CN102260670A - Method for excavating low-abundance expression gene and system identification splicing variants by using reverse transcription technique combining nested PCR (Polymerase Chain Reaction) with gene-specific primers (GSP), and application thereof - Google Patents
Method for excavating low-abundance expression gene and system identification splicing variants by using reverse transcription technique combining nested PCR (Polymerase Chain Reaction) with gene-specific primers (GSP), and application thereof Download PDFInfo
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Abstract
The invention discloses a method for excavating a low-abundance expression gene and system identification splicing variants by using a reverse transcription technique combining nested PCR (Polymerase Chain Reaction) with gene-specific primers (GSP), and application thereof. The method is based on reverse transcription PCR of 3'GSP1, gradient PCR is utilized to search for the best annealing temperature of each gene for each primer pair combination, and whether it is a new splicing variant is judged on the basis of GSP nested PCR sequencing and sequence analysis. The method is simple and easy to implement, has the advantages of favorable repetitiveness, high stability and strong reliability, greatly saves the experimental cost, can be applied to excavation of eucaryote low-abundance expression gene under different physiopathological states, and identification and detection of high/low-abundance expression gene splicing variants, and can be used for excavating low-abundance expression gene and identifying and detecting splicing variants of high expression gene and low-abundance expression gene. The method can be used for detecting both known splicing variants of targeted genes and unknown splicing variants of genes, and has wide application prospects.
Description
Technical field
The invention belongs to biological technology application, particularly relate to a kind of method of utilizing nest-type PRC in conjunction with the low abundance expressing gene of reverse transcription technology mining and system's evaluation splice variant of gene specific primer, this method can be applicable in the correlative study and assessment of hanging down abundance expressing gene and the expression of alternative splicing variant under the varying environment.
Background technology
The research report, higher eucaryote has 95% gene generation alternative splicing approximately.Gene is expressed specific splicing form under specific environment, the latter is the biomarker of some disease often, also may be the reason that causes these diseases, therefore find that as much as possible the alternative splicing form of important gene is significant aspect biology and medical research.But because some this abundance of gene transcription is low unusually, this has brought certain difficulty just for the exon composition at the kind of organizing definite special splice variant under specified phase, physiology or the pathological conditions and special transcript.Simultaneously, more and more evidences shows that low abundance expressing gene plays a part very important in the important biomolecule process, for example low abundance expresses the gene splicing variant and cytodifferentiation, metabolism and phenotypic alternation etc. have confidential relation, and may be an important motivating force in the organic evolution process, yet because the low transcript of low abundance expressing gene and high heterogeneous, the understanding that causes people to express gene transcripts for low abundance also is in a starting stage.
In order deeply to be familiar with the 26S Proteasome Structure and Function of low abundance expressing gene, develop the multiple technologies method at present and sought and identify low abundance expressing gene and splice variant thereof, mainly comprised expressed sequence tag (EST) prediction, the sequential analysis of genetic expression (SAGE), the RNA-Seq prediction, mRNA difference shows PCR (DD-PCR), representative variance analysis (RDA), enzyme liberating is subdued, joint is caught and is subdued, the physical removal common sequences, difference is subdued displaying (DSD), restricted mark cDNA scanning, target long-chain polymorphism mark is subdued, suppress subtractive hybridization (SSH), the terminal rapid amplifying (RACE) of cDNA, technology such as SMART-RACE and real-time quantitative PCR.Though these technology can be obtained low abundance expressing gene to a certain extent, but there is himself shortcoming, as false positive height, poor stability, complex steps, workload is big, the cycle is long, cost is high excessively, make it be difficult to generally apply, thereby still do not have the effective ways that excavate low abundance expressing gene and system's evaluation splice variant at present.
Summary of the invention
The object of the present invention is to provide a kind of be used to excavate the gene specific primer that low abundance expressing gene and system identify splice variant (gene-specific primer, GSP).
Gene specific primer provided by the present invention (GSP), be to finish by the primer-design software design of primer-design software or other similar functions, gene specific primer (GSP) comprises 3 ' GSP (near 3 ' end) and 5 ' GSP (near 5 ' end), and requires as far as possible near two ends.Except the prerequisite condition of primer institute of general PCR, gene specific primer (GSP) also needs to satisfy the requirement of gene specific (non-simple sequence alignment level) on thermodynamic characteristics.
The gene specific primer (GSP) that is used to excavate the different splice variants of low abundance expressing gene and system identified gene is in the pairing full length cDNA sequence scope of the known splice variant of gene to be identified, seek possible candidate's primer zone, and design 3 ' GSP with the form of DNA hybridization probe (hybridization probe) and reverse transcription primer, 3 ' the GSP that will design then is directly used in transcriptive process,reversed and nest-type PRC reaction, identifies splice variant in order to excavate low abundance expressing gene and system.
Specifically, the design of described gene specific primer (GSP) is finished automatically by the Primer3 program, and the programdesign main thought is as follows:
1) from NCBI GenBank database, obtain the sequence of a certain gene splicing variant, and according to the GenBank data-base recording or use splign, gmap, softwares such as blat, blast to resolve its gene structure;
2) in the full length cDNA sequence scope, seek possible candidate's primer zone;
3) be candidate's primer marking (Score), form by three parts, i.e. S=S
Basic+ S
End+ S
Genome
A) this mark of primer quality-base (S
Basic): give a mark according to conventional primer quality evaluation system, promptly primer length be that 18～25bp, GC content are about 50%, about 58 ℃ of Tm, high more with the primer marking that these parameters are approaching more;
B) primer and gene two ends are apart from mark (S
End): promptly high more according to the requirement of GSP the closer to two ends marking, give a mark respectively at 5 ' GSP and 3 ' GSP;
C) full genome primer specificity control mark (S
Genome): from the possible primer target locating point of the full genome scanning of thermodynamic (al) angle, site few more (except that target gene) marking is high more;
4), and get the highest preceding 5 primers of marking as the primer that is used to excavate low abundance expressing gene and system's evaluation splice variant according to marking situation ordering candidate primer.
In the method for design of said gene special primer (GSP), usually provide preceding 5 primers alternative in the step 4), if, can not obtain 5 alternative primers through after the screening, so then reduce the primer screening standard according to practical situation, 1-4 bar primer gets final product as alternative before selecting.
Specifically, preferably at wild-type Caenorhabditis elegans N
2The nucleotide sequence of 5 ' GSP1 of the known splice variant design of the glb-18 gene of strain system is shown in sequence in the sequence table 1, the nucleotide sequence of 3 ' GSP1 is shown in sequence in the sequence table 2, the nucleotide sequence of 3 ' GSP2 is shown in sequence in the sequence table 3, and the nucleotide sequence of 3 ' GSP3 is shown in sequence in the sequence table 4; Shown in sequence in the sequence table 5, the nucleotide sequence of 5 ' GSP6 is shown in sequence in the sequence table 6 at the nucleotide sequence of 5 ' GSP5 of the known splice variant design of mouse BDNF gene, and the nucleotide sequence of 3 ' GSP4 is shown in sequence in the sequence table 7.
What second purpose of the present invention provided a kind of simple and easy to do, good reproducibility, stability height, good reliability, cost savings type utilizes the method for nest-type PRC in conjunction with the low abundance expressing gene of reverse transcription technology mining and system's evaluation splice variant of gene specific primer (GSP).
Low abundance expressing gene of excavation provided by the present invention and system identify the method for splice variant, are to utilize the reverse transcription technology of nest-type PRC in conjunction with described gene specific primer (GSP).
The described reverse transcription technology of utilizing nest-type PRC in conjunction with gene specific primer (GSP) is to participate in reverse transcription directly by the gene specific primer (GSP) of different abundance genes, carries out the method for nest-type PRC then.
Specifically, low abundance expressing gene of described excavation and system identify that the method for splice variant can may further comprise the steps:
1) based on the reverse transcription PCR (RT-PCR) of 3 ' GSP1;
2) utilize grads PCR to seek the optimum annealing temperature of every pair of combination of primers of each gene;
3) based on the nest-type PRC of GSP;
4) the nest-type PRC amplified production is checked order (comprising that gel reclaims, connects, transforms, clones, chooses the clone and shakes bacterium, bacterium liquid PCR evaluations, upgrading grain, enzyme and cut the step of identifying and checking order);
5) based on the sequence results analysis of information biology, take a decision as to whether new splice variant.
In aforesaid method, 20 μ l reverse transcription systems and reverse transcription method based on 3 ' GSP1 in the described step 1) are: (1). total RNA 1 μ g, corresponding gene 10pmol/L 3 ' GSP1 1 μ l, RNase Free H
2O polishing to 12 μ l, behind the mixing, 65 ℃ of 5min place on ice then immediately; (2) .5 * RT buffer 4 μ l, dNTP Mixture 2 μ l, RNase inhibitor 1 μ l, ThermoScript II 1 μ l; Carry out reverse transcription by following program behind solution mixing in will (1) and (2): 30 ℃ of 10min earlier, 40 ℃ of 40min then, 85 ℃ of 5min again, last 4 ℃ stop.
Described step 2) 12.5 μ l reaction systems of grads PCR are in: ddH
2O 9.187 μ l, 10 * Ex Taq buffer, 1.25 μ l, dNTP MIxture 1.0 μ l, Ex Taq 0.063 μ l, each 0.25 μ l of upstream and downstream primer, template (being the RT-PCR product of step 1)) 0.5 μ l; Reaction conditions is: 95 ℃ of pre-sex change 5min; 95 ℃ of 45s → different gradient annealing temperatures (gradient is followed successively by 55.0 ℃, 55.5 ℃, 56.8 ℃, 58.8 ℃, 60 ℃, 61.1 ℃, 62 ℃, 63.3 ℃, 65.7 ℃, 67.9 ℃) each 45s → 72 ℃ 1min, totally 35 circulations then; 72 ℃ are extended 5min; Last 4 ℃ stop.
In the described step 3) at wild-type Caenorhabditis elegans N
2The nest-type PRC of the glb-18 gene of strain system is divided into three-wheel to carry out, and reaction system is 12.5 μ l, comprising: ddH
2O 9.187 μ l, 10 * Ex Taq buffer, 1.25 μ l, dNTP Mixture 1.0 μ l, Ex Taq 0.063 μ l, each 0.25 μ l of upstream and downstream primer, template 0.5 μ l; In the first round nest-type PRC reaction system, template is a reverse transcription product, and the upstream and downstream primer is respectively 5 ' GSP1 and 3 ' GSP1; Second takes turns in the nest-type PRC reaction system, and template is the solution after first round nest-type PRC product dilutes 10 times, and the upstream and downstream primer is respectively 5 ' GSP1 and 3 ' GSP2; In the third round nest-type PRC reaction system, template is second to take turns the solution after 10 times of the nest-type PRC product dilutions, and the upstream and downstream primer is respectively 5 ' GSP1 and 3 ' GSP3; Reaction conditions is: 95 ℃ of pre-sex change 5min of elder generation; 95 ℃ of 45s → optimum annealing temperatures are (according to the amplification Caenorhabditis elegans N that is groped then
2The annealing temperature of the glb-18 gene of strain system determines that three-wheel is 60 ℃) 45s → 72 ℃ 1min, 35 circulations; 72 ℃ are extended 5min; Last 4 ℃ stop.
Be divided into five at the nest-type PRC of mouse BDNF gene in the described step 3) and take turns and carry out, reaction system is 12.5 μ l, comprising: ddH
2O 9.187 μ l, 10 * Ex Taq buffer, 1.25 μ l, dNTP Mixture 1.0 μ l, Ex Taq 0.063 μ l, each 0.25 μ l of upstream and downstream primer, template 0.5 μ l; In the first round nest-type PRC reaction system, template is a reverse transcription product, and the upstream and downstream primer is respectively 5 ' GSP4 and 3 ' GSP2; Second takes turns in the nest-type PRC reaction system, and template is the solution after first round nest-type PRC product dilutes 10 times, and the upstream and downstream primer is respectively 5 ' GSP4 and 3 ' GSP3; In the third round nest-type PRC reaction system, template is second to take turns the solution after 10 times of the nest-type PRC product dilutions, and the upstream and downstream primer is respectively 5 ' GSP4 and 3 ' GSP4; In the four-wheel nest-type PRC reaction system, template is the solution after third round nest-type PRC product dilutes 10 times, and the upstream and downstream primer is respectively 5 ' GSP5 and 3 ' GSP4; The 5th takes turns in the nest-type PRC reaction system, and template is the solution after four-wheel nest-type PRC product dilutes 10 times, and the upstream and downstream primer is respectively 5 ' GSP6 and 3 ' GSP4; Reaction conditions is: 95 ℃ of pre-sex change 5min of elder generation; 95 ℃ of 45s → optimum annealing temperatures (, determining that five take turns nest-type PRC and are 62 ℃) 45s → 72 ℃ 1min, totally 35 circulations then according to the annealing temperature of the amplification mouse BDNF gene of being groped; 72 ℃ are extended 5min; Last 4 ℃ stop.
The novel substance that obtains with aforesaid method also belongs to protection scope of the present invention.Described novel substance comprises new low abundance expression gene splicing variant.
Wherein, wild-type Caenorhabditis elegans N
2Strain is that the nucleotide sequence of the new low abundance expression gene splicing variant of glb-18 gene can be shown in sequence in the sequence table 8, and the nucleotide sequence of the new low abundance expression gene splicing variant of mouse BDNF gene can be shown in sequence in the sequence table 9.
The invention provides a kind of method of utilizing nest-type PRC to identify splice variant in conjunction with low abundance expressing gene of gene specific primer (GSP) reverse transcription technology mining and system.The core of this technology at first is the design of GSP, reason be GSP can be special, enrichment efficiently and the gene to be identified that increases, improve the digging efficiency of low abundance expressing gene, next is the nest-type PRC amplification, and reason is the splice variant that the nest-type PRC technology can amplify low abundance expressing gene and gene specifically; The principle of this technology is: 1) based on the reverse transcription PCR (RT-PCR) of 3 ' GSP1, obtain the cDNA of gene to be identified specifically, particularly for low abundance expressing gene, can effectively suppress the amplification of other gene; 2) utilize grads PCR to seek the optimum annealing temperature of every pair of combination of primers of each gene, can guarantee the efficient amplification of low abundance expressing gene; 3) nest-type PRC based on GSP can obtain gene to be identified specifically, but not gene to be identified then can't be amplified out.The present invention can be used for the research of genetic expression under the different physiological statuss of multiple life entity, as intestinal bacteria, yeast, zebra fish, Caenorhabditis elegans, mouse and cell etc., and can be by the low abundance of difference be expressed gene design GSP, adjust and optimize at the difference research demand of different life entities.Present method is simple and easy to do, good reproducibility, stability is high, good reliability, and greatly saved experimental cost, can substitute the existing conventional technology, can be applicable to the excavation and the height of the low abundance expressing gene of eukaryote under the different physiological and pathological states, (described technology not only can be applicable to excavate low abundance expressing gene in the evaluation and discovery of low abundance expression gene splicing variant, more can be used for identifying and finding the splice variant of cance high-expression gene and low abundance expressing gene, in addition, except target is decided the known splice variant of gene, more can be used for the discovery of the unknown splice variant of gene), have a extensive future.
Below in conjunction with specific embodiment the present invention is described in further details.
Description of drawings
Fig. 1 identifies the schema of the method for splice variant for the present invention utilizes nest-type PRC in conjunction with low abundance expressing gene of gene specific primer (GSP) reverse transcription technology mining and system
Fig. 2 is the GSP design (Caenorhabditis elegans glb-18 gene specific primer design information) at the different splice variants of same gene
Fig. 3 is a BDNF gene specific primer design information
Fig. 4 is for strain is the nest-type PRC product electrophoretogram of glb-18 gene at wild-type Caenorhabditis elegans N2
Fig. 5 is the nest-type PRC product electrophoretogram at mouse BDNF gene
Embodiment
Embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Method therefor is ordinary method if no special instructions among the following embodiment.
Embodiment 1: the design that is used to excavate the gene specific primer (GSP) that hangs down abundance expressing gene and system's evaluation splice variant
The design of gene specific primer (GSP) is finished automatically by the Primer3 program, and the programdesign main thought is as follows:
5) from NCBI GenBank database, obtain the sequence of a certain gene splicing variant, and according to the GenBank data-base recording or use splign, gmap, softwares such as blat, blast to resolve its gene structure;
6) in the full length cDNA sequence scope, seek possible candidate's primer zone;
7) be the marking of candidate's primer, form by three parts, i.e. S=S
Basic+ S
End+ S
Genome:
A) this mark of primer quality-base (S
Basic): give a mark according to conventional primer quality evaluation system, promptly about 50%, the Tm of primer length 18～25bp, GC content is about 58 ℃, and is high more with the primer marking that these parameters are approaching more.
B) primer and gene two ends are apart from mark (S
End): promptly high more according to the requirement of GSP the closer to two ends marking, give a mark respectively at 5 ' GSP and 3 ' GSP;
C) full genome primer specificity control mark (S
Genome): from the possible primer target locating point of the full genome scanning of thermodynamic (al) angle, site few more (except that target gene) marking is high more;
8), and get the highest preceding 5 primers (alternative) of marking as the primer that is used to excavate low abundance expressing gene and system's evaluation splice variant according to marking situation ordering candidate primer.Usually provide preceding 5 primers alternative.If after screening, can not obtain 5 alternative primers, so then reduce the primer screening standard according to practical situation, 1-4 bar primer gets final product as alternative before selecting.
Respectively with wild-type Caenorhabditis elegans N
2The glb-18 gene of strain system and the known splice variant of mouse BDNF gene are that example is carried out design of primers (Fig. 2 is the design of primers information (at the GSP design of different splice variant F52A8.4b of same gene and F52A8.4a) of glb-18 gene; Fig. 3 is for being that (1,2,3,4,5 represent the order and the combination of primers of nest-type PRC respectively for the nest-type PRC mode chart of example with one of them splice variant 001048139.1 of BDNF gene; 5G4,5G5,5G6 represent upstream primer respectively; 3G1,3G2,3G3,3G4 represent downstream primer respectively).Primer sequence is as follows:
At wild-type Caenorhabditis elegans N
2The known splice variant of the glb-18 gene of strain system (GenBank number: NM_001136303.1) She Ji special primer:
5 ' GSP1:tgccgtctgctgctcgtcaa (sequence 1 in the sequence table);
3 ' GSP1:ggcggaaaacgattgacacctttca (sequence 2 in the sequence table);
3 ' GSP2:tcctccacaccgtcactgcg (sequence 3 in the sequence table);
3 ' GSP3:tcggtcataatggagagacggtgtt (sequence 4 in the sequence table).
At the known splice variant of mouse BDNF gene (GenBank number: 001048139.1) She Ji special primer:
5 ' GSP5:cgaggttcggctcacaccga (sequence 5 in the sequence table);
5 ' GSP6:agccccagtttggtcccctc (sequence 6 in the sequence table);
3 ' GSP4:gagtcccatgggtccgcaca (sequence 7 in the sequence table).
Embodiment 2: utilize gene specific primer (GSP) in conjunction with the application of nest-type PRC technology in evaluation and the low abundance expressing gene of discovery
Utilize gene specific primer (GSP, the primer that designs with embodiment 1 is an example) in conjunction with the application of nest-type PRC technology in evaluation and the low abundance expressing gene of discovery, concrete grammar may further comprise the steps (schema is as shown in Figure 1):
One, depends on the RNA reverse transcription of 3 ' GSP1
With total RNA (the wild-type Caenorhabditis elegans N that extracts
2Strain system or mouse) be template, reverse transcription goes out corresponding gene cDNA first chain.20 μ l reverse transcription systems and reverse transcription method based on 3 ' GSP1 are (agents useful for same is available from TOYOBO company): (1). total RNA 1 μ g, corresponding gene 10pmol/L 3 ' GSP1 1 μ l, RNase Free H
2O polishing to 12 μ l, behind the mixing, 65 ℃ of 5min place on ice then immediately; (2) .5 * RT buffer 4 μ l, dNTP Mixture 2 μ l, RNase inhibitor 1 μ l, ThermoScript II 1 μ l; Carry out reverse transcription by following program behind solution mixing in will (1) and (2): 30 ℃ of 10min earlier, 40 ℃ of 40min then, 85 ℃ of 5min again, last 4 ℃ stop.Instantaneous centrifugal after ,-20 ℃ of preservations are stand-by.
Two, utilize thermograde PCR to seek the optimum annealing temperature of every pair of combination of primers of each gene
In the experiment respectively at wild-type nematode N
27 low abundance expressing gene (egl-9 (GenBank number: NM_001028661.2 in the strain system, NM_171533.3, NM_001028663.2, NM_001047640.1, NM_001028662.1), hif-1 (GenBank number: NM_075607.4, NM_001028720.2, NM_001028722.2, NM_001028721.1), glb-3 (GenBank number: NM_073431.1), glb-5 (GenBank number: NM_001129381.1, NM_072065.2), glb-8 (GenBank number: NM_059706.2, NM_001136283.1), glb-13 (GenBank number: NM_077678.4), glb-18 (GenBank number: NM_001136303.1, NM_059673.5)) carry out thermograde PCR with the different combination of primers of the low abundance expressing gene BDNF of mouse, the reaction system of every pair of primer PCR is 12.5 μ l, comprising: ddH
2O 9.187 μ l, 10 * Ex Taq buffer, 1.25 μ l, dNTP Mixture 1.0 μ l, Ex Taq 0.063 μ l, each 0.25 μ l of upstream and downstream primer, template (being the RT-PCR product of step 1) 0.5 μ l; Reaction conditions is: 95 ℃ of pre-sex change 5min; 95 ℃ of 45s → different gradient annealing temperatures (gradient is followed successively by 55.0 ℃, 55.5 ℃, 56.8 ℃, 58.8 ℃, 60 ℃, 61.1 ℃, 62 ℃, 63.3 ℃, 65.7 ℃, 67.9 ℃) each 45s → 72 ℃ 1min, totally 35 circulations then; 72 ℃ are extended 5min; Last 4 ℃ stop.After reaction finishes, the PCR product is carried out 2% agarose gel electrophoresis detect, concrete steps are as follows: behind PCR product (12.5 μ l) and 6 * Loading buffer (2.5 μ l) mixing, add and contain GoldView
TMIn 2% sepharose of nucleic acid dye, voltage stabilizing 120V electrophoresis 50min, the sepharose after utilizing the gel imaging instrument with electrophoresis is taken pictures and is preserved.By analyzing gel images, seek the optimum annealing temperature of every pair of combination of primers of each gene.
Three, in conjunction with the nest-type PRC of gene specific primer
At wild-type nematode N
2The nest-type PRC of the glb-18 gene of strain system is divided into three-wheel to carry out, and reaction system is 12.5 μ l, comprising: ddH
2O 9.187 μ l, 10 * Ex Taq buffer, 1.25 μ l, dNTP Mixture 1.0 μ l, Ex Taq 0.063 μ l, each 0.25 μ l of upstream and downstream primer, template 0.5 μ l; In the first round nest-type PRC reaction system, template is a reverse transcription product, and the upstream and downstream primer is respectively 5 ' GSP1 and 3 ' GSP1; Second takes turns in the nest-type PRC reaction system, and template is the solution after first round nest-type PRC product dilutes 10 times, and the upstream and downstream primer is respectively 5 ' GSP1 and 3 ' GSP2; In the third round nest-type PRC reaction system, template is second to take turns the solution after 10 times of the nest-type PRC product dilutions, and the upstream and downstream primer is respectively 5 ' GSP1 and 3 ' GSP3; Reaction conditions is: 95 ℃ of pre-sex change 5min of elder generation; 95 ℃ of 45s → optimum annealing temperatures are (according to the amplification Caenorhabditis elegans N that is groped then
2The annealing temperature of the glb-18 gene of strain system determines that three-wheel is 60 ℃) 45s → 72 ℃ 1min, 35 circulations; 72 ℃ are extended 5min; Last 4 ℃ stop.The PCR product is detected with 2% agarose gel electrophoresis, and concrete steps are as follows: behind PCR product (5 μ l) and 6 * Loading buffer (1.5 μ l) mixing, add and contain GoldView
TMIn 2% sepharose of nucleic acid dye, voltage stabilizing 120V electrophoresis 50min, the sepharose photographic analysis after utilizing the gel imaging instrument with electrophoresis.
Be divided into five at the nest-type PRC of mouse BDNF gene and take turns and carry out, reaction system is 12.5 μ l, comprising: ddH
2O 9.187 μ l, 10 * Ex Taq buffer, 1.25 μ l, dNTP Mixture 1.0 μ l, Ex Taq 0.063 μ l, each 0.25 μ l of upstream and downstream primer, template 0.5 μ l; In the first round nest-type PRC reaction system, template is a reverse transcription product, and the upstream and downstream primer is respectively 5 ' GSP4 and 3 ' GSP2; Second takes turns in the nest-type PRC reaction system, and template is the solution after first round nest-type PRC product dilutes 10 times, and the upstream and downstream primer is respectively 5 ' GSP4 and 3 ' GSP3; In the third round nest-type PRC reaction system, template is second to take turns the solution after 10 times of the nest-type PRC product dilutions, and the upstream and downstream primer is respectively 5 ' GSP4 and 3 ' GSP4; In the four-wheel nest-type PRC reaction system, template is the solution after third round nest-type PRC product dilutes 10 times, and the upstream and downstream primer is respectively 5 ' GSP5 and 3 ' GSP4; The 5th takes turns in the nest-type PRC reaction system, and template is the solution after four-wheel nest-type PRC product dilutes 10 times, and the upstream and downstream primer is respectively 5 ' GSP6 and 3 ' GSP4; Reaction conditions is: 95 ℃ of pre-sex change 5min of elder generation; 95 ℃ of 45s → optimum annealing temperatures are (according to the amplification Caenorhabditis elegans N that is groped then
2The annealing temperature of the glb-18 gene of strain system determines that five take turns nest-type PRC and are 62 ℃) 45s → 72 ℃ 1min, totally 35 circulations; 72 ℃ are extended 5min; Last 4 ℃ stop.The PCR product is detected with 2% agarose gel electrophoresis, and detection method is same as described above.
Experimental result shows that nest-type PRC not only can angle out the purpose fragment of desired length on certain splice variant specifically in conjunction with gene specific primer (GSP) reverse transcription technology, and also has some specific bands also can be angled except the purpose fragment to take out.Fig. 4 and Fig. 5 are respectively at wild-type Caenorhabditis elegans N
2Strain is the experimental result of glb-18 gene and mouse BDNF gene splicing variant.The splice variant of the band that marks with red line among Fig. 4 for angling out successively, the band that green collimation mark goes out is to be the progressively specific band of production decline law.The splice variant of the band that marks with red line among Fig. 5 for angling out successively, the band that green frame and yellow collimation mark go out is to be the progressively specific band of production decline law.
Four, order-checking and sequential analysis
Carrying out gel at purpose band and specific band reclaims, is connected, transforms, clones, chooses the clone and shake bacterium, bacterium liquid PCR evaluations, upgrading grain, enzyme and cut evaluation, check order and sequencing result is analyzed (Biochem Biophys Res Commun, A lentiviral vector with novel multiple cloning sites:stable transgene expression in vitro and in vivo, 2008,371 (3): 546-50; Sheng Wu Gong Cheng Xue Bao, Cloning and sequence analysis of SOCS-2 gene in pig, 2007,23 (6): 1091-6), determine it is known splice variant or new splice variant.At sequencing result, utilize the information biology software analysis after, find wherein corresponding to wild-type Caenorhabditis elegans N
2Strain is the new splice variant that the specific band of glb-18 gene and mouse BDNF gene (specific band in the green frame) promptly is respectively corresponding gene.Wherein, wild-type Caenorhabditis elegans N
2Strain be the nucleotide sequence of new splice variant of glb-18 gene shown in sequence in the sequence table 8, the nucleotide sequence of the new splice variant of mouse BDNF gene is shown in sequence in the sequence table 9.
Five, submit sequence to
At new splice variant, be the GenBank form with Sequin software with the sequence arrangement, submit to the NCBIGenBank database.The GenBank of two new splice variants after the Sequin software processes submits to form as follows:
1, wild-type Caenorhabditis elegans N
2Strain is the new splice variant of glb-18 gene
2, the new splice variant of mouse BDNF gene
Claims (13)
1. be used to excavate the gene specific primer (GSP) of low abundance expressing gene and system's evaluation splice variant, be in the full length cDNA sequence scope of the known splice variant of gene to be identified, to seek possible candidate's primer zone, and design 3 ' GSP with the form of the compatible reverse transcription primer of DNA hybridization probe, 3 ' the GSP that will design then is directly used in reverse transcription and nest-type PRC reaction, in order to excavate the alternative splicing variant of low abundance expressing gene and system's identified gene.
2. gene specific primer according to claim 1 (GSP) is characterized in that: the design of described gene specific primer (GSP) is finished automatically by the Primer3 program, and the programdesign main thought is as follows:
1) from NCBI GenBank database, obtain the sequence of a certain gene splicing variant, and according to the GenBank data-base recording or use splign, gmap, softwares such as blat, blast to resolve its gene structure;
2) in the full length cDNA sequence scope, seek possible candidate's primer zone;
3) be the marking of candidate's primer, form by three parts, i.e. S=S
Basic+ S
End+ S
Genome:
A) this mark of primer quality-base (S
Basic): according to the marking of conventional primer quality evaluation system, promptly about 50%, the Tm of primer length 18～25bp, GC content is about 58 ℃, and is high more with the primer marking that these parameters are approaching more;
B) primer and gene two ends are apart from mark (S
End): promptly high more according to the requirement of GSP the closer to two ends marking, give a mark respectively at 5 ' GSP and 3 ' GSP;
C) full genome primer specificity control mark (S
Genome): from the possible primer target locating point of the full genome scanning of thermodynamic (al) angle, site few more (except that target gene) marking is high more;
4), and get the highest preceding 5 primers (alternative) of marking as the primer that is used to excavate low abundance expressing gene and system's evaluation splice variant according to marking situation ordering candidate primer.
3. gene specific primer according to claim 1 and 2 (GSP), it is characterized in that: provide preceding 5 primers alternative in the described step 4) usually, if after screening, can not obtain 5 alternative primers, so then reduce the primer screening standard according to practical situation, 1-4 bar primer gets final product as alternative before selecting; At wild-type Caenorhabditis elegans N
2The nucleotide sequence that 5 ' GSP1 of the known splice variant design of the glb-18 gene of strain system has sequence 1 in the sequence table, 3 ' GSP1 has the nucleotide sequence of sequence 2 in the sequence table, 3 ' GSP2 has the nucleotide sequence of sequence 3 in the sequence table, and 3 ' GSP3 has the nucleotide sequence of sequence 4 in the sequence table; At the nucleotide sequence that 5 ' GSP5 of the known splice variant design of mouse BDNF gene has sequence 5 in the sequence table, 5 ' GSP6 has the nucleotide sequence of sequence 6 in the sequence table, and 3 ' GSP4 has the nucleotide sequence of sequence 7 in the sequence table.
4. one kind is utilized nest-type PRC to identify the method for splice variant in conjunction with low abundance expressing gene of the reverse transcription technology mining of gene specific primer (GSP) and system, and it is characterized in that: this method has been utilized the reverse transcription technology of nest-type PRC in conjunction with each described gene specific primer (GSP) of claim 1-3.
5. method according to claim 4, it is characterized in that: the described reverse transcription technology of utilizing nest-type PRC in conjunction with gene specific primer (GSP), be to participate in reverse transcription directly, carry out the method for nest-type PRC then by the gene specific primer (GSP) of different abundance genes.
6. method according to claim 5 is characterized in that: said method comprising the steps of:
1) based on the reverse transcription PCR of 3 ' GSP1;
2) utilize grads PCR to seek the optimum annealing temperature of every pair of combination of primers of each gene;
3) based on the nest-type PRC of GSP;
4) order-checking;
5) based on the sequence results analysis of information biology, take a decision as to whether new splice variant.
7. method according to claim 6 is characterized in that: 20 μ l reverse transcription systems and reverse transcription method based on 3 ' GSP1 in the described step 1) are: (1). total RNA 1 μ g, corresponding gene 10pmol/L 3 ' GSP1 1 μ l, RNase Free H
2O polishing to 12 μ l, behind the mixing, 65 ℃ of 5min place on ice then immediately; (2) .5 * RT buffer 4 μ l, dNTP Mixture 2 μ l, RNase inhibitor 1 μ l, ThermoScript II 1 μ l; Carry out reverse transcription by following program behind solution mixing in will (1) and (2): 30 ℃ of 10min earlier, 40 ℃ of 40min then, 85 ℃ of 5min again, last 4 ℃ stop.
8. according to claim 6 or 7 described methods, it is characterized in that: 12.5 μ l reaction systems of grads PCR are described step 2): ddH
2O 9.187 μ l, 10 * Ex Taq buffer, 1.25 μ l, dNTP Mixture 1.0 μ l, Ex Taq 0.063 μ l, each 0.25 μ l of upstream and downstream primer, template (being the RT-PCR product of step 1)) 0.5 μ l; Reaction conditions is: 95 ℃ of pre-sex change 5min; 95 ℃ of 45s → different gradient annealing temperatures (gradient is followed successively by 55 ℃, 55.5 ℃, 56.8 ℃, 58.8 ℃, 60 ℃, 61.1 ℃, 62 ℃, 63.3 ℃, 65.7 ℃, 67.9 ℃) each 45s → 72 ℃ 1min, totally 35 circulations then; 72 ℃ are extended 5min; Last 4 ℃ stop.
9. according to claim 6 or 7 or 8 described methods, it is characterized in that: in the described step 3) at wild-type Caenorhabditis elegans N
2The nest-type PRC of the glb-18 gene of strain system is divided into three-wheel to carry out, and reaction system is 12.5 μ l, comprising: ddH
2O 9.187 μ l, 10 * Ex Taq buffer, 1.25 μ l, dNTP Mixture 1.0 μ l, Ex Taq 0.063 μ l, each 0.25 μ l of upstream and downstream primer, template 0.5 μ l; In the first round nest-type PRC reaction system, template is a reverse transcription product, and the upstream and downstream primer is respectively 5 ' GSP1 and 3 ' GSP1; Second takes turns in the nest-type PRC reaction system, and template is the solution after first round nest-type PRC product dilutes 10 times, and the upstream and downstream primer is respectively 5 ' GSP1 and 3 ' GSP2; In the third round nest-type PRC reaction system, template is second to take turns the solution after 10 times of the nest-type PRC product dilutions, and the upstream and downstream primer is respectively 5 ' GSP1 and 3 ' GSP3; Reaction conditions is: 95 ℃ of pre-sex change 5min of elder generation; 95 ℃ of 45s → optimum annealing temperatures (three-wheel is 60 ℃) 45s → 72 ℃ 1min then, 35 circulations; 72 ℃ are extended 5min; Last 4 ℃ stop.
10. according to claim 6 or 7 or 8 described methods, it is characterized in that: be divided into five at the nest-type PRC of mouse BDNF gene in the described step 3) and take turns and carry out, reaction system is 12.5 μ l, comprising: ddH
2O 9.187 μ l, 10 * Ex Taq buffer, 1.25 μ l, dNTP Mixture 1.0 μ l, Ex Taq 0.063 μ l, each 0.25 μ l of upstream and downstream primer, template 0.5 μ l; In the first round nest-type PRC reaction system, template is a reverse transcription product, and the upstream and downstream primer is respectively 5 ' GSP4 and 3 ' GSP2; Second takes turns in the nest-type PRC reaction system, and template is the solution after first round nest-type PRC product dilutes 10 times, and the upstream and downstream primer is respectively 5 ' GSP4 and 3 ' GSP3; In the third round nest-type PRC reaction system, template is second to take turns the solution after 10 times of the nest-type PRC product dilutions, and the upstream and downstream primer is respectively 5 ' GSP4 and 3 ' GSP4; In the four-wheel nest-type PRC reaction system, template is the solution after third round nest-type PRC product dilutes 10 times, and the upstream and downstream primer is respectively 5 ' GSP5 and 3 ' GSP4; The 5th takes turns in the nest-type PRC reaction system, and template is the solution after four-wheel nest-type PRC product dilutes 10 times, and the upstream and downstream primer is respectively 5 ' GSP6 and 3 ' GSP4; Reaction conditions is: 95 ℃ of pre-sex change 5min of elder generation; 95 ℃ of 45s → optimum annealing temperatures (five take turns be 62 ℃) 45s → 72 ℃ 1min, totally 35 circulations then; 72 ℃ are extended 5min; Last 4 ℃ stop.
11. the excavation of each described method of claim 4-10 low abundance expressing gene under the different physiological and pathological states of eukaryote, and high and low abundance is expressed the evaluation of gene splicing variant and the application in the discovery, it is characterized in that: except target was decided the known splice variant of gene, described method more can be used for the excavation of the unknown splice variant of gene.
12. the novel substance that uses or obtain in each described method of claim 4-10, it is characterized in that: described novel substance comprises new splice variant.
13. novel substance according to claim 14 is characterized in that: described wild-type nematode N
2Strain be the nucleotide sequence of new splice variant of glb-18 gene shown in sequence in the sequence table 8, the nucleotide sequence of the new splice variant of mouse BDNF gene is shown in sequence in the sequence table 9.
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| CN113249512A (en) * | 2021-05-28 | 2021-08-13 | 广西壮族自治区农业科学院 | Method for identifying different cytoplasms of kenaf based on difference of mitochondrial gene transcripts and application |
| CN115725711A (en) * | 2022-08-16 | 2023-03-03 | 深圳市血液中心(深圳市输血医学研究所) | Amplification primer group of blood group antigen coding gene in frozen whole blood, amplification method and genotyping method |
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| CN110268473A (en) * | 2017-02-08 | 2019-09-20 | 微软技术许可有限责任公司 | The design of primers of polynucleotides for being stored fetched |
| CN109609605A (en) * | 2019-01-28 | 2019-04-12 | 西南大学 | The detection method of montage phenomenon in Htt genetic transcription RNA Exon1 |
| CN113249512A (en) * | 2021-05-28 | 2021-08-13 | 广西壮族自治区农业科学院 | Method for identifying different cytoplasms of kenaf based on difference of mitochondrial gene transcripts and application |
| CN115725711A (en) * | 2022-08-16 | 2023-03-03 | 深圳市血液中心(深圳市输血医学研究所) | Amplification primer group of blood group antigen coding gene in frozen whole blood, amplification method and genotyping method |
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