CN107245526A - The gene promoter areas of Hsa miR 17 and its PCR amplifications identification primer sets and reaction system - Google Patents
The gene promoter areas of Hsa miR 17 and its PCR amplifications identification primer sets and reaction system Download PDFInfo
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Abstract
It is main to include the feature of three aspects the invention discloses a set of PCR amplifications that can effectively expand the encoding gene promoters areas of Hsa miR 17 and Sanger sequencing identifications primer and its supporting reaction system：（1）5 ' special tailing PCR primers, sequence is as shown in SEQ ID NO.1 and 2；（2）The final concentration of DMSO of volume ratio 3~5% special PCR reaction systems are with the addition of in standard PCR amplification system；（3）A set of forward and reverse to identify the Sanger sequencing primer systems for expanding purpose fragment correct sequence twice, sequence is as shown in SEQ ID NO.3~10.The primer system and reaction system of the present invention can effectively expand the encoding gene promoters area fragments of Hsa miR 17, and the correct sequence of whole section of amplified fragments can be identified, for promoting the correlative study of the expression regulations of Hsa miR 17 that there is practical significance, with good application value.
Description
Technical field
The invention belongs to biological technical field.Hsa-miR-17 encoding genes can be effectively expanded more particularly, to a set of
The PCR amplifications of promoter region and Sanger sequencing identifications primer and its supporting reaction system.
Background technology
MicroRNA（miRNA）It is the small molecule single stranded RNA that a class endogenous, non-coding, length are about 18~25bp, its
By with complementary mRNA 3 ' non-translational regions complete or being not fully integrated and regulating and controlling the degraded and translation of corresponding mRNA, so that
Regulate and control corresponding protein expression.MiRNA not only take part in the regulation and control of the biological processes such as cell differentiation, increment, apoptosis, also with
Generation, the development of tumour are closely related.Promoter region refers to be located at the section of DNA sequence that structural gene 5 ' holds upstream, difference turn
The record factor regulates and controls the expression of corresponding gene by being combined with specific promoter area, so as to realize to corresponding physiology or pathologic process
Regulation.Therefore miR-96 gene promoter region is studied to the interaction between specific transcription factor for illustrating corresponding miRNA's
Expression and regulation mechanism and then to clear its pathology, physiological function significant.
Luciferase reporter gene experiment is to detect transcription factor interacts with target gene promoter region DNA one
Classic Experiments method is planted, its core is need to build the luciferase reporter gene of insertion specific gene promoter region DNA fragmentation
Plasmid, and the premise for building plasmid is then the DNA fragmentation for obtaining promoter region.Therefore effective start is obtained using PCR amplifications
Sub-district DNA fragmentation has important practical value to the functional study for carrying out related gene.But in very polygenic promoter
There is the special gene structure on such as CpG islands in area, and these special gene structures often cause the mispairing of primer or cause office
The formation of portion's secondary structure fails so as to cause PCR to expand.So which kind of method to realize having for promoter region DNA fragmentation by
Effect PCR amplifications and identification are the problem of correlative study work need to often be faced.
Expressed and related to prognosis in addition, Hsa-miR-17 is high in the kinds of tumors tissue such as glioma, leukaemia, because
This Hsa-miR-17 is an important potential oncogene.Hsa-miR-17 has also assisted in mammary cancer chemotherapy response and non-small simultaneously
The targeted therapy resistance of cell lung cancer, so it is also a potential anti-tumor medicine target spot.But Hsa-miR-17
Expression regulation and correlation function are not yet illustrated completely, therefore as described above, obtain effective Hsa-miR-17 promoter regions DNA
Fragment has important practical value to the functional study for carrying out Hsa-miR-17, the correlative study for promoting Hsa-miR-17
With practical significance.
The content of the invention
The technical problem to be solved in the present invention is that the defect and deficiency for overcoming above-mentioned prior art can effectively expand there is provided a set of
Increase PCR amplifications and Sanger sequencing identifications primer and its supporting reaction system in Hsa-miR-17 encoding gene promoters area.
An object of the present invention is to provide a kind of PCR that can effectively expand Hsa-miR-17 encoding gene promoters area and expanded
Increase primer.
Another object of the present invention is to provide a kind of PCR amplifications that can effectively expand Hsa-miR-17 encoding gene promoters area
System.
Another object of the present invention be to provide one kind can forward and reverse Sanger sequencing identifications Hsa-miR-17 encoding genes open
The primer system of mover region sequence.
Another object of the present invention is to provide Hsa-miR-17 gene promoter region sequences.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
The pcr amplification primer thing in Hsa-miR-17 encoding gene promoters area, upstream and downstream can be effectively expanded the invention provides a kind of
Primer is as shown in SEQ ID NO.1 and 2.
The PCR amplification system in Hsa-miR-17 encoding gene promoters area can be effectively expanded present invention also offers a kind of,
The DMSO of final concentration of volume ratio 3~5% is added in standard PCR amplification system.
Preferably, the PCR amplification system is：The μ l of Green Mix 7.5,10 μM of upstream and downstream primers each 1 μ l, 3~5%
DMSO H2The 1 μ l of μ l, DNA of O 4.5.
Can forward and reverse Sanger sequencing identifications Hsa-miR-17 encoding gene promoters area sequence present invention also offers one kind
The primer system of row, including four positive sequencing primers and four reverse sequencing primers, sequence is respectively such as SEQ ID NO.3~10
It is shown.
In summary, the invention provides a set of PCR expansions that can effectively expand Hsa-miR-17 encoding gene promoters area
Increase and Sanger sequencing identifications primer and its supporting reaction system, it is main to include the feature of three aspects：
（1）5 ' special tailing PCR primers；（2）Final concentration of 3~5% DMSO spy is with the addition of in standard PCR amplification system
Different PCR reaction systems；（3）It is a set of forward and reverse to identify the Sanger sequencing primer bodies for expanding purpose fragment correct sequence twice
System.
First, it is specific as follows present invention uses the tailing pcr amplification primer thing of special 5 '：
Sense primer（SEQ ID NO.1）：
5′-GAACCAGATCTTGGAATTCTTCGAAGGCTACGCGGAGAATC-3′（Underscore part is tailed primer sequence
Row）,
Anti-sense primer（SEQ ID NO.2）：
5′-GCTCGGTACCAAGCGCTACCGGTTACCTGCACTGTAAGCACTTT-3′（Underscore part is tailed primer sequence
Row）.
Secondly, present invention uses the DMSO that final concentration of 3~5% are with the addition of in standard PCR amplification system.
It can be realized by the PCR amplification system that 5 ' tailing PCR primers and the DMSO containing final concentration 3~5% are used in combination
The Successful amplification in Hsa-miR-17 encoding gene promoters area.
Finally, the present invention realizes the Hsa- to Successful amplification using forward and reverse Sanger sequencing identifications primer system
The complete sequence determination identification of miR-17 encoding gene promoters area fragment.
Forward and reverse Sanger sequencing identifications primer system includes following component：
Positive sequencing primer：
Positive sequencing primer 1（SEQ ID NO.3）：5′-TGCCATCAGGACCACA-3′；
Positive sequencing primer 2（SEQ ID NO.4）：5′-TAGATTTGGACGGTGGTA-3′；
Positive sequencing primer 3（SEQ ID NO.5）：5′-TGGCTTATGCAGTTTACG-3′；
Positive sequencing primer 4（SEQ ID NO.6）：5′-CCCATTAGGGATTATGCT-3′；
Reverse sequencing primer：
Reverse sequencing primer 1（SEQ ID NO.7）：5′-TGCTACAAGTGCCTTCAC-3′；
Reverse sequencing primer 2（SEQ ID NO.8）：5′-CTGGAAGTGGTGGCTCT-3′；
Reverse sequencing primer 3（SEQ ID NO.9）：5′-TGCATAAGCCAGTTTCC-3′；
Reverse sequencing primer 4（SEQ ID NO.10）：5′-AGAGTAAGAGTCGAGCAATAC-3′.
Therefore, present invention also offers a kind of method that can effectively expand Hsa-miR-17 encoding gene promoters area, bag
Include following steps：
S1. using sample DNA as template, entered using pcr amplification primer thing described in claim 1 and contained in performing PCR amplification, amplification system
The DMSO of volumetric concentration 3~5%；
S2. amplified production electrophoresis obtains the amplified production for having 1608bp purpose bands；
S3. amplified production step S2 obtained is purified, and PCR primer does sequencing reaction after purification, obtains Hsa-miR-17 codings
Gene promoter region sequence.
Wherein it is preferred to, the amplification system that PCR described in step S1 is expanded is：The μ l of Green Mix 7.5,10 μM of upstream and downstream
Primer each 1 μ l, 3~5% DMSO H2The 1 μ l of μ l, DNA of O 4.5.
Preferably, the reaction condition that PCR described in step S1 is expanded：94℃3min；94 DEG C of 30s, 53~62 DEG C of 30s, 72 DEG C
45s, totally 40 circulation；72℃7 min.
Preferably, the method purified described in step S3：Take the μ l of PCR primer 5 to add the 2 prosperous promise SAP enzymatic mixtures in μ l Beijing to mix
It is even, 37 DEG C of 1h, 80 DEG C of 15min.
Preferably, the reaction system of sequencing reaction described in step S3：The μ l of purified pcr product 3, Bigdye3.1 1 μ l, sequencing
Primer（5pM/μl）2μl.
Preferably, the reaction condition of sequencing reaction described in step S3：96℃1 min；96℃10s、50℃5s、60℃4
Min, 25 circulations；12 DEG C of constant temperature；Thereafter sodium acetate ethanol purification sequencing reaction product, upper machine sequencing.
A kind of kit that can effectively expand Hsa-miR-17 encoding gene promoters area also should be in protection model of the invention
Within enclosing, the kit includes the primer shown in above-mentioned SEQ ID NO.1~10.
Preferably, the kit also includes DMSO.
Preferably, the kit also includes reagent needed for standard PCR amplification.
The application method of kit is as described above.
In addition, the Hsa-miR-17 gene promoter region sequences that obtain of the present invention also should protection scope of the present invention it
Interior, particular sequence is as shown in SEQ ID NO.11.
The invention has the advantages that：
It is of the invention compared with Standard PCR reaction system can not effectively expand Hsa-miR-17 encoding gene promoters area fragment
Primer system and reaction system can realize effective amplification to this purpose fragment and can identify that the sequence of whole section of amplified fragments is correct
Property.Therefore, technical scheme has practical significance for the correlative study of promotion Hsa-miR-17 expression regulations, and
PCR system design of primers thinking and system optimization strategy that the present invention is provided can also be applied to the DNA pieces of other high and low G/C contents
Section amplification, with good application value.
Brief description of the drawings
Fig. 1 is Hsa-miR-17 gene promoter region sequence features.
Fig. 2 is Hsa-miR-17 gene promoter areas pcr amplification product electroresis appraisal figure；M is Beijing Tiangeng 1KB DNA
Marker；NTC is negative control；1 to 6 to represent annealing temperature respectively be respectively 47 DEG C, 50 DEG C, 53 DEG C, 56 DEG C, 59 DEG C and 62
℃。
Fig. 3 is Hsa-miR-17 gene promoter areas pcr amplification product sequencer map.
Fig. 4 is Hsa-miR-17 gene promoter areas PCR amplification system specific outcome figure；Note：M is Beijing Tiangeng 1KB
DNA Marker；NTC is negative control；PC is positive control；1~3 represents 3 nude mouse tissue DNA samples respectively.
Fig. 5 is 10 cell sample Hsa-miR-17 gene promoter areas pcr amplification product electroresis appraisal figures；Note：M is north
Capital Tiangeng 1KB DNA Marker；NTC is negative control.
Embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
Limit in any form.Unless stated otherwise, the reagent of the invention used, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following examples agents useful for same and material are purchased in market.
Retrieval, signature analysis and design of primers near the Hsa-miR-17 gene promoter areas of embodiment 1
1st, in UCSC websites（http://genome.ucsc.edu/）Search obtains Hsa-miR-17 upstream region of gene 2000bp and started
Sub-district DNA sequence dna；Oligo7 software analysis sequence signatures are utilized after download sequence；Oligo7 software analysis finds Hsa-miR-17
The average G/C content of upstream region of gene 2000bp promoter regions DNA sequence dna is 48%, but preceding 500bp and the G/C content of rear 1500bp sequences
Respectively 80.8% and 37.1%, show the chimeric sequences that the target sequence is high and low G/C content DNA fragmentation, with particularity.Do
The melting temperature analysis of target sequence also in feature low after preceding height, is shown in Fig. 1.
2nd, PCR upstream and downstream primers are separately designed in high and low G/C content DNA fragmentation junctional area and downstream starting transcription site
Afterwards, whole low G/C content promoter region is covered, increase design on this basis holds the tailing that with the addition of joint sequence to draw a pair 5 '
Thing；And sequencing identification primer includes forward and reverse two sets of primer systems, primer is often covered respectively containing 4 primers, each bar primer uniformly divides
Cloth is analyzed before, during and after target sequence by sequence assembly, and the sequencing primer system can survey logical whole section of target sequence.
The primer refers to table 1, table 2.BLAST analyses show that primer specificity is good.
The Hsa-miR-17 gene promoter areas specific PCR amplimer of table 1
The Hsa-miR-17 gene promoter areas Sanger sequencing primers of table 2
The structure of the Hsa-miR-17 gene promoter areas PCR amplification system of embodiment 2
1st, non-small cell lung cancer cell strain HCC827 genomic DNAs are extracted, micro-spectrophotometer determines nucleic acid concentration.With
HCC827 cell genomic dnas are sample, in different annealing temperature and dimethyl sulfoxide (DMSO)（Dimethyl sulfoxide, DMSO）
Conventional amplification primer and the amplification of 5 ' tailing amplimer performing PCRs of table 1,1.5% Ago-Gel electricity are utilized respectively under concentration conditions
Swimming identification pcr amplification product.
Reaction system：Green Mix 7.5 μ l, 10 μM of upstream and downstream primers each 1 μ l, H2O or DMSO H2O(9.9%,
16.5%) 4.5μl, DNA 1μl.DMSO final concentration is respectively volume ratio 0%, 3%, 5% in the system
Reaction condition：94℃3min；94 DEG C of 30s,（47,50,53,56,59,62）DEG C 30s, 72 DEG C of 45s, totally 40 circulation；72℃
7 min。
2nd, result：Standard PCR primer amplification purpose fragment failed under various conditions；Tailed primer adds without DMSO
Added-time failed amplification purpose fragment under each annealing temperature；Tailed primer is respectively in DMSO final concentration of 3% and annealing temperature
Degree is at 53 DEG C~62 DEG C and DMSO final concentration of 5% and annealing temperature can Successful amplification purpose fragments, production at 53 DEG C~59 DEG C
The clear single goal bands of the visible 1608bp of thing electrophoresis（See Fig. 2）.
Meanwhile, it as a result also illustrate that the DMSO that final concentration volume ratio 3~5% is added in amplification reaction system is used in combination 5 '
Tailed primer can realize the Successful amplification in Hsa-miR-17 encoding gene promoters area.
In summary, experiment is demonstrated, and DMSO is not added with amplification system, and PCR reactions are unsuccessful；Without using tailed primer,
Even if adding, PCR reactions after DMSO are also unsuccessful.Therefore, DMSO addition is coordinated to be PCR system success using tailed primer
The key of amplification.
The Hsa-miR-17 gene promoter areas pcr amplification product sequencing identification of embodiment 3
The reaction tube PCR primer of final concentration of 5% collocation, 59 DEG C of the annealing temperatures of DMSO does product sequencing identification in selection example 2.
Respective reactor pcr amplification product is purified：Take the μ l of PCR primer 5 to add the prosperous promise SAP enzymatic mixtures in 2 μ l Beijing to mix, 37 DEG C
1h, 80 DEG C of 15min.PCR primer does sequencing reaction after purification：Reaction system：The μ l of purified pcr product 3, Bigdye3.1 1 μ l, survey
Sequence primer（5pM/μl）2μl.Reaction condition：96℃1 min；96 DEG C of 10s, 50 DEG C of 5s, 60 DEG C of 4 min, 25 circulations；12 DEG C of perseverances
Temperature.Thereafter sodium acetate ethanol purification sequencing reaction product, upper machine sequencing.
Sequencing gained PCR primer sequence is consistent completely with target sequence, points out 5 ' tailing PCR designed by the invention to expand
Primer is using specific supporting PCR reaction systems（Add DMSO final concentration of 5% Standard PCR reaction system）In the case of, into
Work(realizes the PCR amplifications to Hsa-miR-17 gene promoter areas DNA fragmentation（See Fig. 3）.
The Performance Evaluation of the Hsa-miR-17 gene promoter areas PCR amplification system of embodiment 4
Being utilized respectively by negative control, positive control and laboratory sample of water, HCC827 cell DNAs and 3 nude mouse tissue DNA should
The set up Hsa-miR-17 gene promoter areas PCR amplification system of invention carries out specificity experiments.Separately do the detection of the system
Lower limit and repeatability assess experiment：By 40 ng/ μ l concentration HCC827 cell DNAs by 2 times of doubling dilutions, 5 concentration formation samples
Series, the sample series are expanded using the PCR system set up, and each sample is all provided with 3 repeating pipes.
Specificity experiments result is shown：Positive and negative control has no and visible purpose amplified band, 3 nude mouse tissues respectively
Sample amplification is showed no purpose amplified band, and all samples are showed no non-specific amplification band, shows PCR system specificity
It is good.Refer to Fig. 4.
Monitoring lower-cut and repeated experiment result are shown：The Hsa-miR-17 gene promoter areas PCR amplification bodies set up
Tie up to each reaction tube in the range of the ng/ μ l of sample genomic dna concentration 10~40（Containing repeating pipe）PCR primer electroresis appraisal
See single, clear purpose amplified band；And in the range of 1.25~5ng/ μ l 3 repeating pipes of each sample gradient fail entirely see or
Single, clear purpose amplified band is had no completely；Result above shows that PCR system repeatability is good, and detection sensitivity is reachable
10ng/μl。
The cell sample Hsa-miR-17 gene promoter area PCR of embodiment 5 are expanded
10 cells are expanded using the Hsa-miR-17 gene promoter areas PCR system set up（Lung cancer HCC827, H1650,
MSTO211H cells are purchased from Shanghai cell institute of the Chinese Academy of Sciences, and G6, B2, C10, C8, E5 are monoclonal beggar's cell of HCC827 cells；
Lung cancer H460 cells, glioma U251 and T98G cell are that this laboratory preserves cell）Sample gene group DNA, 1.5% agar
PCR primer is identified in sugared gel electrophoresis, wherein randomly selecting the pcr amplification product row Sanger sequencing identifications of 1 sample again.
As a result show, the Hsa-miR-17 gene promoter areas PCR amplification system Successful amplification set up using the present invention
10 cell sample genomic DNAs, the visible single clear purpose amplified band of product electroresis appraisal, are shown in Fig. 5；All PCR primers
Sequencing, sequencing result and target amplification sequence are completely the same.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Attached tumour hospital of Guangzhou medical university
<120>Hsa-miR-17 gene promoter areas and its PCR amplifications identification primer sets and reaction system
<130>
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 41
<212> DNA
<213>PCR expands sense primer
<400> 1
gaaccagatc ttggaattct tcgaaggcta cgcggagaat c 41
<210> 2
<211> 44
<212> DNA
<213>PCR expands anti-sense primer
<400> 2
gctcggtacc aagcgctacc ggttacctgc actgtaagca cttt 44
<210> 3
<211> 16
<212> DNA
<213>Positive sequencing primer 1
<400> 3
tgccatcagg accaca 16
<210> 4
<211> 18
<212> DNA
<213>Positive sequencing primer 2
<400> 4
tagatttgga cggtggta 18
<210> 5
<211> 18
<212> DNA
<213>Positive sequencing primer 3
<400> 5
tggcttatgc agtttacg 18
<210> 6
<211> 18
<212> DNA
<213>Positive sequencing primer 4
<400> 6
cccattaggg attatgct 18
<210> 7
<211> 18
<212> DNA
<213>Reverse sequencing primer 1
<400> 7
tgctacaagt gccttcac 18
<210> 8
<211> 17
<212> DNA
<213>Reverse sequencing primer 2
<400> 8
ctggaagtgg tggctct 17
<210> 9
<211> 17
<212> DNA
<213>Reverse sequencing primer 3
<400> 9
tgcataagcc agtttcc 17
<210> 10
<211> 21
<212> DNA
<213>Reverse sequencing primer 4
<400> 10
agagtaagag tcgagcaata c 21
<210> 11
<211> 1560
<212> DNA
<213>Hsa-miR-17 gene promoter region sequences
<400> 11
ggctacgcgg agaatcgcag ggccgcgctc ccccttgtgc gacatgtgct gccggcccgg 60
gctccatgag cgtggcgggc actttgcagt ctcgggtgtt cctgcccggt cttctgttcc 120
taaactgcag caaagggaaa aggaactgaa aaaggcaggc tcgtcgttgc aatatcacca 180
aaagagaaaa ttaacggcat gccatcagga ccacagcagt tggagaaaca actctttatc 240
ccggcttgca gccacgaggt cttgattggg ggaggggtgg tgaagaatag tctgtgggct 300
gctttttttt tttcctttta ctggagctgt acagtggagt cggtgattgc tgctgatcat 360
aatcaagtat tttaggagct tatttagaca tgtatctgat agctaaggat ttttcaactt 420
tattctctta cgtatttttc aactgtaaat tattgggctt ttaaatcctg ctagtattgc 480
tcgactctta ctctcacaaa tggatggaat taattgctgt taggaggttg gaaaatagca 540
aatatagatt tggacggtgg tagtaatttt gagcaaataa tgttttatct tttttttcct 600
tatttttccc tattccagtc atacacgtgg acctaactgc accagtagct tttctgagaa 660
tacttgctga aaaggaagtt ttctggaatg ggtaagtgta ttctgatttt cttgaacttt 720
tcttaaaaac aaatttttct tgctattaaa gttgaataaa taggattggt ttcttagaga 780
gtaaaagtag gtgtttcttt ctttagacaa tgtacctttt ctgaaaaact aactcattaa 840
gtacggattt gctaatttta aggtagtaaa attacagtgt aaatattcct gtacattttt 900
ggaaactggc ttatgcagtt tacgaaatat aattttagac cctcttttaa gttgggtgat 960
aaagtagata taacctgaga tgatagattt aaacaggata tttacgttct gctacaattg 1020
actgataaca cttgaagtgt agtctgaaca gtaattttgt taatcatttc aacaagtatt 1080
tgctaagtgg aagccagaag aggaggaaaa tgttttgcca cgtggatgtg aagatttcct 1140
ctaaaaggta cacatggact aaattgcctt taaatgttcc aaaattagtt ctcatttatt 1200
tgcagtctca ttttgttttg ttttttttct ctatgtgtca atccatttgg gagaggccag 1260
ccattggaag agccaccact tccagtgcta gttggatggt tggttatgat tgccttctgt 1320
aaagaattct taaggcataa atacgtgtct aaatggacct catatctttg agataattaa 1380
actaattttt tcttccccat tagggattat gctgaatttg tatggtttat agttgttaga 1440
gtttgaggtg ttaattctaa ttatctattt caaatttagc aggaaaaaag agaacatcac 1500
cttgtaaaac tgaagattgt gaccagtcag aataatgtca aagtgcttac agtgcaggta 1560
Claims (10)
1. a kind of can effectively expand the pcr amplification primer thing in Hsa-miR-17 encoding gene promoters area, it is characterised in that upstream and downstream
Primer is as shown in SEQ ID NO.1 and 2.
2. a kind of can effectively expand the PCR amplification system in Hsa-miR-17 encoding gene promoters area, it is characterised in that in routine
The DMSO of final concentration of volume ratio 3~5% is added in PCR amplification system.
3. PCR amplification system according to claim 2, it is characterised in that amplimer used is as claimed in claim 1.
4. PCR amplification system according to claim 2, it is characterised in that amplification system is：The μ l of Green Mix 7.5,
10 μM of upstream and downstream primers each 1 μ l, 3~5% DMSO H2The 1 μ l of μ l, DNA of O 4.5.
5. it is a kind of can forward and reverse Sanger sequencing identifications Hsa-miR-17 encoding gene promoters region sequences primer system, its
It is characterised by, including four positive sequencing primers and four reverse sequencing primers, sequence is respectively such as the institute of SEQ ID NO.3~10
Show.
6. a kind of method that can effectively expand Hsa-miR-17 encoding gene promoters area, it is characterised in that comprise the following steps：
S1. using sample DNA as template, entered using pcr amplification primer thing described in claim 1 and contained in performing PCR amplification, amplification system
The DMSO of volumetric concentration 3~5%；
S2. amplified production electrophoresis obtains the amplified production for having 1608bp purpose bands；
S3. amplified production step S2 obtained is purified, and PCR primer does sequencing reaction after purification, obtains Hsa-miR-17 codings
Gene promoter region sequence.
7. a kind of can effectively expand the kit in Hsa-miR-17 encoding gene promoters area, it is characterised in that comprising having the right
It is required that primer system described in pcr amplification primer thing described in 1 and/or claim 5.
8. kit according to claim 7, it is characterised in that also include DMSO.
9. kit according to claim 7, it is characterised in that also include reagent needed for standard PCR amplification.
10. Hsa-miR-17 gene promoter region sequences, it is characterised in that sequence is as shown in SEQ ID NO.11.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636333A (en) * | 2016-09-28 | 2017-05-10 | 广东凯普生物科技股份有限公司 | PDGFRA gene mutation detection kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636333A (en) * | 2016-09-28 | 2017-05-10 | 广东凯普生物科技股份有限公司 | PDGFRA gene mutation detection kit |
Non-Patent Citations (7)
| Title |
|---|
| ACCESSION:AL162375: "Human DNA sequence from clone RP11-282D2 on chromosome 13, complete sequence", 《GENBANK》 * |
| ACCESSION：NC 00013: "Homo sapiens chromosome 13，GRCh38.p13 Primary Assembly", 《GENBANK》 * |
| FRANK VANDENBUSSCHE, DAVID J. LEFEBVRE, ILSE DE LEEUW等: "Laboratory validation of two real-time RT-PCR methods with 5′-tailed", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| OBRADOVIC, JASMINA; JURISIC等: "Optimization of PCR Conditions for Amplification of GC-Rich EGFR Promoter Sequence", 《JOURNAL OF CLINICAL LABORATORY ANALYSIS》 * |
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