CN106676096A - Whole set of reagents and method for cloning full-length gene - Google Patents
Whole set of reagents and method for cloning full-length gene Download PDFInfo
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- CN106676096A CN106676096A CN201510756147.8A CN201510756147A CN106676096A CN 106676096 A CN106676096 A CN 106676096A CN 201510756147 A CN201510756147 A CN 201510756147A CN 106676096 A CN106676096 A CN 106676096A
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Abstract
The invention discloses a whole set of reagents and a method for cloning a full-length gene. The whole set of reagents for cloning the full-length gene comprises an adaptor A and a universal primer; the adaptor A is an adaptor which comprises a sense strand an antisense strand, the sense strand is a single chain DNA shown in a sequence 1, the antisense strand is a single chain DNA shown in a sequence 2, and the sequence 2 and reverse direction of 22th-37th site of the sequence 1 are complementary; the universal primer is a single chain DNA shown in 1st-26st site of the sequence 1. Experiments prove that the whole set of reagents and the method for cloning the full-length gene can be used for amplification till 5' and 3' terminal of a target gene, that is the full length of the gene. At the same time, the reagents and the method can be used for amplification of multiple target genes at the same time. The whole set of reagents and the method for cloning the full-length gene can be applied to clone of unknown gene, starting of amplification, acquisition of unknown flanking sequences, identification of insertion sites, and the like.
Description
Technical field
The present invention relates to be used for the reagent set and method of Cloning of full length gene in biological technical field.
Background technology
Gene is the general name of the specific nucleotide sequence with hereditary effect on intracellular DNA molecular, is have heredity effect
The DNA molecular fragment answered.Gene Handling protein synthesis, are the Different Individual performances of different plant species and same species
Go out the basic reason of different character.Gene passes to the next generation by DNA replication dna and cell division hereditary information,
And expressed hereditary information by controlling the synthesis of protein.Therefore, research of the people to organism is all concentrated on
For the research of gene function.And the premise for studying the function of gene is must to obtain candidate gene, that is, need advanced
The clone of row candidate gene.
Along with the development of Modern Molecular Biotechnology, the ways and means of many acquisition new genes, such as collection of illustrative plates are occurred in that
Technology, transposon tagging, mRNA differential display techniques, two subtracting techniques and cDNA library triage techniqueses etc.,
But said method has the shortcomings that length experimental period, technical step are loaded down with trivial details and workload is big mostly.CDNA quickly expands end
Increasing technology (rapid amplification of cDNA ends, RACE) is a kind of PCR-based from low-abundance transcription
The effective ways of the ends of 5' and 3 ' of rapid amplifying cDNA in this, the side of the full length gene clone to be widely used at present
Method.
However, in RACE technologies, obtaining 3 ' ends of gene relatively easily, but obtain 5 ' ends of gene just
It is extremely difficult, often there is serious non-specific amplification, increased the work of many colony screenings.In addition, base
Because of the acquisitions at 5 ' ends, the RACE test kits of purchase 5 ' are needed, but 5 ' RACE test kits of different company's research and development
Principle is not quite similar, and price is very expensive.Be badly in need of at present it is a kind of can be in the method for simple and quick clone gene total length.
The content of the invention
The technical problem to be solved is the total length for how cloning genes of interest.
To solve above-mentioned technical problem, present invention firstly provides for the reagent set of Cloning of full length gene.
Reagent set for Cloning of full length gene provided by the present invention, is made up of joint A and universal primer；
The joint that the joint A is made up of positive chain and reverse strand, the positive chain of the joint A is shown in sequence 1
Single stranded DNA, the single stranded DNA being reversely linked as shown in sequence 2 of the joint A, the of sequence 2 and sequence 1
22-37 positions reverse complemental；
Single stranded DNA of the universal primer shown in the 1-26 positions of sequence 1.
Wherein, sequence 1,2,3 and 4 from the direction of the 1st to last 1 be corresponding single stranded DNA from 5 '
Hold to the direction at 3 ' ends.
In above-mentioned reagent set, 5 ' ends of the reverse strand of the joint A can be phosphorylated modification.
In above-mentioned reagent set, the source of the gene can be plant, animal, microorganism or virus.
In above-mentioned reagent set, the joint A and the universal primer can independent packagings.
To solve above-mentioned technical problem, present invention also offers the joint A or described universal primers.
To solve above-mentioned technical problem, present invention also offers for the system of Cloning of full length gene.
System for Cloning of full length gene provided by the present invention, including the reagent set and for Cloning of full length base
Other reagents of cause and/or instrument.
In said system, described other reagents and/or instrument for Cloning of full length gene can be extraction RNA, synthesis
The chains of cDNA first or the second chain, the connection of DNA fragmentation, the DNA fragmentation containing sticky end is carried out end-filling,
To reagent and/or instrument needed for DNA fragmentation plus dATP, DNA purification and/or PCR amplifications.
In said system, the source of the gene is plant, animal, microorganism or virus.
To solve above-mentioned technical problem, present invention also offers the method for clone's genes of interest.
The method of clone's genes of interest provided by the present invention, including following 1) -3)：
1) double-strand cDNA is obtained by template of biological total serum IgE；
2) to step 1) double-strand cDNA carry out end-filling successively, plus dATP and connect the joint A, obtain
Connection product containing joint A；
3) with step 2) connection product as template, enter performing PCR with the special primer and the universal primer of genes of interest
Amplification, obtains genes of interest.
In said method, step 1) may also include purification is carried out to double-strand cDNA.
In said method, step 2) may also include purification is carried out to the connection product containing joint A.
In said method, it is described 1) may include it is following 11) and 12)：
11) reverse transcription is carried out by template of biological total serum IgE, obtains the first chain cDNA；
12) with step 11) the first chain cDNA as templated synthesis the second chain cDNA, obtain double-strand cDNA.
The operational flowchart of said method is as shown in Figure 2.
Said method step 2) in, described end-filling system can use the Quick Blunting kit of NEB companies
Carry out；The system of described plus dATP may include：DATP, plus A buffer, plus the polymerase of dATP, described dATP
Concentration be 5mM, concentration of the polymerase in described reaction system be 0.5U/ μ l；Described joint A
Linked system include：Joint A, ATP and ligase, the concentration of described joint A is 10 μM, described ATP
Concentration be 1.7mM, concentration of the ligase in the linked system be 0.5U/ μ l.
Said method step 3) in, described PCR system includes：Primer, polymerase mixture, and step 2)
In the connection product that obtains.The concentration of described primer is 0.2 μM, and the polymerase mixture is in the PCR bodies
Shared 50% volume in system.
RNA described in said method can be the source of any monoploid, diploid or polyploid biology or non-biological material
RNA, the such as RNA of animal, plant, microorganism or virus.
To solve above-mentioned technical problem, present invention also offers the reagent set, the joint A, described general drawing
The application of thing or the system in gene cloning.
In above-mentioned application, the gene can derive from plant, animal, microorganism or virus.
In the present invention, applicant proposed a kind of new technique of brand-new gene cloning, even if using artificial synthetic linker,
Simultaneously 5 ' and 3 ' RLM-RACEs are carried out, then carry out the new technique of total length amplification.It is demonstrated experimentally that what the present invention was provided
Linkers A, is connectable to the two ends of double stranded cDNA fragment, and the cloning process with the present invention is complete with for cloning
The reagent set of long gene, successfully 5 ' and 3 ' ends of genes of interest are arrived in amplification, and further successfully amplification is arrived
Full length sequence.Meanwhile, the present invention can simultaneously carry out the amplification of multiple genes of interest.The invention provides one kind side
Just, efficient, special gene clone method, obtains in unknown gene clone, the amplification for starting, unknown flanking sequence
Take, the aspect such as the identification of insertion point is respectively provided with wide application prospect.
Description of the drawings
Fig. 1 is the structural representation of joint A.
Fig. 2 is gene cloning schematic flow sheet.
Fig. 3 is the electrophoretogram of the Broussonetia papyrifera blade total serum IgE for extracting.
Fig. 4 is the agarose gel electrophoresiies testing result of 3 ' ends and 5 ' end amplified productions.Wherein, swimming lane M is molecule
Amount standard, swimming lane 1 is the agarose gel electrophoresiies testing result of 5 ' end amplified productions, and swimming lane 2 is that 3 ' end amplifications are produced
The agarose gel electrophoresiies testing result of thing.
Fig. 5 is the agarose gel electrophoresiies testing result of the product that PCR amplifying target genes full-length cDNAs are obtained.Wherein,
Swimming lane M is molecular weight standard.
Specific embodiment
The present invention is further described in detail with reference to specific embodiment, the embodiment for being given only for
The present invention is illustrated, rather than in order to limit the scope of the present invention.
Experimental technique in following embodiments, if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, commercially obtain.
Method described in following embodiments is if no special instructions conventional method, and nucleotide sequence used is by Shanghai
Invitrogen biotech firms synthesize.Agents useful for same is if no special instructions the product of NEB companies, and water used is super
Pure water.
The method for cloning genes of interest with the reagent set for Cloning of full length gene, including following 1) -3)：
1) double-strand cDNA is obtained by template of biological total serum IgE；
2) to step 1) double-strand cDNA carry out end-filling, plus dATP and jointing A successively, contained
The connection product of joint A；
3) with step 2) connection product as template, with the special primer and universal primer of genes of interest enter performing PCR expand
Increase, obtain genes of interest.
Wherein, for the reagent set of Cloning of full length gene, it is made up of joint A and universal primer；
The joint that joint A is made up of positive chain and reverse strand, the positive chain of joint A is the single stranded DNA shown in sequence 1,
The 22-37 positions reverse complemental of the single stranded DNA being reversely linked as shown in sequence 2 of joint A, sequence 2 and sequence 1；
Single stranded DNA of the universal primer shown in the 1-26 positions of sequence 1.
Below by taking the Gene A of Broussonetia papyrifera as an example, the method for clone gene total length is specifically described.
Embodiment 1, the reagent set for Cloning of full length gene clone A
1. the extraction of total serum IgE
Broussonetia papyrifera blade is experiment material used in this experiment, carries out the extraction of total serum IgE.It is solidifying using 1% agarose after extraction
Gel electrophoresis detection, as a result as shown in Figure 3.As a result show, the RNA for being extracted there are two obvious electrophoretic bands, from upper
28S RNA and 18S RNA are followed successively by under, show to obtain that purity is higher, more complete total serum IgE.
The synthesis of the chains of 2.cDNA first
Total serum IgE with the extraction of above-mentioned steps 1 uses PrimeScript as templateTM 1st Strand cDNA
Synthesized Kit test kits (Takara companies) and with reference to the requirement of kit specification, reversion synthesis its
One chain cDNA.Reaction system and reaction condition are as follows：Oligo-dT (10pmol/ μ l) 1 μ l, Total RNA
(≤1 μ g) 2 μ l, dNTP Mixture (10mmol/l each) 1.0 μ l, 5 × Buffer 4.0 μ l, RNase
The μ l of Inhibitor (40U/ μ l) 0.5, PrimeScript RTase (200U/ μ l) 0.5 μ l, RNase-free
distilled water 11μl；65℃5min,42℃45min,70℃15min.By the first chain of synthesis
CDNA be stored in -20 DEG C it is standby.
The synthesis of the chains of 3.cDNA second and purification
The synthesis of the chains of cDNA second：After the synthesis of first chain is finished, the synthesis of the second chain is directly carried out.In the synthesis of the first chain
Be separately added into the μ l of 2.5 × the second chain buffer 40 in system, the μ l of archaeal dna polymerase I 23, the μ l of RNase H 0.8, plus
Water is placed 2 hours to 100 μ l at 25 DEG C；70 DEG C, ice bath is placed in after 10min, obtains double-strand cDNA.
The purification of double-strand cDNA：The medium volume of system after the synthesis of the second chain adds Fen Lv Fang isoamyl alcohols (Fen
Chloroform in isoamyl alcohol the volume ratio of phenol, chloroform and isoamyl alcohol be 25:24:1), after mixing, 12000g, from
Heart 3min；Supernatant is transferred to a clean centrifuge tube, plus the ethanol of 2 times of volumes, the sodium acetate of 1/10 volume, -20 DEG C
Place 30min after 12000g, centrifugation 15min collect precipitation, wash precipitation using 70% ethanol, be dried, with sterilize go from
Sub- water dissolution.
4. double-stranded cDNA ends blunting, plus dATP
Double-strand cDNA after purification in above-mentioned steps 3 carries out end-filling.Reaction system and reaction condition are as follows：
The μ l of purified product 30,10 × Blunting Buffer 5 μ l, the μ l of 1mM dNTP 10, Quick Blunting kit
Enzyme Mix, 1 μ l, plus H2The μ l of O 9, the μ l of total system 50；Reaction condition：30 DEG C of incubation 30min.To anti-
Product is answered to carry out purification recovery using the common DNA product purification kit of TIANGEN Biotech (Beijing) Co., Ltd..
Then recovery product is carried out plus dATP, reaction system and condition are as follows：Recovery product 22 μ l, 100mM dATP 1
μ l, 10 × NEB Buffer 23 μ l, Klenow exo-(NEB) (50,000units/ml) 0.3 μ l, H2O 3.7
μ l, the μ l of cumulative volume 30；Reaction condition：Fully mix, 37 DEG C of incubation 30min, 75 DEG C of 20min heat inactivations.
Purification recovery is carried out using Tiangeng Product Purification Kit to product, is washed using the elution buffer dissolving of 30 μ l
It is de-, it is recycled product.
5. the connection of joint A
By the recovery product of above-mentioned steps 4, joint A is added, reaction system and condition are specific as follows:The μ l of recovery product 30,
The μ l of 100mM dATP 1, NEB Buffer 22 μ l, 10 μM of joint A 2.5 μ l, (1000u) T4DNA Ligase
0.5 μ l, plus H2The μ l of O 15, the μ l of cumulative volume 50, connect 2 hours, 65 DEG C of 20min of connection product under room temperature (or 16 DEG C),
Connection enzymatic activity inactivation.Purification recovery is carried out using Tiangeng Product Purification Kit to coupled reaction product, using 30 μ l
Elution buffer dissolving eluting.
6th, PCR amplifications purpose fragment
The product that purification in above-mentioned steps 5 is reclaimed, for the PCR reactions of genes of interest amplification, purpose PCR for obtaining
Product.
5 ' end amplification reaction systems and condition are as follows：Purification is reclaimed in step 5 product 3 μ l, GSP Primer
(gene-specific primer) 1 μ l, universal primer Primer are (single-stranded shown in the 1-26 positions of sequence 1
DNA) 1 μ l, 2 × Phusion PCR Master Mix 25 μ l, H2The μ l of O 20, altogether 50 μ l, reaction condition
For：First 98 DEG C of denaturations 1min；Then 98 DEG C of 10s, 60 DEG C of 30s, 72 DEG C of 60s, totally 35 circulations；
Last 72 DEG C of extensions 5min.It is applied to the GSP Primer (i.e. the special primer of Broussonetia papyrifera Gene A) of 5 ' end amplifications
Sequence be 5 '-CTAGCTGTAGTGGTAGTGGTAGTAGT-3 '.
3 ' end amplification reaction systems and condition are as follows：Purification is reclaimed in step 5 product 3 μ l, GSP Primer
(gene-specific primer) 1 μ l, universal primer Primer are (single-stranded shown in the 1-26 positions of sequence 1
DNA) 1 μ l, 2 × Phusion PCR Master Mix 25 μ l, H2The μ l of O 20, altogether 50 μ l, reaction condition
For：First 98 DEG C of denaturations 1min；Then 98 DEG C of 10s, 60 DEG C of 30s, 72 DEG C of 60s, totally 35 circulations；
Last 72 DEG C of extensions 5min.It is applied to the GSP Primer (i.e. the special primer of Broussonetia papyrifera Gene A) of 3 ' end amplifications
Sequence be 5 '-CCCTACTAGCTGCAGTACTCTTGCAGAG-3 '.
After reaction terminates, 1% agarose gel electrophoresiies detection is carried out to pcr amplification product, as a result as shown in Figure 4.Its
In, swimming lane M divides for the DNA of TRANS2000DNA molecular weight standards (Beijing Quanshijin Biotechnology Co., Ltd)
Sub- amount standard, swimming lane 1 and 2 is respectively the pcr amplification product of 5 ' end amplifications and 3 ' end amplifications.Reclaim and purification
Pcr amplification product, is connected on PMD-18T carriers, and connection product conversion bacillus coli DH 5 alpha competence is thin
Born of the same parents, screening positive clone carries out the identification of bacterium solution PCR, and the plasmid for extracting positive colony is sequenced, and sequencing result is entered
Row BLAST is analyzed.
Using the overlay region between 5 ' and 3 ' end fragments of above-mentioned acquisition, by the purpose that the splicing of Contig softwares is obtained
The full length cDNA sequence of gene, and the special primer of following Broussonetia papyrifera Gene A is designed according to genes of interest full length cDNA sequence：
Primer 1：5 '-ATCATCACTTCACTGTTTCAGCTCCAACTAAC-3 ',
Primer 2：5′-TTCATTCCCACATATACCATAGTCACCA-3′.
With above-mentioned steps it is 2-in-1 into the first chain cDNA as template, with primer 1 and primer 2 enter performing PCR amplification.PCR is expanded
Product carries out 1% agarose gel electrophoresiies detection, as a result as shown in Figure 5.Wherein, swimming lane M is TRANS2000DNA molecules
The DNA molecular amount standard of amount standard (Beijing Quanshijin Biotechnology Co., Ltd), swimming lane 1 is pcr amplification product.Knot
Fruit shows that Jing PCR amplifications obtain the fragment of the specific amplification of genes of interest.Simultaneously purification product is reclaimed, is connected
It is connected on PMD-18T carriers, connection product conversion bacillus coli DH 5 alpha competent cell, screening positive clone carries out bacterium
Liquid PCR identifies that the plasmid for extracting positive colony is sequenced.Sequencing result shows, the pcr amplification product and above-mentioned spelling
Connect the sequence for obtaining identical in amplification part, be the sequence of purpose gene.
Claims (10)
1. it is used for the reagent set of Cloning of full length gene, is made up of joint A and universal primer；
The joint that the joint A is made up of positive chain and reverse strand, the positive chain of the joint A is shown in sequence 1
Single stranded DNA, the single stranded DNA being reversely linked as shown in sequence 2 of the joint A, the of sequence 2 and sequence 1
22-37 positions reverse complemental；
Single stranded DNA of the universal primer shown in the 1-26 positions of sequence 1.
2. reagent set according to claim 1, it is characterised in that：The 5 ' of the reverse strand of the joint A are last
End is phosphorylated modification.
3. joint A or described universal primers described in claim 1 or 2.
4. the system of Cloning of full length gene, including reagent set described in claim 1 or 2 and for Cloning of full length are used for
Other reagents of gene and/or instrument.
5. system according to claim 4, it is characterised in that：Described other reagents for Cloning of full length gene
And/or instrument be extract RNA, synthesis the chains of cDNA first or the second chain, the connection of DNA fragmentation, to containing sticky end
DNA fragmentation carry out end-filling, to the reagent needed for DNA fragmentation plus dATP, DNA purification and/or PCR amplifications and
/ or instrument.
6. reagent set according to claim 1 and 2 or the system described in claim 4 or 5, its feature
It is：The source of the gene is plant, animal, microorganism or virus.
7. the method for cloning genes of interest, including following 1) -3)：
1) double-strand cDNA is obtained by template of biological total serum IgE；
2) to step 1) double-strand cDNA carry out successively end-filling, plus dATP and connection claim 1 or 2 in
The joint A, obtains the connection product containing joint A；
3) with step 2) connection product as template, with general described in the special primer and claim 1 of genes of interest
Primer enters performing PCR amplification, obtains genes of interest.
8. method according to claim 7, it is characterised in that：It is described 1) including it is following 11) and 12)：
11) reverse transcription is carried out by template of biological total serum IgE, obtains the first chain cDNA；
12) with step 11) the first chain cDNA as templated synthesis the second chain cDNA, obtain double-strand cDNA.
9. the method according to claim 7 or 8, it is characterised in that：It is described biological for plant, animal, micro- life
Thing or virus.
10. reagent set described in claim 1 or 2, the joint A or described universal primers, claim 4 or
Application of the system described in 5 in gene cloning.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110283883A (en) * | 2019-05-08 | 2019-09-27 | 湖南农业大学 | A kind of primer and method for unknown RNA mycoviruses genomic clone |
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| CN101255429A (en) * | 2008-03-28 | 2008-09-03 | 浙江大学 | Cabbage pollen wall growth related gene PGBc2 and separation method thereof |
| CN101319217A (en) * | 2008-03-28 | 2008-12-10 | 浙江大学 | Male-fertility correlated gene BcPW1 of celery cabbage and process for the separation |
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2015
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|---|---|---|---|---|
| US5789206A (en) * | 1995-07-07 | 1998-08-04 | Myriad Genetics, Inc. | Method for ligating adaptors to nucleic acids which methods are useful for obtaining the ends of genes |
| WO2001005980A1 (en) * | 1999-07-14 | 2001-01-25 | Pioneer Hi-Bred International, Inc. | Compositions and methods for fumonisin detoxification |
| CN101255429A (en) * | 2008-03-28 | 2008-09-03 | 浙江大学 | Cabbage pollen wall growth related gene PGBc2 and separation method thereof |
| CN101319217A (en) * | 2008-03-28 | 2008-12-10 | 浙江大学 | Male-fertility correlated gene BcPW1 of celery cabbage and process for the separation |
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| A. CHENCHIK ET AL.: "Full-Length cDNA Cloning and Determination of mRNA 5¢ and 3¢Ends by Amplification of Adaptor-Ligated cDNA.", 《BIOTECHNIQUES》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110283883A (en) * | 2019-05-08 | 2019-09-27 | 湖南农业大学 | A kind of primer and method for unknown RNA mycoviruses genomic clone |
| CN110283883B (en) * | 2019-05-08 | 2023-03-14 | 湖南农业大学 | Primer and method for unknown RNA fungal virus genome cloning |
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Application publication date: 20170517 |