CN101864413A - 5'-RACE joint sequence addition method as well as joint sequence and amplification method of 5' end unknown gene complete encoding sequence - Google Patents
5'-RACE joint sequence addition method as well as joint sequence and amplification method of 5' end unknown gene complete encoding sequence Download PDFInfo
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Abstract
The invention discloses a 5'-RACE joint sequence addition method as well as a joint sequence and an amplification method of an 5' end unknown gene complete encoding sequence , and relates to a joint sequence addition method as well as a joint sequence and an amplification method of a gene complete encoding sequence, overcoming the defects of no selectivity to the integrity of mRNA 5' terminal information, high template complexity, and the like of the traditional joint sequence addition method. The 5'-RACE joint sequence addition method comprises the following steps of: (1) designing a specific reverse transcription primer and a joint sequence according to a target gene and a target species; and (2) adding the joint sequence by reverse transcription. The joint sequence is shown as SEQ ID NO:10. The unknown gene complete encoding sequence at the 5' end comprises the following steps of: (1) designing a primer; (2) carrying out reverse transcription and adding a joint; (3) carrying out nested PCR (Polymerase Chain Reaction) amplification; and (4) sequencing and analyzing. The 5'-RACE joint sequence addition method has the advantages of selectivity to the integrity of the mRNA 5'end information, high gene cloning efficiency, and the like.
Description
Technical field
The present invention relates to the amplification method of a kind of joint sequence addition method and joint sequence and gene complete encoding sequence.
Background technology
If want the only known gene of sequence of 3 ' end, one side of clones coding district, need to obtain earlier the unknown nucleotide sequence of 5 ' end, one side, and then obtain complete encoding sequence.Nowadays, the RACE technology is the best approach according to the known partial sequence amplification of gene unknown fragment.(rapid amplification of cDNA ends, RACE) put it briefly is exactly combination by reverse transcription and PCR to the RACE technology, the technology of amplification gene unknown fragment.Utilize the known array of gene coding region 3 ' end, the technology that obtains 5 ' end unknown nucleotide sequence is called 5 '-RACE, is the most common technique of 5 ' end unknown gene complete encoding sequence of nowadays increasing.
5 '-RACE ultimate principle: at first obtain to comprise the RNA sample of goal gene mRNA, and utilize the primer of gene specific to carry out reverse transcription, obtain the corresponding cDNA of goal gene; 3 ' terminal (corresponding mRNA sequence 5 ' end) at cDNA adds specific sequence, and the particular sequence that this section is added on the cDNA is called joint (adaptor) usually, and the synthetic simultaneously primer that is complementary with the adaptor sequence is called anchor primer; Utilize gene known array design gene-specific primer then, carry out pcr amplification, obtain the fragment of unknown portions with the anchor primer pairing; The sequencing fragment that amplification is obtained again can obtain the sequence of goal gene unknown portions.
Addition means by joint sequence can be divided into 3 big classes at present: terminal enzyme (DNA) method (terminaldeoxynucleotidyl transferase method, TdT method), joint connection method (adaptor ligation) and oligonucleotide cap method (oligo caping method).In recent years some technology of amplification coding district's 3 ' one side and 5 ' one side unknown fragment simultaneously occurred, and released the reagent corresponding box, as GeneRacer (Invitrogen company), SMART RACE (Takara-BD company) etc.The ultimate principle of these technology is with the initial reverse transcription of oligo (dT), 3 ' end and 5 ' end at the global cDNA product of complete mRNA reverse transcription gained add 3 '-adaptor and 5 '-adaptor respectively, make all complete cDNA all have adaptor at two ends, become the common template that comprises all gene informations.Utilize the anchor primer collocation of the special primer and a certain end of goal gene again, then can utilize with a common template and obtain different genes, different terminal unknown nucleotide sequence.
Adopt terminal enzyme (DNA) method and joint connection method to have a common problem, can all add adaptor at the 3 ' end of all cDNA of reverse transcription gained exactly, can't distinguish the complete and incomplete mRNA of 5 ' end, also can't distinguish reverse transcription carry out fully with incomplete cDNA, therefore can not guarantee the complete of mRNA5 ' final word.Like this, if will obtain the information of the complete 5 ' end of gene coding region, often need to carry out " reverse transcription-interpolation adaptor-PCR-order-checking " process more than once, need the new reverse transcriptase primer of sequences Design at every turn, just need to carry out many wheel RACE according to last time order-checking gained.In addition, these two kinds of methods all need earlier the reverse transcription product to be carried out purifying, could add adaptor, and cDNA can lose in purge process, therefore needs the relatively large cDNA of preparation.
Though oligonucleotide cap method can optionally increase to the complete mRNA of 5 ' end, has solved terminal enzyme (DNA) method and the joint connection method problem to mRNA integrity non-selectivity.But oligonucleotide cap method exists and need carry out the multistep operation to RNA, between the enzyme reaction in each step, all need again RNA is carried out purifying, and RNA is easy to be degraded, therefore be easy to lose a large amount of RNA, thereby reduced the efficient of RACE work after this, for the lower mRNA of abundance, even be difficult to obtain amplified production.
Utilize common template as parent material, though but high-throughput ground carries out gene clone, if only need the unknown fragment by 5 '-RACE amplification minority, 5 ' end unknown gene coding region, common template also is not suitable for.Reason has following 2 points:
At first, the complicacy of common template is very high.Common template does not have selectivity to gene, and the global cDNA of all genes all is added with adaptor, comprises the cDNA that all gene pairss are answered in theory.In the template of complexity, goal gene cDNA content is relatively low, and for expressing the lower gene of abundance, clone's difficulty is then bigger.And template is complicated more, and easy more generation non-specific amplification in the PCR reaction influences the amplification efficiency of purpose fragment in the PCR reaction, and non-specific amplified production also can disturb the selection of purpose band.
Secondly, in the common template preparation process, the possibility of information loss is bigger.This be because, common template requires all very high to the integrity of RNA and reverse transcription reaction, the incomplete cDNA of chain extension can not be added adaptor in the reverse transcription product of imperfect mRNA and the reverse transcription reaction; And the chain extension process of reverse transcription reaction is easy to take place premature termination, if mRNA template to be extended is longer, or has complicated secondary structure, and then the possibility of premature termination is bigger.Reverse transcription reaction need extend to 5 ' end from the 3 ' end of mRNA always during the preparation common template, for the long gene in coding region, is difficult to obtain the global cDNA of gene.These all can increase by the workload of 5 '-RACE, and may influence 5 '-RACE result.
In addition, primer design and use all are subjected to the intensive restriction in existing all 5 '-RACE methods.The terminal enzyme (DNA) method can only be added a string identical Nucleotide, common template can only use same set of adaptor, though the adaptor in joint connection method and the oligonucleotide cap method can design voluntarily, but because the efficient of the effect length ligation of chain, the adaptor that therefore also can only add limited length (is generally 15～30bp).So just can't use the adaptor and the corresponding anchor primer that are suitable for purpose species, goal gene most, even contain the nonspecific binding site of anchor primer in the portion gene sequence in the template probably.Need be when pcr amplification with anchor primer and gene specific primer collocation, the researchist just must be according to the characteristics design gene specific primer of anchor primer, will dwindle the range of choice of gene specific primer like this, the primer that is suitable for goal gene most often can't be directly used in amplification.The handiness of design of primers is limited, just makes the researchist need spend big workload and inquires into the PCR condition, influences gene clone efficient.
Summary of the invention
The objective of the invention is does not have selectivity for the integrity to mRNA 5 ' final word that solves existing joint sequence addition method existence; Poor to the goal gene specific aim; Reverse transcription reaction is insufficient; The joint sequence design is dumb; The cost height is operated not easy; And the design of the amplification method center tap sequence of existing 5 ' end unknown gene complete encoding sequence and anchor primer is dumb; The cost height is operated not easy; Defectives such as the cloning efficiency of unknown fragment is low; And provide the amplification method of a kind of 5 '-RACE joint sequence addition method and joint sequence and 5 ' end unknown gene complete encoding sequence.
The present invention 5 '-RACE joint sequence adds according to the following steps:
One, according to goal gene purpose of design gene specific reverse transcriptase primer, according to goal gene place species gene stack features designed joint sequence, 3 ' tip designs of joint sequence has at least 3 successive guanines;
Two, in the reverse transcription system, add joint and goal gene specificity reverse transcriptase primer,, realized that 5 '-RACE joint sequence adds then with there being the active reversed transcriptive enzyme of terminal enzyme (DNA) to carry out reverse transcription.
5 '-RACE joint sequence addition method principle can add several dC (being generally 2～4 cytosine(Cyt)s) at institute's synthetic cDNA end as shown in Figure 1 continuously when the chain extension reaction of reverse transcription proceeds to 5 ' the terminal cap sequence of mRNA; Hold the joint sequence that has oligo (dG) to change primer owing in the reverse transcription system, add 3 ' in advance as template, so when reverse transcription is near completion, the oligo (dG) of joint sequence 3 ' end can match with the dC of cDNA end, there is the active reversed transcriptive enzyme of terminal enzyme (DNA) that the template conversion then takes place, with the joint sequence is that template is proceeded chain extension, thereby obtain the strand cDNA fragment that 3 ' end has added joint, realized that 5 '-RACE joint sequence adds.
The present invention 5 '-RACE joint sequence addition method has the following advantages:
1. selective to the integrity of mRNA 5 ' final word.Got rid of of the interference of the incomplete fragment of information to clone's process.
2, the initial reverse transcription of application target gene specific reverse transcriptase primer is targeted to goal gene.The starting template complexity is low, got rid of independent basis because of interference.
3, reverse transcription reaction is abundant.Premature termination takes place when avoiding rate reverse transcription reaction chain extension effectively.
4, the flexible design of adaptor.Can design long joint sequence, but design length is greater than the joint sequence of 40bp, to strengthen the specificity of adaptor.
5. cost is low, and is easy and simple to handle.The inventive method operation steps is few, subsequent P CR specificity and amplification efficiency height, and use laboratory common agents and equipment to finish.
6, the inventive method is finished the interpolation of joint sequence automatically by reversed transcriptive enzyme.
Joint sequence of the present invention is for being applicable to 5 '-RACE joint sequence of wild soybean (Glycine soja), and this 5 '-RACE joint sequence is 5 '-ATAGCAGTGTGATACCATGAGACGGGCACATTACGGCTCGGGG-3 '.
The present invention's 5 ' end unknown gene complete encoding sequence increases according to the following steps:
One, according to goal gene and purpose species purpose of design gene specific reverse transcriptase primer, goal gene specificity nest-type PRC primer and joint sequence, and according to joint sequence design nido anchor primer, 3 ' tip designs of joint sequence has at least 3 successive guanines;
Two, in the reverse transcription system, add joint and goal gene specificity reverse transcriptase primer,, obtain the strand cDNA fragment that 3 ' end has added joint sequence then with there being the active reversed transcriptive enzyme of terminal enzyme (DNA) to carry out reverse transcription;
Three, holding the strand cDNA fragment of having added joint with 3 ' is template, carries out the nest-type PRC amplification with goal gene specificity nest-type PRC primer and nido anchor primer;
Four, the nest-type PRC amplified fragments that step 3 is obtained checks order, and utilizes software analysis again, and it is terminal to 5 ' terminal sequence to obtain goal gene 3 ', promptly obtains the complete encoding sequence of the unknown goal gene of 5 ' end after the checking; The designed nido anchor primer of step 1 no visible non-specific amplification product in the species gene group at goal gene place or cDNA wherein.
The amplification method of the present invention's 5 ' end unknown gene complete encoding sequence has the following advantages:
1, selective to the integrity of mRNA 5 ' final word.Have only the cDNA that fully extends to mRNA 5 ' end just can be added joint sequence and in the PCR reaction, obtain amplification, got rid of the interference of information incomplete fragment, only by obtaining to comprise the sequence of goal gene coding region 5 ' terminal complete information once taking turns RACE to clone's process.
2, the initial reverse transcription of application target gene specific reverse transcriptase primer is targeted to goal gene.Can not take a turn for the worse in theory record reaction of the mRNA of other gene, thus in the reverse transcription product based on the cDNA of goal gene correspondence, the starting template complexity is low, can get rid of independent basis because of interference, improve the specificity and the efficient of PCR reaction among the RACE.
3, reverse transcription reaction is abundant.Goal gene specificity reverse transcriptase primer makes the chain extension contraction in length of reverse transcription reaction near the 5 ' end of mRNA, and the risk of premature termination takes place when having reduced the reverse transcription reaction chain extension, helps making reverse transcription reaction fully to extend to mRNA 5 ' end; And in reverse transcription reaction, therefore the record reaction because the mRNA of other gene can not take a turn for the worse also can guarantee the reverse transcription efficient of goal gene mRNA, makes the complete mRNA of goal gene be become cDNA and to add and go up adaptor by reverse transcription intactly.
4, adaptor and primer design are flexible.The inventive method can design different anchor primers at different species, and the possibility that non-specific amplification takes place in this species gene group or cDNA anchor primer is significantly reduced.For different genes, can use different anchor primers, anchor primer can be arranged in pairs or groups with the nest-type PRC amplimer of the best.Because adaptor introduces with the form of reverse transcription template, therefore do not relate to ligation, therefore also looser to the limitation of length of adaptor, can design long adaptor, thereby design more paired nido anchor primer with it, so that primer screening and PCR condition are inquired into.
5. cost is low, and is easy and simple to handle.The inventive method operation steps is few, PCR specificity and amplification efficiency height, and use laboratory common agents and equipment to finish, and do not need RNA or cDNA are carried out operations such as purifying the sample loss that can avoid the rapid operation of multistep to cause.
6. the cloning efficiency height of unknown fragment.The inventive method is taken turns RACE, the operation of several simple steps and minority PCR reaction several times by one, can finish the amplification of 5 ' end unknown nucleotide sequence, obtains complete coding region 5 ' terminal sequence information.The inventive method is specially adapted to the amplification of individual gene 5 ' one side unknown fragment, and whole cloning efficiency all is higher than existing 5 ' end unknown gene complete encoding sequence amplification method.Because template is targeted to gene, and the primer design collocation flexibly, so the PCR condition is also inquired into than being easier to; And amplified production comprises the complete information of goal gene coding region 5 ' end, do not need to repeat the RACE process.
7, the main process of the inventive method is finished automatically by reversed transcriptive enzyme, does not need operations such as purifying, and the interpolation of joint sequence directly occurs in the reverse transcription reaction same system afterwards.
Description of drawings
Fig. 1 is the present invention 5 '-RACE joint sequence addition method schematic diagram.Fig. 2 is goal gene known portions sequence B last pxrd analysis figure as a result in the embodiment 18.Fig. 3 is first round pcr amplification figure as a result in the embodiment 18, " M " swimming lane standard specimen is Marker among Fig. 3, " 1 " swimming lane standard specimen is for being the pcr amplification product of primer with GSP-1 and AP1, " 2 " swimming lane standard specimen is for being the pcr amplification product of primer with GSP-1, and " 3 " swimming lane standard specimen is for being the pcr amplification product of primer with AP1.Fig. 4 second takes turns pcr amplification figure as a result in the embodiment 18, " M " swimming lane standard specimen is Marker among Fig. 4, " 1 " swimming lane standard specimen is for being the pcr amplification product of primer with GSP-2 and AP2, " 2 " swimming lane standard specimen is for being the pcr amplification product of primer with GSP-2, and " 3 " swimming lane standard specimen is for being the pcr amplification product of primer with AP2.Fig. 5 is third round pcr amplification figure as a result in the embodiment 18, " M " swimming lane standard specimen is Marker among Fig. 5, " 1 " swimming lane standard specimen is for being the pcr amplification product of primer with GSP-3 and AP3, " 2 " swimming lane standard specimen is for being the pcr amplification product of primer with GSP-3, and " 3 " swimming lane standard specimen is for being the pcr amplification product of primer with AP3.Fig. 6 is clear band between 750bp～1000bp and existing EST (PTE-234) splicing in embodiment 18 nest-type PRCs, and analyzes splicing figure as a result by BlastX.Fig. 7 is that the goal gene total length is verified primer PCR figure as a result in the embodiment 18, and " M " swimming lane standard specimen is Marker among Fig. 7, and " 1 " swimming lane standard specimen is total cDNA, and " 2 " swimming lane standard specimen is a water.Fig. 8 is the ORF of final gained gene order in embodiment 18 proof tests figure that predicts the outcome.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment 5 '-RACE joint sequence adds according to the following steps:
One, according to goal gene purpose of design gene specific reverse transcriptase primer, according to goal gene place species gene stack features designed joint sequence, 3 ' tip designs of joint sequence has at least 3 successive guanines;
Two, in the reverse transcription system, add joint and goal gene specificity reverse transcriptase primer,, realized that 5 '-RACE joint sequence adds then with there being the active reversed transcriptive enzyme of terminal enzyme (DNA) to carry out reverse transcription.
Present embodiment has realized the interpolation of 5 '-RACE joint sequence when finishing reverse transcription.
The present embodiment method can obtain the strand cDNA fragment that 3 ' end has added joint.
Embodiment two: the difference of present embodiment and embodiment one is: goal gene specificity reverse transcriptase primer that step 1 is designed and the binding site of mRNA and this goal gene cDNA3 ' termination are near.Other step and parameter are identical with embodiment one.
The binding site of goal gene specificity reverse transcriptase primer and mRNA is the closer to the 5 ' end (being goal gene cDNA3 ' end) of mRNA, can make the chain extension contraction in length of reverse transcription reaction, reduce the risk of reverse transcription reaction premature termination, helped making reverse transcription reaction fully to extend to the 5 ' end of mRNA.
Embodiment three: the difference of present embodiment and embodiment one is: the distance of goal gene specificity reverse transcriptase primer that step 1 is designed and the binding site of mRNA and this goal gene cDNA3 ' end is 30～200bp.Other step and parameter are identical with embodiment one.
Embodiment four: the difference of present embodiment and embodiment one is: the distance of goal gene specificity reverse transcriptase primer that step 1 is designed and the binding site of mRNA and this goal gene cDNA3 ' end is 30.Other step and parameter are identical with embodiment one.
Embodiment five: the difference of one of present embodiment and embodiment one to four is: 3 ' tip designs of the joint sequence that step 1 is designed has 3,4,5,6,7 or 8 successive guanines.Other step and parameter are identical with one of embodiment one to four.
Embodiment six: present embodiment 5 ' end unknown gene complete encoding sequence increases according to the following steps:
One, according to goal gene and purpose species purpose of design gene specific reverse transcriptase primer, goal gene specificity nest-type PRC primer and joint sequence, and according to joint sequence design nido anchor primer, 3 ' tip designs of joint sequence has at least 3 successive guanines;
Two, in the reverse transcription system, add joint and goal gene specificity reverse transcriptase primer,, obtain the strand cDNA fragment that 3 ' end has added joint sequence then with there being the active reversed transcriptive enzyme of terminal enzyme (DNA) to carry out reverse transcription;
Three, holding the strand cDNA fragment of having added joint with 3 ' is template, carries out the nest-type PRC amplification with goal gene specificity nest-type PRC primer and nido anchor primer;
Four, the nest-type PRC amplified fragments that step 3 is obtained checks order, and utilizes software analysis again, and it is terminal to 5 ' terminal sequence to obtain goal gene 3 ', promptly obtains the complete encoding sequence of the unknown goal gene of 5 ' end after the checking; The designed nido anchor primer of step 1 no visible non-specific amplification product in the species gene group at goal gene place or cDNA wherein.
Present embodiment goal gene specificity nest-type PRC primer quantity can be some, and preferable range is 2～4.
The Tm value of nido anchor primer and goal gene specificity nest-type PRC primer should be more approaching in the present embodiment, and the difference of nido anchor primer and the mutual Tm value of goal gene specificity nest-type PRC primer should be less than 1 ℃; And features such as the length of nido anchor primer and goal gene specificity nest-type PRC primer and G/C content should be similar.
Goal gene specificity reverse transcriptase primer, goal gene specificity nest-type PRC primer, joint sequence and nido anchor primer sequence can freely design in the present embodiment step 1.Can design its distinctive corresponding primer at different species.Can first designed joint sequence, again according to its design nido anchor primer; Also can design many nido anchor primers earlier, at last the anchor primer sequence be combined, design 1 joint sequence that contains the binding site of each bar anchor primer.Because joint sequence is not participated in enzyme reaction directly by template interpolations of changing the mechanism, therefore its length there is not strict restriction, as if wanting to design more nido anchor primer, can increase the length of joint sequence.5 '-RACE that nido anchor primer that design is finished and joint sequence can be applied to other gene of biology of the same race.
The present embodiment method can be used different joint sequences and nido anchor primer at different plant species, is chosen in the nido anchor primer of no visible non-specific amplification product among this species gene group or the cDNA; At different genes, can be according to selected goal gene specificity reverse transcriptase primer and goal gene specificity nest-type PRC design of primers joint sequence and nido anchor primer, better PCR condition easy to use.Because joint sequence is introduced with the form of reverse transcription template, therefore do not relate to ligation, so the limitation of length of butt junction fragment is also looser, can designs long joint sequence, reach more paired nido anchor primer with it, so that the screening of primer and PCR condition are inquired into.
CDNA 3 ' the terminal joint sequence that adds that the present embodiment method is directly fully being extended in the reverse transcription process, therefore the integrity to cDNA has had selectivity, have only when the mRNA 5 ' of goal gene terminal complete, and reverse transcription reaction extends the cDNA that is obtained when complete and just can be added joint sequence.Use again according to the nido anchor primer and the collocation of goal gene specificity nest-type PRC primer of joint sequence design and carry out PCR, wherein have only the cDNA that has added joint sequence just can be amplified.Increase by nido or heminested PCR, got rid of the interference of non-specific amplification product, can obtain unknown cDNA 5 ' the end parts fragment of goal gene.
The chain extension time of present embodiment method steps two reverse transcription reactions is long, and elongating temperature will be tried one's best high within the scope that enzyme can bear, and can help opening the secondary structure of mRNA, and the cDNA chain is extended smoothly.
The present embodiment step 4 can adopt the BlastX supervisor that sequence is analyzed.
Embodiment seven: the difference of present embodiment and embodiment six is: the 5 ' termination of goal gene specificity reverse transcriptase primer that step 1 is designed and the binding site of RNA and this goal gene mRNA is near; And specific nest-type PRC primer of goal gene that step 1 is designed and binding site and this goal gene cDNA3 ' termination of cDNA are near.Other step and parameter are identical with embodiment six.
The specific nest-type PRC primer of the goal gene that designs in the present embodiment should be able to well be arranged in pairs or groups with the nido anchor primer.
The binding site of present embodiment goal gene specificity reverse transcriptase primer and mRNA is the closer to the 5 ' end (being goal gene cDNA3 ' end) of mRNA, can make the chain extension contraction in length of reverse transcription reaction, reduce the risk of reverse transcription reaction premature termination, helped making reverse transcription reaction fully to extend to the 5 ' end of mRNA.
When purpose of design gene specific reverse transcriptase primer, just make its binding site and unknown portions approaching as far as possible, only stay the binding site of goal gene nest-type PRC primer, and one section required known array of enough follow-up sequence assembly gets final product.Goal gene specificity reverse transcriptase primer length to be extended in reverse transcription reaction can be shortened so as far as possible, improve the sufficient possibility of reverse transcription reaction chain extension.In addition, because the reverse transcriptase primer of gene specific only combine with the mRNA of goal gene in theory and extend, therefore make in the reverse transcription product based on the cDNA of goal gene correspondence, complexity is low, and the PCR that helps after this reacts.
Embodiment eight: the difference of present embodiment and embodiment six is: the distance of the 5 ' end of goal gene specificity reverse transcriptase primer that step 1 is designed and the binding site of RNA and this goal gene mRNA is 30～200bp.Other step and parameter are identical with embodiment six.
Embodiment nine: the difference of present embodiment and embodiment six is: the distance of the 5 ' end of goal gene specificity reverse transcriptase primer that step 1 is designed and the binding site of RNA and this goal gene mRNA is 40～180bp.Other step and parameter are identical with embodiment six.
Embodiment ten: the difference of present embodiment and embodiment six is: the distance of the 5 ' end of goal gene specificity reverse transcriptase primer that step 1 is designed and the binding site of RNA and this goal gene mRNA is 50～150bp.Other step and parameter are identical with embodiment six.
Embodiment 11: the difference of present embodiment and embodiment six is: the distance of the 5 ' end of goal gene specificity reverse transcriptase primer that step 1 is designed and the binding site of RNA and this goal gene mRNA is 60～100bp.Other step and parameter are identical with embodiment six.
Embodiment 12: the difference of one of present embodiment and embodiment six to 11 is: the designed joint sequence length of step 1 is greater than 40bp.Other step and parameter are identical with one of embodiment six to 11.
Long more helping more of present embodiment center tap sequence length designed more nido anchor primer, and can reduce the probability that non-specific amplification takes place in this species gene group or cDNA the nido anchor primer.
Embodiment 13: the difference of one of present embodiment and embodiment six to 12 is: the designed nido anchor primer of step 1 is 2～10.Other step and parameter are identical with one of embodiment six to 12.
The Tm value of control nido anchor primer is not less than 58 ℃ in the present embodiment.
Present embodiment can be carried out prerun to each nido anchor primer by single primer PCR before the amplifying target genes fragment, filter out the primer of no visible non-specific amplification product: with each nido anchor primer respectively to the genomic dna of purpose species or total cDNA of gene non-selectivity is carried out the PCR of single primer, abandon the primer of single primer amplification serious (being that single primer PCR obtains clear band or smear), to improve the efficient of nest-type PRC; Being retained in single primer PCR in the purpose species does not have the anchor primer of visible amplified production, can continue on for the clone of other gene in the same species.
Embodiment 14: the difference of one of present embodiment and embodiment six to 12 is: the designed nido anchor primer of step 1 is 3～5.Other step and parameter are identical with one of embodiment six to 12.
Embodiment 15: the difference of one of present embodiment and embodiment six to 14 is: the designed goal gene specificity nest-type PRC primer of step 1 is 2～4.Other step and parameter are identical with one of embodiment six to 14.
Will be in the present embodiment apart from unknown portions (cDNA3 ' end) goal gene specificity reverse transcriptase primer called after GSP-RT farthest, because the chain extension temperature of reverse transcription reaction reaches as high as 45 ℃ usually, therefore, the Tm value of control GSP-RT is about 50 ℃.The gene nest-type PRC primer of each clauses and subclauses near GSP-RT one side called after GSP-1, GSP-2 successively ... GSP-n, select the higher nest-type PRC primer of Tm value, the Tm value of primer is not less than 58 ℃.
The present embodiment reverse transcription reaction carries out the dilution of suitable multiple with product after finishing, as the template of PCR reaction.Each bar nido anchor primer and goal gene specificity nest-type PRC primer can arbitrarily be arranged in pairs or groups, but note relative position: earlier carry out pcr amplification with the nido anchor primer of apart from each other and goal gene Auele Specific Primer and goal gene nest-type PRC primer, again with the template of product, use instead at a distance of primer time far away and carry out nido or heminested PCR as next round PCR.When carrying out nest-type PRC, the band that reclaims last round of PCR gained respectively is as template as far as possible at every turn, if last round of PCR obtains the band of disperse, or band hardly as seen, also can directly this product be diluted certain multiple as template.
Because fragment sequence the unknown to be amplified, therefore the primer of design might have the not only non-specific binding site in a place on the purpose fragment, therefore, take turns the contrast that all will have single primer PCR among the PCR, to get rid of the product that single primer amplification takes place certain bar primer at each.Also can before the clone formally begins, carry out single primer PCR to the reverse transcription product respectively, filter out the primer that single primer PCR does not have visible amplified production with each bar primer.
Embodiment 16: the difference of one of present embodiment and embodiment six to 15 is: goal gene specificity nest-type PRC primer that step 1 is designed and the Tm value between the nido anchor primer differ less than 1 ℃.Other step and parameter are identical with one of embodiment six to 15.
The Tm value of goal gene specificity nest-type PRC primer and nido anchor primer is more approaching in the present embodiment, and non-specific amplification does not take place in each primer substantially in template, is convenient to like this when the clone different goal gene nest-type PRC primers arbitrarily be arranged in pairs or groups with different anchor primers.
Embodiment 17: the difference of one of present embodiment and embodiment six to 16 is: following steps are adopted in checking in the step 4: known array in the sequencing result of step 3 nest-type PRC amplified fragments and the goal gene is spliced, the sequence that splicing is obtained is compared and integrity analysis then, at splicing institute calling sequence two ends design primer, confirm the splicing result again by PCR and order-checking.Other step and parameter are identical with one of embodiment six to 16.
Present embodiment reclaims amplified production behind the amplified production that obtains the goal gene unknown portions, is connected with cloning vector, can learn the sequence of unknown fragment by order-checking.With sequencing result and known array splicing, can infer the complete sequence of gene coding region.Can analyze the exactness and the integrity of institute's calling sequence by BlastX.Again at sequence assembly result's two ends design primer, the genomic dna of purpose species or the cDNA that do not have a gene Selection are carried out pcr amplification, the product that obtains is reclaimed once more, checks order, obtain true, the complete sequence in goal gene coding region.
Embodiment 18: present embodiment is carried out the gene complete encoding sequence amplification according to known 3 '-EST (the genbank accession number is DT083046, shown in SEQ ID NO:1) in the wild soybean:
One, according to goal gene and purpose species purpose of design gene specific reverse transcriptase primer, goal gene specificity nest-type PRC primer and joint sequence, and according to joint sequence design nido anchor primer, 3 ' tip designs of joint sequence has 4 successive guanines;
Two, use Trizol to extract the total RNA of wild soybean blade, get the total RNA of 1 μ g, use the Powerscript reversed transcriptive enzyme of Takara-BD company to carry out reverse transcription, reverse transcription reaction is initial with gene specific primer GSP-RT, add joint in reaction system, elongating temperature is 45 degree; The Powerscript reversed transcriptive enzyme has the terminal enzyme (DNA) activity, optionally adds joint sequence on the cDNA that fully extends to mRNA 5 ' end;
Three, an above step 3 ' the end strand cDNA fragment of having added joint is template, carries out nest-type PRC with goal gene specificity nest-type PRC primer and nido anchor primer and increases; Three-step approach is all adopted in the PCR reaction: 94 ℃ of sex change, landing approach annealing, 72 ℃ of extensions, totally 30 circulations; Wherein annealing temperature drops to primer Tm-10 ℃, 0.5 ℃ of each circulation reduction from primer Tm+5 ℃; The reverse transcription product is diluted 5 times as template, is that primer carries out first round pcr amplification (first round pcr amplification result as shown in Figure 3) with GSP-1 and AP1; Again with 100 times of first round PCR product dilutions as template, be that primer carries out second and takes turns pcr amplification (second takes turns the pcr amplification result as shown in Figure 4) with GSP2 and AP2; Taking turns the recovery of PCR product and dilute 100 times as template second again, is that primer carries out third round pcr amplification (third round pcr amplification result as shown in Figure 5) with GSP3 and AP3;
Four, the nest-type PRC amplified fragments that step 3 is obtained checks order, and utilizes software analysis again, and it is terminal to 5 ' terminal sequence to obtain goal gene 3 ', promptly obtains the goal gene complete encoding sequence after the checking; The designed nido anchor primer of step 1 no visible non-specific amplification product in the species gene group at goal gene place or cDNA wherein.
Known 3 '-EST (being named as PTE-234) of the wild soybean of selecting for use in the present embodiment (Glycine soja), result through the BlastX programanalysis shows that this sequence has comprised goal gene coding region 3 ' end, apart from also the have an appointment aminoacid sequence of 270amino acids (amino acid) of 5 ' end, the nucleotide sequence the unknown of (base pair) is promptly arranged＞800basepair, as shown in Figure 2.
3 of goal gene specificity nest-type PRC primers have been designed in the present embodiment step 1, will be apart from unknown portions (cDNA3 ' end) goal gene specificity reverse transcriptase primer called after GSP-RT farthest, goal gene specificity nest-type PRC primer is near goal gene specificity reverse transcriptase primer one side called after GSP-1, GSP-2 and GSP-3 successively.
GSP-RT：5’-CCTCCTCTAACAATCAAG-3’；
GSP-1：5’-CTAACAATCAAGTTTAGCAAGAAATCC-3’；
GSP-2：5’-CCTCTAGCAACCTATTCAACTTTCTG-3’；
GSP-3：5’-AGATTATGGAACTCTGCCTTTATGTC-3’。
Designed 3 nido anchor primers in the present embodiment step 1, the Tm value of nido anchor primer and goal gene specificity reverse transcriptase primer, goal gene specificity nest-type PRC primer should be more approaching in the present embodiment.From holding nido anchor primer called after AP1～AP3 successively near cDNA 5 '.
AP1：5’-ATAGCAGTGTGATACCATGAGACG-3’；
AP2：5’-GTGTGATACCATGAGACGGGC-3’；
AP3：5’-GGCACATTACGGCTCGGG-3’。
The joint sequence that designs in the present embodiment step 1 is 5 '-ATAGCAGTGTGATACCATGAGACGGGCACATTACGGCTCGGGG-3 '.
Select the Powerscript reversed transcriptive enzyme of Takara-BD company in the present embodiment step 2 for use, the Powerscript enzyme is after extending, can add 3 dC (cytosine(Cyt)) at the synthetic cDNA of institute chain 3 ' end, so 3 ' tip designs of joint sequence is 4 successive dG (guanine).This primer is applicable to the cDNA that is added 3～4 dC after extending.
Through the operation of present embodiment step 3,3 product bands have clearly been obtained." 2 " in Fig. 3～5 and " 3 " swimming lane are the single primer amplification result when wheel primer that PCR uses, wherein do not have visible list primer extension product, show that the non-specific amplification of single primer does not almost take place for goal gene specificity nest-type PRC primer and anchor primer.Therefore, present embodiment joint sequence and 3 nido anchor primers are fit to continue on for the amplification of other gene 5 ' end unknown fragment in the wild soybean.
Present embodiment has the clear band of a size between 750bp～1000bp in the band of 3 nest-type PRC gained, relatively conform to expected results.Reclaim this band order-checking back and existing EST (PTE-234) splicing, and analyze splicing by BlastX, analyze the splicing result as shown in Figure 6, what preliminary proof was obtained is the sequence of goal gene unknown portions, and has comprised the complete 5 ' end in coding region.
So far, begin, passed through 1 reverse transcription reaction and 3 PCR reactions altogether, promptly obtained the segmental amplification of purpose by the preparation of total RNA.Sequencing result shows that this amplification is correct, complete.
Proof test:
Complete encoding sequence according to splicing prediction of result gene, at the two ends, coding region of prediction design total length checking primer, total cDNA with wild soybean is a template, carry out pcr amplification, obtained the big or small band (as shown in Figure 7) that meets expected results, the gained band is checked order, it is complete to have obtained the goal gene coding region, definite sequence (goal gene is GU474544 in the genbank accession number), this complete sequence BlastX analytical results is consistent with the BlastX result (Fig. 6) of splicing institute calling sequence, and comprised complete opening code-reading frame (open reading frame, ORF), ORF predicts the outcome as shown in Figure 8.
Embodiment 19: the present embodiment joint sequence is for being applicable to 5 '-RACE joint sequence of wild soybean (Glycine soja), and this 5 '-RACE joint sequence is 5 '-ATAGCAGTGTGATACCATGAGACGGGCACATTACGGCTCGGGG-3 '; And designed and the corresponding nido anchor primer of present embodiment 5 '-RACE joint sequence, nido anchor primer sequence is shown in SEQ ID NO:7～9.
Sequence table
＜110〉Northeast Agricultural University
＜120〉amplification method of 5 '-RACE joint sequence addition method and joint sequence and 5 ' end unknown gene complete encoding sequence
<160>10
<210>1
<211>186
<212>DNA
＜213〉Glycine (Glycine Willd)
<400>1
gggtactcga?gctgatattg?atatgaaaga?cataaaggca?gagttccata?50
atctatatgg?ggtttcttta?ccccagaaag?ttgaataggt?tgctagaggg?100
agctacaagg?atttcttgct?aaacttgatt?gttagaggag?gctaattgaa?150
gaaattgaaa?agagaacaaa?aacttggaag?atcaac 186
<210>2
<211>945
<212>DNA
＜213〉Glycine (Glycine Willd)
<400>2
atggctttca?atcaagagtt?agaagctgta?acccaagctt?tctcagggca?50
tggagtggat?gagaaatcat?tggtaacatt?actgggaaaa?tgggatcctc?100
tggagagaga?atctttcagg?aagaagacac?cacatttgtt?cagtgaagat?150
catgaacgcc?attttcagag?gtgggatgat?caatatgtct?gtcttctcaa?200
gcatgaattc?gtgcgtttta?agaatgctgt?ggtgctttgg?tccatgcacc?250
cttgggaaag?agatgcccgt?ttagtaaaag?aggccctaaa?gaagggtcca?300
aatgcatatg?gagtgctgat?tgaggtttca?tgcactagat?cctcagaaga?350
gctattggga?gctaggaagg?cataccattc?cctctttgac?cactccattg?400
aagaagatgt?tgcctctcac?atccatggca?ttgaaagaaa?gctattggtt?450
gccctactaa?gtgcctatag?atacgaagga?acaaaggtta?aagatgacac?500
tgcaaaatca?gaggccaaaa?tcctttccaa?tgcaattaag?aatgctcata?550
agaagcctat?tagtgaggat?gatgaagtga?caaggatatt?ggcaacaaga?600
agcaaactac?atctccaagc?agtttacaag?cactataaag?agatatctgg?650
caagaacctt?gatgaggatc?ttgatgattt?aagatttaaa?gaggctgtgc?700
aatgcctttg?taccccacaa?acatatttca?gcaaggtttt?gaatgcagca?750
ttgcgaattg?atgtggacaa?gaatactaag?aaatcgctta?ctcgtgccgt?800
tgttactcga?gctgatattg?atatgaaaga?cataaaggca?gagttccata?850
atctatatgg?ggtttcttta?ccccagaaag?ttgaagaggt?tgctagaggg?900
agctacaagg?atttcttgct?aaacttgatt?gttagaggag?gctaa 945
<210>3
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉goal gene specificity reverse transcriptase primer GSP-RT
<400>3
cctcctctaa?caatcaag?18
<210>4
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉goal gene specificity nest-type PRC primer GSP-1
<400>4
ctaacaatca?agtttagcaa?gaaatcc?27
<210>5
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉goal gene specificity nest-type PRC primer GSP-2
<400>5
cctctagcaa?cctattcaac?tttctg?26
<210>6
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉goal gene specificity nest-type PRC primer GSP-3
<400>6
agattatgga?actctgcctt?tatgtc?26
<210>7
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nido anchor primer AP1
<400>7
atagcagtgt?gataccatga?gacg?24
<210>8
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nido anchor primer AP2
<400>8
gtgtgatacc?atgagacggg?c?21
<210>9
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nido anchor primer AP3
<400>9
ggcacattac?ggctcggg?18
<210>10
<211>43
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the nido anchor primer and at the joint sequence of wild soybean design
<400>10
atagcagtgt?gataccatga?gacgggcaca?ttacggctcg?ggg?43
Claims (10)
1.5 '-RACE joint sequence addition method is characterized in that 5 '-RACE joint sequence adds according to the following steps:
One, according to goal gene purpose of design gene specific reverse transcriptase primer, according to goal gene place species gene stack features designed joint sequence, 3 ' tip designs of joint sequence has at least 3 successive guanines;
Two, in the reverse transcription system, add joint and goal gene specificity reverse transcriptase primer,, realized that 5 '-RACE joint sequence adds then with there being the active reversed transcriptive enzyme of terminal enzyme (DNA) to carry out reverse transcription.
2. 5 '-RACE joint sequence addition method according to claim 1 is characterized in that binding site and this goal gene cDNA3 ' termination of goal gene specificity reverse transcriptase primer that step 1 is designed and mRNA is near.
3. 5 '-RACE joint sequence addition method according to claim 1 is characterized in that 3 ' tip designs of the joint sequence that step 1 is designed has 3,4,5,6,7 or 8 successive guanines.
4. joint sequence is applicable to 5 '-RACE joint sequence of wild soybean (Glycine soja) to it is characterized in that this 5 '-RACE joint sequence is 5 '-ATAGCAGTGTGATACCATGAGACGGGCACATTACGGCTCGGGG-3 '.
5.5 ' amplification method of end unknown gene complete encoding sequence, it is characterized in that 5 ' end unknown gene complete encoding sequence increases according to the following steps:
One, according to goal gene and purpose species purpose of design gene specific reverse transcriptase primer, goal gene specificity nest-type PRC primer and joint sequence, and according to joint sequence design nido anchor primer, 3 ' tip designs of joint sequence has at least 3 successive guanines;
Two, in the reverse transcription system, add joint and goal gene specificity reverse transcriptase primer,, obtain the strand cDNA fragment that 3 ' end has added joint sequence then with there being the active reversed transcriptive enzyme of terminal enzyme (DNA) to carry out reverse transcription;
Three, holding the strand cDNA fragment of having added joint with 3 ' is template, carries out the nest-type PRC amplification with goal gene specificity nest-type PRC primer and nido anchor primer;
Four, the nest-type PRC amplified fragments that step 3 is obtained checks order, and utilizes software analysis again, and it is terminal to 5 ' terminal sequence to obtain goal gene 3 ', promptly obtains the complete encoding sequence of the unknown goal gene of 5 ' end after the checking; The designed nido anchor primer of step 1 no visible non-specific amplification product in the species gene group at goal gene place or cDNA wherein.
6. the amplification method of gene complete encoding sequence according to claim 5 is characterized in that the binding site of specific nest-type PRC primer of the designed goal gene of step 1 and cDNA and this goal gene cDNA3 ' termination are near.
7. the amplification method of gene complete encoding sequence according to claim 5 is characterized in that the designed joint sequence length of step 1 is greater than 40bp.
8. according to the amplification method of claim 5,6 or 7 described gene complete encoding sequences, it is characterized in that the designed nido anchor primer of step 1 is 2～10; The designed goal gene specificity nest-type PRC primer of step 1 is 2～4.
9. the amplification method of gene complete encoding sequence according to claim 8 is characterized in that the Tm value between the designed goal gene specificity nest-type PRC primer of step 1 differs less than 1 ℃.
10. according to the amplification method of claim 5,6,7 or 9 described gene complete encoding sequences, it is characterized in that in the step 4 that checking adopts following steps: known array in the sequencing result of step 3 nest-type PRC amplified fragments and the goal gene is spliced, the sequence that splicing is obtained is compared and integrity analysis then, at splicing institute calling sequence two ends design primer, confirm the splicing result again by PCR.
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Cited By (4)
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| CN102660538A (en) * | 2012-05-04 | 2012-09-12 | 厦门大学 | Method for promoting gene cloning efficiency |
| CN103468670A (en) * | 2012-06-08 | 2013-12-25 | 上海欧孚生物医药科技有限公司 | Full-length cDNA nucleic acid linear amplification method and kit |
| CN107779500A (en) * | 2017-10-25 | 2018-03-09 | 上海药明生物技术有限公司 | A kind of sequence measurement and primer sequence of quick obtaining Rat hybridoma cell monoclonal antibodies sequence |
| CN117904179A (en) * | 2024-01-26 | 2024-04-19 | 甘肃农业大学 | Application of GhANN gene in regulation and control of cold resistance and heat resistance of upland cotton |
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| US5962272A (en) * | 1996-01-03 | 1999-10-05 | Clontech Laboratories, Inc. | Methods and compositions for full-length cDNA Cloning using a template-switching oligonucleotide |
| CN1385539A (en) * | 2002-05-24 | 2002-12-18 | 中山大学 | Garrupa growth hormone gene, carrier having same, strains and production and use for expressino products |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660538A (en) * | 2012-05-04 | 2012-09-12 | 厦门大学 | Method for promoting gene cloning efficiency |
| CN103468670A (en) * | 2012-06-08 | 2013-12-25 | 上海欧孚生物医药科技有限公司 | Full-length cDNA nucleic acid linear amplification method and kit |
| CN103468670B (en) * | 2012-06-08 | 2016-01-20 | 上海欧孚生物医药科技有限公司 | Full-length cDNA nucleic acid linear amplification method and test kit |
| CN107779500A (en) * | 2017-10-25 | 2018-03-09 | 上海药明生物技术有限公司 | A kind of sequence measurement and primer sequence of quick obtaining Rat hybridoma cell monoclonal antibodies sequence |
| CN117904179A (en) * | 2024-01-26 | 2024-04-19 | 甘肃农业大学 | Application of GhANN gene in regulation and control of cold resistance and heat resistance of upland cotton |
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