CN100404676C - Method for configuring CDMA library and its special primer - Google Patents
Method for configuring CDMA library and its special primer Download PDFInfo
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- CN100404676C CN100404676C CNB2004100006818A CN200410000681A CN100404676C CN 100404676 C CN100404676 C CN 100404676C CN B2004100006818 A CNB2004100006818 A CN B2004100006818A CN 200410000681 A CN200410000681 A CN 200410000681A CN 100404676 C CN100404676 C CN 100404676C
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Abstract
The present invention discloses a method for building a cDNA library and special primers thereof. The aim of the present invention is to provide a method for building a cDNA library without endonuclease and DNA ligase and two pairs of special primers thereof. One pair of the special primers for building the cDNA library provided by the present invention are SEQ ID No. 1 and SEQ ID No. 2 in the sequence list, and the other pair of the primers are SEQ ID No. 3 and SEQ ID No. 4 in the sequence list. The method for building the cDNA library comprises the following steps: (1) synthesizing the first cDNA chain by using total RNA as a template and SEQ ID No. 1 and SEQ ID No. 2 in the sequence list as primers; (2) synthesizing double-chain cDNA by using SEQ ID No. 3 and SEQ ID No. 4 in the sequence list; (3) connecting the double-chain cDNA synthesized in step (2) with a carrier by using a recombination system of lambda phage insertion/excision specific sequences, and leading the carrier into a host to obtain a cDNA library. The method and the special primers of the present invention have extensive adaptability, and are suitable for any sources of biological sample RNA.
Description
Technical field
The present invention relates to the method and the primer special thereof in a kind of construction cDNA library in the gene engineering technology field.
Background technology
The structure in cDNA library is a molecular biology, functional genomics, indispensable technology in the excellent resources gene isolation clone research.The template that its Poly (A) mRNA that makes up general employing purifying is a ThermoScript II is synthesized first chain, use archaeal dna polymerase (Polymerase) to synthesize the cDNA two strands subsequently, for synthetic cDNA two strands is cloned in the carrier, usually at two different restriction enzyme sites of its terminal connection, so that clone's cDNA has directivity.All there is following problem in these methods: can not guarantee cDNA synthetic total length, thereby cDNA library difficult quality guarantee; For synthetic cDNA two strands is cloned in the carrier, must make operation very loaded down with trivial details at two different restriction enzyme sites of its terminal connection; During connection the higher representativeness in library that makes of the selectivity deviation of cDNA length is reduced; All to relate to the use of endonuclease and dna ligase, and complex operation step, need the experimental technique personnel to have consummate operating skill; Need with a large amount of Poly (A) mRNA, also the needs of incompatibility flux more can not be built the storehouse to the valuable sample of trace; Because operation is too loaded down with trivial details, can not high pass quantize the needs of the develop rapidly of incompatibility functional genomics.
Along with the develop rapidly of functional genomics, the structure in cDNA library is proposed the more requirement of Gao Gengxin, comprise (1) use the total RNA of trace as template in order to avoid the mRNA purifying, the more important thing is make micro-valuable sample (even unicellular) build Kucheng for may; (2) operation steps is simplified so that automatization and flux.
In view of above problem, different solutions are studied out in succession.The LD PCR method synthetic double chain cDNA of Clontech company can solve the big problem of Poly (A) mRNA consumption, but can not guarantee total length (the Chenchik A of cDNA, Zhu YY, Diatchenko L, Li R, Hill J, and Siebert is and use of high-quality cDNA from small amount of total RNA bySMART PCR.In Gene Cloning and Analysis by RT-PCR.Edited by Paul Siebertand James Larrick.BioTechique Books P.1998.Generation, natiek, MA); The GatewayTechnology of Invi trogen company has solved when connecting the selectivity offset issue to cDNA length, but do not separate the problem (Ohara O and Temple is cDNA libraryconstruction assisted by the in vitro recombination reaction.Nucleic AcidResearch.vol.29 G.2001.Directional, No.4 e22) of never using dna ligase fully.In a word, also there is not a kind of cDNA library constructing method to overcome the above problems fully now.
The innovation and creation content
The purpose of this invention is to provide a kind of primer special that does not need the construction cDNA library of endonuclease and dna ligase.
Totally two pairs of primer specials provided by the present invention, wherein, a pair of primer is used for the synthetic of cDNA article one chain, is the SEQ ID № 1 (attB2 (dT) in the sequence table
25) and SEQ ID № 2 (attB1 (rG)
3); Another is used for the synthetic of cDNA second chain to primer, is SEQ ID № 3 (attB1) in the sequence table and SEQ ID № 4 (attB2).
SEQ ID № 1 (attB2 (dT) in the sequence table
25) by 51 based compositions, be with 25 (d) T at 3 ' end, tail end has designed the VN sequence, to improve the specificity that starts guiding, and in its 5 ' tip designs attB2 recombining reaction distinguished sequence, make the clone operations that does not relate to endonuclease and dna ligase become possibility; SEQ ID № 2 (attB1 (rG)
3) by 28 based compositions, finish the reverse transcription of mRNA afterwards at a cDNA chain 3 ' terminal characteristic (the Chenchik A that continues to prolong three C bases based on RNase H-ThermoScript II, ZhuYY, Diatchenko L, Li R, Hill J, and Siebert is and use ofhigh-quality cDNA from small amount of total RNA by SMART PCR.In Gene CloningandAnalysisby RT-PCR.Edited by Paul Siebert and James Larrick.BioTechiqueBooks P.1998.Generation, natiek, MA), in primer 3 ' tip designs three rG (non-deoxynucleotide G), these three rG and a cDNA chain 3 ' terminal three C base complementrities that prolong, make RNase H-ThermoScript II in synthetic, carry out the template conversion automatically, and recombining reaction distinguished sequence attB1 is fixed on a cDNA chain 3 ' end.
SEQ ID № 3 in the sequence table and SEQ ID № 4 are by 29 based compositions, and SEQ ID № 3 contains the attB1 sequence, and SEQ ID № 4 contains the attB2 sequence.
Second purpose of the present invention provides the method for synthetic cDNA first chain of the total length of a kind of cDNA of guaranteeing.
The method of synthetic cDNA first chain provided by the present invention is to be template with total RNA (Total RNA), is the synthetic cDNA article one chain of primer with SEQ ID № 1 in the sequence table and SEQ ID № 2.
Wherein, the building-up reactions of described cDNA article one chain is carried out in two steps, earlier with the 1 synthetic cDNA article one chain of the SEQ ID № in the sequence table, and then carries out the template conversion reaction of cDNA article one chain in synthesizing with SEQ ID № 2.The building-up process of cDNA article one chain as shown in Figure 1, the building-up reactions step of cDNA article one chain is as follows:
Following reagent is sequentially added among little test tube:
RNA sample (1 microgram total amount RNA)
AttB2 (dT)
25Primer (10uM) 1 microlitre
DEPC-H
2O to 5 microlitre
Mixing and with solution centrifugal to managing at the end, place 72 ℃ of water-baths 2 minutes, quick ice bath 2 minutes afterwards, reagent below adding again:
5 * the first chains synthesize damping fluid 2 microlitres
DTT (20mM) 1 microlitre
DNTP (10mM) 1 microlitre
SuperScript II ThermoScript II 1 microlitre
Reaction volume 10 microlitres
Mixing, centrifugal, place 42 ℃, after reaction is carried out 60 minutes, add following reagent and carry out the template conversion reaction:
AttB1 (rG)
3Primer (10uM) 1 microlitre
5 * the first chains synthesize damping fluid 0.5 microlitre
SuperScript II ThermoScript II 1 microlitre
Reaction volume 12.5 microlitres
Mixing, centrifugal, place 42 ℃, afterreaction finished in 60 minutes.
This method was decomposed into for two steps with cDNA first chain synthesis reaction and template conversion reaction, but still in same reaction tubes, carry out, like this, first chain is synthetic finish after, carry out the template conversion reaction again, avoid reverse transcription not finish template before conversion effectively, thereby improved the total length of cDNA greatly.
The 3rd purpose of the present invention provides a kind of method that does not need the structure full-length cDNA library of endonuclease and dna ligase fully.
The method that does not need the construction cDNA library of endonuclease and dna ligase fully provided by the present invention may further comprise the steps:
1) being template with total RNA (Total RNA), is the synthetic cDNA article one chain of primer with SEQ ID № 1 in the sequence table and SEQ ID № 2;
2) with SEQ ID № 3 and SEQ ID № 4 synthetic double chain cDNAs in the sequence table;
3) with step 2) the double-stranded cDNA of synthetic is connected in the carrier with lambda particles phage insertions/excision distinguished sequence recombination system, with described carrier importing host, obtains the cDNA library.
Wherein, the building-up reactions of the article one of cDNA described in step 1) chain is carried out in two steps, earlier with the 1 synthetic cDNA article one chain of the SEQ ID № in the sequence table, and then carries out the template conversion reaction of cDNA article one chain in synthesizing with SEQ ID № 2.The building-up process of cDNA article one chain as shown in Figure 1, the building-up reactions step of cDNA article one chain is as follows:
Following reagent is sequentially added among little test tube:
RNA sample (1 microgram total amount RNA)
AttB2 (dT)
25Primer (10uM) 1 microlitre
DEPC-H
2O to 5 microlitre
Mixing and with solution centrifugal to managing at the end, place 72 ℃ of water-baths 2 minutes, quick ice bath 2 minutes afterwards, reagent below adding again:
5 * the first chains synthesize damping fluid 2 microlitres
DTT (20mM) 1 microlitre
DNTP (10mM) 1 microlitre
SuperScript II ThermoScript II 1 microlitre
Reaction volume 10 microlitres
Mixing, centrifugal, place 42 ℃, after reaction is carried out 60 minutes, add following reagent and carry out the template conversion reaction:
AttB1 (rG)
3Primer (10uM) 1 microlitre
5 * the first chains synthesize damping fluid 0.5 microlitre
SuperScriptII ThermoScript II 1 microlitre
Reaction volume 12.5 microlitres
Mixing, centrifugal, place 42 ℃, afterreaction finished in 60 minutes.
Step 1) was decomposed into for two steps with cDNA first chain synthesis reaction and template conversion reaction, but still in same reaction tubes, carry out, like this, first chain is synthetic finish after, carry out the template conversion reaction again, avoid reverse transcription not finish template before conversion effectively, thereby improved the total length of cDNA greatly.
Because 5 ' and the 3 ' end that utilizes SEQ ID № 1 in the sequence table and SEQ ID № 2 attB1 and attB2 sequence to be separately fixed at first chain, the synthetic of cDNA two strands becomes very simple, as shown in Figure 2, the synthetic of cDNA two strands can adopt pcr amplification to realize.
Carry out quality examination after the building-up reactions of cDNA two strands finishes, adopt 0.75% agarose gel electrophoresis usually.Enter library construction then, to build the storehouse the same with routine, and before making up the library, the cDNA that amplification is good must pass through molecular sieve (chromatography or agarose gel electrophoresis) and remove and synthesize incomplete small segment cDNA.The cDNA that reclaims just can be for library construction.
Wherein insertion/the excision of lambda particles phage described in step 3) distinguished sequence recombination system comprises recombinant clone enzyme and carrier, described carrier contains attP1 and the special recombination site of attP2, described recombinant clone enzyme and carrier all have the commercial reagents box, are that lambda particles phage inserts enzyme (integrase) and pDONR207 as the BP Clonase of Invitrogen.The structure in cDNA library carries out according to program shown in Figure 3.So far, the cDNA library is structured among the plasmid vector.Afterwards, with above reaction solution transformed into escherichia coli,, obtain the cDNA library as intestinal bacteria DH10B competent cell.
SEQ ID № 1 in the sequence table and SEQ ID № 2 these two primers, the basis of not only having established LD PCR synthetic double chain cDNA, and recombinant clone organically integrated, make the clone operations that does not relate to endonuclease and dna ligase become possibility.The present invention decomposes cDNA first chain synthesis reaction and template conversion reaction, carry out (but still in same reaction tubes) in two steps, like this, first chain is synthetic finish after, carry out the template conversion reaction again, avoid reverse transcription not finish template before conversion effectively, thereby guaranteed the total length of cDNA.
The present invention is based on RNase H
-Characteristic of ThermoScript II (Clark JM.1988.Novel non-templatednucleotide addition reaction catalyzed by prokaryotic and eucaryotic DNApolymerases.Nucleic Acid Research.16:9677-9686) and lambda particles phage insertion/excision (integration/excision) distinguished sequence recombination system (site-specific recombinationsystem, Bauer CE, Gardner JF, Gumport RI.1985.Extent of sequence homologyrequired for bacteriophage lambda site-specific recombination.J.Mol.Biol.181:187-197), the synthetic primer of cDNA and the construction process in cDNA library have been designed, make the synthetic and library construction of cDNA more fast and simple, the total length of library cDNA is protected, owing to do not need the intervention of endonuclease and dna ligase fully, simplify procedures greatly, be convenient to that high pass quantizes and the needs of the develop rapidly of adaptive functions genomics; In addition, this method also has following advantage: the employing recombination method in carrier, need not make operation easier at its terminal necessary two different restriction enzyme sites that connect the double-stranded directed cloning of synthetic cDNA; Only need the total RNA of trace, need be with a large amount of Poly (A) mRNA, the needs of accommodating fluxization more make micro-valuable sample even the single celled Kucheng of building be possible; The insertion of cDNA has directivity; Recombination frequency height (＞10
6Clone/μ g cDNA); Since adopt recombinant clone, little to the selectivity deviation of cDNA length, make the library of building have higher representativeness; The excisionase system of application lambda particles phage can shuttle back and forth building library and be transferred to other carrier, as the flux distribution of expression vector for genetic expression.Method of the present invention and primer special suitability thereof are very wide, can be used for any source biological sample RNA, will provide a kind of new strong gene clone technology for the china natural resources gene discovers and uses.
Description of drawings
Fig. 1 is the synthetic synoptic diagram of cDNA article one chain
Fig. 2 is the synthetic synoptic diagram of cDNA two strands
Fig. 3 is the structure synoptic diagram in recombinant cDNA library
Fig. 4 is the electrophoresis detection figure of synthetic salt mustard leaf cDNA
Fig. 5 detects the electrophorogram in salt mustard leaf cDNA library for PCR
Embodiment
The structure in embodiment 1, salt mustard (Thellungiella halophila) salt inducer blade cDNA library
Salt mustard salt inducer blade (L) cDNA is synthetic.Total amount RNA from three week size plant with the Trizol reagent of Invitrogen from the blade extracting, be template with the total amount RNA of 1 microgram as reverse transcription, SuperScriptII reagent and the instruction manual thereof of reverse transcription reaction employing Invitrogen.Use above inverse transcription reaction liquid 1 microlitre as the pcr amplification template then, with the ExTaq enzyme reagent amplifying doulbe-chain cDNA of TaKaRa company.The fragment that the cDNA of amplification reclaims behind agarose gel electrophoresis greater than 500bp is used to build the storehouse.The cDNA library construction uses BP Clonase and pDONR207 reagent and the instruction manual thereof of Invitrogen.Concrete steps are as follows:
The building-up reactions step of cDNA article one chain is as follows: following reagent is sequentially added among little test tube of one 0.5 milliliter:
RNA sample (1 microgram total amount RNA)
AttB2 (dT)
25Primer (10uM) 1 microlitre
DEPC-H
2O to 5 microlitre
Mixing and with solution centrifugal to managing at the end, put pipe in 72 ℃ of water-baths 2 minutes, quick ice bath 2 minutes afterwards, reagent below adding again:
5 * the first chains synthesize damping fluid 2 microlitres
DTT (20mM) 1 microlitre
DNTP (10mM) 1 microlitre
SuperScript II ThermoScript II 1 microlitre
Reaction volume 10 microlitres
Mixing, centrifugal, place 42 ℃, after reaction is carried out 60 minutes, add following reagent and carry out the template conversion reaction:
AttB1 (rG)
3Primer (10uM) 1 microlitre
5 * the first chains synthesize damping fluid 0.5 microlitre
SuperScript II ThermoScript II 1 microlitre
Reaction volume 12.5 microlitres
Mixing, centrifugal, place 42 ℃, afterreaction finished in 15 minutes.
Wherein, the synthetic damping fluid (Invitrogen) of 5 * the first chains
The cDNA first chain synthesis reaction liquid 2 microlitres
10 * damping fluid (TaKaRa), 10 microlitres
DNTP 8 microlitres
AttB1 (10uM) 2 microlitres
AttB2 (10uM) 2 microlitres
ExTaq enzyme 1 microlitre
DH
2O 75 microlitres
Reaction volume 100 microlitres
Mixing, centrifugal, place on the PCR instrument and react by following condition,
95 ℃ of 1 circulations in 2 minutes
95 ℃ of 5 second
64 ℃ of 10 second
72 ℃ of 20 circulations in 6 minutes
72 ℃ of 1 circulations in 10 minutes
Reaction is got 5 microlitre reaction product after finishing, and the agarose gel electrophoresis with 0.75% carries out quality examination, the result as shown in Figure 4, L is a reaction product, M is Marker, cDNA is distributed between 1 to 1.5kb mostly, it is extremely successful to illustrate that cDNA synthesizes.The fragment that the cDNA of amplification reclaims behind agarose gel electrophoresis greater than 500bp is used to build the storehouse.Adopt BP Clonase and the pDONR207 of Invitrogen to carry out following reaction:
CDNA 10 microlitres that reclaim (gram in the 100-200)
5 * BP clone enzyme buffer liquid, 4 microlitres
PDONR207 (gram/microlitre 2 microlitres in 150
BP clone enzyme 4 microlitres
Reaction capacity 20 microlitres
Mixing, centrifugal, place 25 ℃ and spend the night (16 hours).After reaction finishes, add 2 microlitre proteolytic ferment K, place 37 ℃, reacted 15 minutes.So far, the cDNA library is structured among the plasmid vector.Wherein, 5 * BP clone enzyme buffer liquid (Invitrogen).Then, with above BP clone enzyme reaction solution transformed into escherichia coli DH10B competent cell and obtain the cDNA library according to a conventional method, every microgram cDNA obtains about 3 * 10
6Individual clone.Library cDNA inserts fragment PCR augmentation detection result as shown in Figure 5, and its size is all more than 500bp.
Sequence table
<160>4
<210>1
<211>51
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(50)
＜223〉v=a, c or g,
<220>
<221>misc_feature
<222>(51)
＜223〉n=a, c, g, or t
<400>1
accactttgt?acaagaaagc?tgggtttttt?tttttttttt?tttttttttv?n 51
<210>2
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(26，27，28)
<223>n＝rg
<400>2
acaagtttgt?acaaaaaagc?aggctnnn 28
<210>3
<211>29
<212>DNA
＜213〉artificial sequence
<400>3
ggggacaagt?ttgtacaaaa?aagcaggct 29
<210>4
<211>29
<212>DNA
＜213〉artificial sequence
<400>4
ggggaccact?ttgtacaaga?aagctgggt 29
Claims (5)
1. the primer special in a construction cDNA library, totally two pairs, a pair of primer is SEQ ID NO:1 and the SEQ ID NO:2 in the sequence table, is used for synthetic cDNA first chain; Another is SEQ ID NO:3 and SEQ ID NO:4 in the sequence table to primer; Wherein, the building-up reactions of cDNA article one chain is carried out in two steps, is template with total RNA earlier, with the synthetic cDNA article one chain of SEQ ID NO:1, and then carries out the template conversion reaction of cDNA article one chain in synthesizing with SEQ ID NO:2.
2. the method in a construction cDNA library may further comprise the steps:
1) is template with total amount RNA,, and then carries out the template conversion reaction of cDNA article one chain in synthesizing with SEQ ID NO:2 with the synthetic cDNA article one chain of SEQ ID NO:1;
2) with SEQ ID NO:3 and SEQID NO:4 synthetic double chain cDNA in the sequence table;
3) with step 2) the double-stranded cDNA of synthetic is connected in the carrier with lambda particles phage insertions/excision distinguished sequence recombination system, with described carrier importing host, obtains the cDNA library; Described lambda particles phage insertion/excision distinguished sequence recombination system comprises recombinant clone enzyme and carrier pDONR207.
3. method according to claim 2 is characterized in that: described recombinant clone enzyme is BP Clonase.
4. according to claim 2 or 3 described methods, it is characterized in that: described host is intestinal bacteria.
5. method according to claim 4 is characterized in that: described intestinal bacteria are intestinal bacteria DH10B.
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| CN101914524B (en) * | 2010-07-23 | 2011-12-21 | 南京农业大学 | Method for extracting microRNA precursor cDNA from cDNA library synthesized from small RNA |
| CN101974786B (en) * | 2010-09-09 | 2013-03-13 | 浙江省农业科学院 | Method for constructing eukaryote cDNA library and special primers thereof |
| CN109137086A (en) * | 2018-10-16 | 2019-01-04 | 梁耀极 | A kind of banking process of the full length mRNA sequencing of improvement |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1414106A (en) * | 2001-10-24 | 2003-04-30 | 北京大学 | Method of clone gene and its application |
| WO2003035839A2 (en) * | 2001-10-24 | 2003-05-01 | Dgi Biotechnologies, Inc. | Target specific screening andits use for identifying target binders |
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2004
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1414106A (en) * | 2001-10-24 | 2003-04-30 | 北京大学 | Method of clone gene and its application |
| WO2003035839A2 (en) * | 2001-10-24 | 2003-05-01 | Dgi Biotechnologies, Inc. | Target specific screening andits use for identifying target binders |
Non-Patent Citations (4)
| Title |
|---|
| cDNA文库构建方法的进展. 张霖等人.生命的化学，第22卷第6期. 2002 |
| cDNA文库构建方法的进展. 张霖等人.生命的化学，第22卷第6期. 2002 * |
| Suppression subtractive hybridization : a methodforgenerating differentially regulated or tissue - sepecificcDNAprobe and libraries. DIACHENKO等人.Proe Ncad Acad Sci USA,Vol.93 No.12. 1996 |
| Suppression subtractive hybridization : a methodforgenerating differentially regulated or tissue - sepecificcDNAprobe and libraries. DIACHENKO等人.Proe Ncad Acad Sci USA,Vol.93 No.12. 1996 * |
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