CN106636074A - 3'RACE (rapid amplification of cDNA ends) method for acquiring complete 3' end sequence as 3' end having repetitive sequence - Google Patents
3'RACE (rapid amplification of cDNA ends) method for acquiring complete 3' end sequence as 3' end having repetitive sequence Download PDFInfo
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Abstract
The invention discloses a 3'RACE (rapid amplification of cDNA ends) method for acquiring a complete 3' end sequence as a 3' end having a repetitive sequence. The method comprises the following steps: 1) tailing mRNA with terminal transferase, so that a connector sequence is added: tailing the mRNA with dCTP or dGTP, so that a single-base repetitive sequence of 16-22bp is added; 2) synthesizing first chain cDNA: conducting reverse transcription on the tailed mRNA by taking an anchor primer having oligodG or oligodC as a connector sequence; and 3) conducting 3'RACE amplification: with the cDNA having the connector sequence as a template, an internal specific primer GSP2 of the template which is designed in accordance with a RACE primer design principle as well as a corresponding nested primer NGSP2 thereof as forward primers of the amplification, and a complementary sequence (AUAP) of the connector sequence as a reverse amplification primer, conducting 3'RACE on the cDNA sequence having the connector. With the application of the 3'RACE method provided by the invention, the problem that a complete end sequence cannot be acquired in 3'RACE due to a special structure (having the A repetitive sequence) of the sequence can be solved.
Description
Technical field
The present invention relates to biological technical field, obtains more particularly to a kind of being directed in the case that 3 ' ends carry A repetitive sequences
Obtain 3 ' RACE methods of completely 3 ' end sequences.
Background technology
CDNA ends rapid amplifying technology (Rapid Amplification of cDNA Ends, RACE) is to combine PCR
Technology, the method that intact end is obtained using the known portions of cDNA sequence.The method extensively should with its unique advantage
For multiple research fields.RACE technologies have passed through so far from proposition and constantly improve.Wherein, forefathers are had found in mRNA
When reverse transcription is into first cDNA chain, primer usually with any position random incorporation of poly (A), affect reverse transcription result.
Therefore to solve problems, researcher introduces two degeneracy nucleotides [5 '-oligo at 3 ' ends of oligo (dT) primer
(dT) 16-30MN-3 ', M=A/G/C, N=A/G/C/T] locking primer (lock clocking primer) is become, it is fixed
Position is correctly carried out in the starting point of poly (A) tail with guaranteeing reverse transcription.Based on this, in order to simplify experimental implementation flow process, improve effect
The quality of rate and reverse transcription product, many Reagent Companies develop respective distinctive reverse transcription primer and anchor primer in succession, such as
The existing SMARTer of CLONTECH companiesTMRACE kits etc..
Although the improved method for having derived many by classical RACE now, each method has what its correspondence was adapted to
RNA species, but for some have the species of special mRNA sequence structure, 3 ' RACE technologies still suffer from an experiment primer
The number of drawbacks such as, partial reaction system low with template matching efficiency be unstable, causes its result a variety of incomplete by-products occur
Thing and affect subsequent experimental.For example, there is the A repetitive sequences of approximate poly (A) in some eukaryote mRNA sequences, it is this
There is " internal priming " phenomenon when using the conventional RACE of reverse transcription primer 3 ', often in mRNA sequence
(when there is the phenomenon, the sequence for obtaining does not have terminator codon, is commonly called as in the industry " false tail "), namely be difficult to be obtained by 3 ' RACE
Obtain 3 ' end sequences completely.For this sequence, current common practice is to redesign another primer, sees whether also go out
Existing vacation tail, if occurred again, then tries, therefore, for 3 ' RACE of this structure, wasting time and energy uncertain can reach
Expected Results.
The content of the invention
The main object of the present invention is to solve some species because 3 ' ends of its mRNA sequence have A repetitive structures, causes to make
The problem of the complete fragment in end cannot be obtained with 3 ' RACE.
The technical scheme that the present invention is provided is as follows：
One kind obtains 3 ' RACE methods of complete 3 ' end sequence in the case of carrying A repetitive sequences for 3 ' ends, including
Following steps：
1) tailing is carried out to mRNA using terminal enzyme (DNA), increases by the first joint sequence：MRNA is entered with dCTP or dGTP
Row tailing, increases 16-22bp single base repetitive sequences, and the 16-22bp single bases repetitive sequence is the first joint sequence；
2) synthesis of the first chain cDNA
With the anchor primer of the second joint sequence with oligodG or oligodC, the mRNA after tailing is inverted
Record；Described the second joint sequence and the first joint sequence is complementary, and length is identical；Anchor primer total length is 36-44 base.
3) 3 ' RACE amplifications are carried out
CDNA with belt lacing sequence as template, design specific primer GSP2 (template segments inside specific primer),
NGSP2 (the specific nested primer in template segments inside) is the forward primer of amplification, while design anchor corresponding with joint sequence
Primer (AUAP) is determined as the reverse primer of amplification, 3 ' RACE are carried out to the cDNA sequence of belt lacing.
In the preferred embodiment, step 1) tailing, increase 18-20bp single base repetitive sequences.
In the preferred embodiment, step 1) in dCTP or dGTP concentration be 10mM.
In the preferred embodiment, step 1) carry out terminating reaction within 30 seconds with 80 DEG C of reactions after tailing.
The present invention carries out tailing to mRNA according to cDNA ends rapid amplifying know-why first with terminal enzyme (DNA), increases
Plus one section of C or G joint sequence, the inventive method can reduce 3 ' end tool A repetitive structures mRNA sequence reverse transcription synthesize first
By the A repetitive structures of approximate poly (A) in primer oligo (dT) random incorporation sequence to experiment during the chain RACE of cDNA and 3 '
The impact for bringing, so as to obtain end complete sequence.Solving 3 ' RACE caused by the special construction institute because of sequence cannot obtain
The problem of whole end sequence.
Description of the drawings
Fig. 1 is the present invention plus the RACE of joint cDNA 3 ' amplification principle schematics.
Fig. 2 is control group and experimental group electrophoresis result
Fig. 3 is control group and experimental group sequence alignment result.
Specific embodiment
Embodiment 1
With Kandelia candel mangrove (Kandelia obovata) Fe-SOD (Fe-Superoxide
Dismutase) gene is subjects, is named as FeSOD1.Extract autumn eggplant total serum IgE and after quality testing, ultralow temperature (-
80 DEG C) save backup.
Embodiment 2
Control group：Thermo Scientific RevertAid First Strand cDNA are utilized by universal method
Synthesis Kit (Fermentas EU) reverse transcription is cDNA.
Embodiment 3
Experimental group：(1) tailing process is carried out to mRNA using the terminal enzyme (DNA) of TaKaRa companies.
Following reactant liquor is prepared in microcentrifugal tube, full dose is 50ul (table 1).
The preparation of the TdT reactant liquors of table 1
Reactant liquor is mixed, 37 DEG C are reacted 30 minutes after slight oscillatory.80 DEG C of reactions, 30 seconds terminating reactions.
(2) using Thermo Scientific RevertAid First Strand cDNA Synthesis Kit
(Fermentas EU) kit carries out reverse transcription experiment.The preparation of concrete reactant liquor is as shown in table 2.
Following reactant liquor (table 2) is prepared in microcentrifugal tube.
The preparation of the reactant liquor of table 2
Note：AP3-GGCCACGCGTCGACTAGTACGGGGGGGGGGGGGGGGGG
65 DEG C are reacted 5 minutes, open RNA secondary structures, are easy to primer to combine.React 1 minute on ice.
4. following reactant liquor (table 3) is prepared in microcentrifugal tube.
The preparation of the inverse transcription reaction liquid of table 3
5. reactant liquor is mixed, 25 DEG C are reacted 5 minutes after slight oscillatory.
6. 50 DEG C of reactions synthesize cDNA in 60 minutes.It is last to react 15 minutes terminating reactions, 4 DEG C of of short duration preservations with 70 DEG C.
2.2 3 ' the primer information and amplification system (table 4-6) of RACE amplifications
The amplimer information of table 4
With the mRNA after tailing as template, 3 ' RACE amplifications are carried out.
The RACE amplification systems of table 53 '
* note：The first round uses GSP primers, and with first round product as template, NGSP is primer amplification to the second wheel
The amplification program of table 6
3 ' RACE products are reclaimed, cloning and sequencing.
3 ' RACE product electrophoresis results
From electrophoretogram (Fig. 2) as can be seen that carrying out being expanded after tailing to mRNA, the band for being obtained is big compared with control group
300bp or so.Additionally, from control group strip analysis, band clearly becomes clear at 250-500bp, illustrate that this is in PCR reactions and locate
In dominant binding site, so as to cause the binding site of 700bp or so weakened.And tailing process is carried out to mRNA, can obtain
Complete fragment sequence.
Respectively control group is reclaimed, be connected after purification in pMD18-T carriers (TaKaRa) with the product of experimental group, and converted
Bacillus coli DH 5 alpha carries out TA clones and the detection of bacterium solution PCR, 20 positive colony is respectively selected at random and send sequencing.
Sequencing result is analyzed
Sequencing result shows that 20 positive colony of control group are the fragment of 280bp or so, 3 ' cDNA terminal deletions.
And experimental group obtains fragment of the length for 548bp.Sequence alignment analysis are carried out to sequencing result using DNAMAN6.0 softwares, and
With transcript profile sequencing result as checking, (Fig. 3) is should be apparent that, can by carrying out the method for tailing reverse transcription again to mRNA
To reduce during reverse transcription because of the ends of cDNA 3 ' deficient phenomena caused by primer erroneous combination mRNA chain.Therefore can be with by the method
Obtain 3 ' and hold complete sequence.
<110>Xiamen University
<120>One kind is for 3 ' ends with 3 ' the RACE methods that complete 3 ' end sequence is obtained in the case of A repetitive sequences
<160>4
<210>1
<211>38
<212>DNA
<213>Artificial sequence
<400>1
GGCCACGCGT CGACTAGTAC GGGGGGGGGG GGGGGGGG 38
<210>2
<211>20
<212>DNA
<213>Artificial sequence
<400>2
CATGAGCAAA GACACGCTGG 20
<210>3
<211>20
<212>DNA
<213>Artificial sequence
<400>3
GTTGGCAATG CAGTCAATCC 20
<210>4
<211>20
<212>DNA
<213>Artificial sequence
<400>4
GGCCACGCGT CGACTAGTAC 20
Claims (4)
1. it is a kind of to hold with 3 ' the RACE methods that complete 3 ' end sequence is obtained in the case of A repetitive sequences, including such as 3 '
Lower step：
1) tailing is carried out to mRNA using terminal enzyme (DNA), increases by one section of joint sequence：MRNA is carried out with dCTP or dGTP adding
Tail, increases 16-22bp single base repetitive sequences；
2) synthesis of the first chain cDNA
Reverse transcription is carried out to the mRNA after tailing with the anchor primer with oligod G or oligod C；
3) 3 ' RACE amplifications are carried out
CDNA with belt lacing sequence as template, according to RACE design of primers principles design template inside specific primer GSP2 and
Its corresponding nested primer NGSP2 is the forward primer of amplification, while the complementary series AUAP of designed joint sequence, length 16-
22bp, as reverse amplimer, to the cDNA sequence of belt lacing 3 ' RACE is carried out.
2. a kind of being directed in the case that 3 ' ends carry A repetitive sequences as claimed in claim 1 obtains complete 3 ' end sequence
3 ' RACE methods, it is characterised in that：Step 1) tailing, increase 18-20bp single base repetitive sequences.
3. a kind of being directed in the case that 3 ' ends carry A repetitive sequences as claimed in claim 1 obtains complete 3 ' end sequence
3 ' RACE methods, it is characterised in that：Step 1) in dCTP or dGTP concentration be 10mM.
4. a kind of being directed in the case that 3 ' ends carry A repetitive sequences as claimed in claim 1 obtains complete 3 ' end sequence
3 ' RACE methods, it is characterised in that：Step 1) carry out terminating reaction within 30 seconds with 80 DEG C of reactions after tailing.
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| CN201710100037.5A CN106636074B (en) | 2017-02-23 | 2017-02-23 | For 3 ' ends with 3 ' the RACE methods for obtaining complete 3 ' end sequence in the case where A repetitive sequence |
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| CN201710100037.5A CN106636074B (en) | 2017-02-23 | 2017-02-23 | For 3 ' ends with 3 ' the RACE methods for obtaining complete 3 ' end sequence in the case where A repetitive sequence |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107760678A (en) * | 2017-10-31 | 2018-03-06 | 安徽省农业科学院水产研究所 | The amplification method of 3 ' RACE adapter-primers and 3 ' end unknown gene sequences |
| WO2019090482A1 (en) * | 2017-11-07 | 2019-05-16 | 北京优乐复生科技有限责任公司 | Second-generation high-throughput sequencing library construction method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101514353A (en) * | 2009-02-19 | 2009-08-26 | 上海交通大学 | Clone method of SGAE label 3' end cDNA segment |
| WO2012013932A1 (en) * | 2010-07-29 | 2012-02-02 | University Court Of The University Of St Andrews | Improved race |
-
2017
- 2017-02-23 CN CN201710100037.5A patent/CN106636074B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101514353A (en) * | 2009-02-19 | 2009-08-26 | 上海交通大学 | Clone method of SGAE label 3' end cDNA segment |
| WO2012013932A1 (en) * | 2010-07-29 | 2012-02-02 | University Court Of The University Of St Andrews | Improved race |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107760678A (en) * | 2017-10-31 | 2018-03-06 | 安徽省农业科学院水产研究所 | The amplification method of 3 ' RACE adapter-primers and 3 ' end unknown gene sequences |
| WO2019090482A1 (en) * | 2017-11-07 | 2019-05-16 | 北京优乐复生科技有限责任公司 | Second-generation high-throughput sequencing library construction method |
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