to separation sequence does not have selectivity, as long as being obtained in that the thing of high-quality genomic DNA
Plant and can be carried out test.
4th, the present invention have developed 5 butt joints, and introduce blunt end cloning joint, extreme enrichment restricted enzyme can
Scope is selected, guarantee test result can be completed to greatest extent.
5th, in same reaction system, enzyme action and connection are carried out simultaneously, by designing jointing base sequence, are made
Genome sequence after enzyme action is listed in restriction enzyme site original after connection top connection and disappears, and effectively prevents from again cutting connection top connection
Come.
6th, carry out to judge whether purpose fragment is false positive according to nested PCR product electrophoresis result.
Description of the drawings
Fig. 1 is the schematic flow sheet of the present invention；
Fig. 2 is the joint and its corresponding restriction enzyme site schematic diagram of the design of the present invention；
Underscore part is jointing and the sticky end of corresponding restriction endonuclease complementary pairing, is caused after the completion of coupled reaction
Former joint disappears.
Fig. 3 is that case study on implementation of the present invention separates alfalfa MBF1c gene flanking sequence nested PCR amplification schematic diagrams.
BamHI, EcoRI, HindIII, SmaI, XhoI indicate for enzyme and jointing used in the reaction,
Each group of short-term indicates that the nest-type PRC used in correspondence reaction system reacts, and what A was indicated takes turns PCR primer for the 2nd, and what B was indicated is
Third round PCR primer.Because alfalfa Herba Medicaginiss are polyploid plant, the MBF bases of multiple copies are there may be in its genome
Cause, from electrophoresis result it may be seen that BamHI, HindIII and SmaI this 3 individuality ties up to the MBF flanking sequences of alfalfa
Effect is preferable in separation.
Specific embodiment
This combination is embodied as case
Primer synthesizes and joint preparation：Primer synthesis is in the synthesis of Ying Jun companies, and sequence is as follows：
Adapter-primer：AF 5'- GTAATACGACTCACTATAGGGCACGCGTGGT -3',
AER (EcoRI) 5'- AATTACCACGCGTGCTG -3',
AHR (HindIII) 5'- AGCTACCACGCGTGCTG -3',
ABR (BamHI/BglII) 5'- GATCACCACGCGTGCTG -3',
AXR (XhoI) 5'- TCGAACCACGCGTGCTG -3',
APR (blunt-ended) 5'- ACCACGCGTGCTG -3',
Nest-type PRC primer：AD1 5'- GTAATACGACTCACTATAGGGC -3',
AD2 5'- ACTATAGGGCACGCGTGGT -3',
MBFR1 5'- TAGTTTTAGGGATTAGGAGC -3',
MBFR2 5'- GTTGACGGCTTTCTCATCTT -3',
MBFR3 5'–TTCTTGCGGATAACGACTGG–3'。
Joint is prepared and carried out in PCR instrument, is prepared using denaturing gradient method for annealing, and system is as follows：AF(10μmol)10μ
L, A*R (10 μm of ol) 10 μ l, (A*R refers in 5 reverse primers such as AER, ABR) mix homogeneously carries out joint and prepares instead
Should, response procedures are as follows：94 DEG C of 3min, 70 DEG C of 10min, 60 DEG C of 10min, 50 DEG C of 10min, 30 DEG C of 10min, 25 DEG C
10min, terminates.
Alfalfa extracting genome DNA：Adopt with reference to Molecular Cloning:A Laboratory guide (Sha's nurse Brooker, Huang Peitang is translated)
The method of 2%CTAB extracts alfalfa genomic DNA.
(1) alfalfa WL440 culture 7-14d in fertile soil (bud) are sowed,
(2) 100-500mg fresh leaf tissue samples are taken, with liquid nitrogen grinding,
(3) 1ml 2%CTAB, 65 DEG C of incubation 30-60min are added,
(4) 12000g, 10min, 25 DEG C, take 800-1000 μ l supernatants into new pipe, add 200 μ l chloroforms, 25 DEG C, are hanging down
30min is shaken on straight concussion shaking table,
(5) 10000g, 10min, 25 DEG C, take 700-800 μ l supernatants into new pipe, add the dehydrated alcohol of 2 times of volumes, mix
- 20 DEG C of placement 20min after even,
(6) 12000g, 10min, 25 DEG C, remove supernatant, add the ethanol of 1ml 75%,
(7) 10000g, 5min, 4 DEG C, remove supernatant, are air-dried 15-30min,
(8) 20-30 μ l dd H are added2O 50-60 DEG C dissolve 5min.
Enzyme action coupled reaction：Take EcoR I, BamH I, Hind III, Xho I, Sma I (Fermentas) enzyme action Herba Medicaginiss base respectively
Because of a group DNA, 37 DEG C of 0.5～2h of enzyme action, endonuclease reaction system is as follows：
Every kind of restricted enzyme individually digests a genomic DNA, and the buffering that restriction endonuclease is reacted is noted when using
Liquid correspondence.
Coupled reaction is carried out after endonuclease reaction is complete, 4 DEG C of connection 16h, system is as follows：
Nest-type PRC reacts：To dilute 20～50 times of enzyme action connection product as template, with AD1 and MBFR1 as primer, match somebody with somebody
Put PCR reactant mixtures (giving birth to work 2 × Taq PCR reaction kits in Shanghai)：
Response procedures are：94 DEG C of 3min, 94 DEG C of 30s, 72 DEG C of 3min, 7 circulations, 94 DEG C of 30s, 68 DEG C of 3min, 32
Individual circulation, 72 DEG C of 5min, 25 DEG C of 5min, reaction terminates.
To dilute 50～100 times of first round PCR primer as template, the second wheel nested PCR amplification is carried out, reaction system is such as
Under：
Response procedures are：94 DEG C of 3min, 94 DEG C of 30s, 72 DEG C of 3min, 5 circulations, 94 DEG C of 30s, 68 DEG C of 3min, 25
Individual circulation, 72 DEG C of 5min, 25 DEG C of 5min, reaction terminates.Third round nest-type PRC reaction with second wheel, (template for dilution 50～
100 times of the second wheel PCR primer, primer is AD2 and MBFR3).
Electrophoresis detection nested PCR product：Take the 2nd wheel respectively carries out horizontal gel electrophoresis with the 3rd wheel nested PCR product, takes the 2nd
Wheel and the 3rd wheel have a substantially amplification, and the 3rd wheel product is slightly less than the band of the 2nd wheel and is reclaimed and (give birth to work glue reclaim reagent in Shanghai
Box), as shown in figure 3, (Ying Jun companies) is sequenced by sequence is obtained, the sequence for obtaining is the flank of alfalfa MBF1c
Sequence.
The case study on implementation being not limited to described in specific embodiment of the present invention, those skilled in the art are according to this
Bright technical scheme draws other embodiments, also belongs to the technological innovation scope of the present invention.The technology of obvious this area
Personnel the present invention can be carried out it is various change and modification without departing from the spirit and scope of the present invention.So, if of the invention
These modification and modification belong in the range of the claims in the present invention and its equivalent technologies, then the present invention is also intended to change comprising these
Including dynamic and modification.
SEQUENCE LISTING
Sequence table
<110>Guizhou Province Grass Industry Research Institute
<120>Based on the method that enzyme action connection separates known array flanking sequence from unknown gene group
<130> nm:
<160> 11
<170> PatentIn version
<210> 1
<211> 31
<212> DNA
<213>Artificial sequence
<220>
<223>Forward primer AF is designed with Primer Premier 5.0 react AD1 primers for preparing joint, nest-type PRC
Binding site.
<400> 1
GTAAT ACGAC TCACT ATAGG GCACG CGTGG T 31
<210> 2
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>It is used to prepare the corresponding joint of EcoRI restriction endonucleases with the design reverse primer AER of Primer Premier 5.0.
<400> 2
AATTA CCACG CGTGC TG 17
<210>3
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>It is used to prepare the corresponding joint of HindIII restriction endonucleases with the design reverse primer AHR of Primer Premier 5.0.
<400> 3
AGCTA CCACG CGTGC TG 17
<210> 4
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>With the design reverse primer ABR of Primer Premier 5.0, for preparing, BamHI/BglII restriction endonucleases are corresponding to be connect
Head.
<400> 4
GATCA CCACG CGTGC TG 17
<210> 5
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>It is used to prepare the corresponding joint of XhoI restriction endonucleases with the design reverse primer AXR of Primer Premier 5.0.
<400> 5
TCGAA CCACG CGTGC TG 17
<210> 6
<211> 15
<212> DNA
<213>Artificial sequence
<220>
<223>With the design reverse primer APR of Primer Premier 5.0, for preparing, the restriction endonucleases of SmaI/EcoR V are corresponding to be connect
Head.
<400> 6
TCGAA CCACG CGTGC 15
<210> 7
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>With Primer Premier 5.0 forward primer AD1 is designed for the reaction of first round nest-type PRC.
<400> 7
GTAAT ACGAC TCACT ATAGG GC 22
<210> 8
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>It is used for the second wheel nest-type PRC reaction with the design forward primer AD2 of Primer Premier 5.0.
<400> 8
ACTAT AGGGC ACGCG TGGT 19
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>With Primer Premier 5.0 reverse primer MBFR1 is designed for the reaction of first round nest-type PRC.
<400> 9
TAGTT TTAGG GATTA GGAGC 20
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>It is used for the second wheel nest-type PRC reaction with the design reverse primer MBFR2 of Primer Premier 5.0.
<400> 10
GTTGA CGGCT TTCTC ATCTT 20
<210> 11
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>With Primer Premier 5.0 reverse primer MBFR3 is designed for the reaction of third round nest-type PRC.
<400> 11
TTCTT GCGGA TAACG ACTGG 20
Claims (2)
- It is 1. a kind of to connect the method that known array flanking sequence is separated from unknown gene group based on enzyme action, it is characterised in that：Bag Include following steps：1) using base pair complementarity principle synthetic primer, 3 PCR are designed according to the target gene of flanking sequence to be obtained Amplification reverse primer, wherein the binding site of 3 primers is all held near the 5 ' of known, according to ordering successively from close to from 5 ' Entitled R1, R2 and R3；One forward primer AF of engineer, it has SEQ ID NO：Nucleotide sequence shown in 1, according to SEQ ID NO：Nucleotide sequence shown in 1, designs two forward primer sequences, according to from 5 ' close to being far named as life successively Entitled AD1 and AD2, AD1 have respectively such as SEQ ID NO with AD2：7 and SEQ ID NO：Nucleotide sequence described in 8；AD1 with R1 carries out the amplification of first round PCR, and AD2 and R2 carries out the second wheel PCR amplifications, and AD2 and R3 carries out the 3rd wheel PCR amplifications, the 1st wheel PCR Product fragment is most long, and the 3rd wheel is most short；The binding site of the 2nd wheel PCR primer takes turns primer in the 1st wheel PCR primer the 1st Binding site inside overlaps with the 1st wheel primer binding site；Equally, the 3rd pair of primer binding site takes turns PCR the 2nd On product, but overlap inside the binding site of the 2nd wheel primer or with the 2nd wheel primer binding site, 3 wheel PCR is produced Thing differs 50-100bp between often taking turns；2) engineer's primer sequence AER, AHR, ABR, AXR, APR, they have respectively such as SEQ ID NO：2, SEQ ID NO：3, SEQ ID NO：4, SEQ ID NO：5, SEQ ID NO：Nucleotide sequence shown in 6, by above-mentioned primer respectively with SEQ ID NO：Primer sequence shown in 1 is sequentially generated and EcoR I, I/Bgl of BamH II, Hind III, Xho I, Sma by degeneration annealing reaction I/EcoR V has the joint of identical sticky end, and the joint can be complementary with the cohesive end of corresponding digestion products, makees in ligase Coupled reaction can be completed with, restriction enzyme site original after the completion of coupled reaction is made by the base outside artificial design cohesive end Disappear；3) using step 2) described in one or more restricted enzyme digest the genome of target species respectively, correspond to respectively Add step 2) the ready joint of institute and ligase, in the following enzyme action side connection of 2-16 DEG C of condition, product is used as flank Sequence separates the 1st template for taking turns PCR；4) by step 3) in obtain template dilute 5-10 times carry out the 1st wheel PCR amplification, with the 1st wheel amplification product dilution 10- 200 times carry out the 2nd wheel amplification for template, and with the 2nd wheel product dilution, 10-100 times carries out the 3rd wheel amplification as template, and enters respectively Row electrophoresis detection, the visible two articles bands for differing 50-100bp between the 2nd wheel and the 3rd wheel；5) by cutting glue reclaim purpose band, and the flanking sequence information that sequencing analysis obtain genes of interest is carried out.
- 2. method according to claim 1, it is characterised in that：Joint is prepared and carried out in PCR instrument, is moved back using denaturing gradient Prepared by ignition method, system is as follows：10 μm of ol10 μ l of AF, 10 μm of ol of A*R 10 μ l；A*R refers in 5 reverse primers, mixes Conjunction uniformly carries out joint and prepares reaction, and response procedures are as follows：94 DEG C of 3min, 70 DEG C of 10min, 60 DEG C of 10min, 50 DEG C of 10min, 30 DEG C 10min, 25 DEG C of 10min, terminate.
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Citations (19)
| Publication number | Priority date | Publication date | Assignee | Title |
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