CN107245526B - Hsa-miR-17 gene promoter region, PCR amplification identification primer group and reaction system thereof - Google Patents

Abstract

The invention discloses a set of PCR amplification and Sanger sequencing identification primers capable of effectively amplifying Hsa-miR-17 coding gene promoter region and a matched reaction system thereof, and the primers mainly comprise the following three characteristics: (1) the sequences of the special 5' tailing PCR primers are shown as SEQ ID NO.1 and SEQ ID NO. 2; (2) adding a special PCR reaction system with final concentration of 3-5% DMSO (dimethyl sulfoxide) into a conventional PCR amplification system; (3) a set of Sanger sequencing primer systems capable of identifying and amplifying the sequence correctness of a target fragment in forward and reverse directions twice is shown in SEQ ID NO. 3-10. The primer system and the reaction system can effectively amplify the promoter region fragment of the Hsa-miR-17 coding gene, can identify the sequence correctness of the whole amplified fragment, have practical significance for promoting the related research of Hsa-miR-17 expression regulation and control, and have good application value.

Description

Hsa-miR-17 gene promoter region, PCR amplification identification primer group and reaction system thereof

Technical Field

The invention belongs to the field of biotechnology. More particularly, relates to a set of PCR amplification and Sanger sequencing identification primers capable of effectively amplifying Hsa-miR-17 coding gene promoter region and a matched reaction system thereof.

Background

MicroRNA (miRNA) is a small-molecule single-stranded RNA which is endogenous and non-coding and has the length of about 18-25 bp, and regulates degradation and translation of corresponding mRNA through complete or incomplete combination with a 3' untranslated region of complementary mRNA, so that expression of corresponding protein is regulated. miRNA not only participates in regulation and control of biological processes such as cell differentiation, proliferation and apoptosis, but also is closely related to generation and development of tumors. The promoter region is a DNA sequence located at the upstream of the 5' end of the structural gene, and different transcription factors are combined with the specific promoter region to regulate the expression of the corresponding gene, so that the regulation of the corresponding physiological or pathological process is realized. Therefore, the research on the interaction between the miRNA gene promoter region and the specific transcription factor has important significance for clarifying the expression regulation mechanism of the corresponding miRNA so as to clear the pathological and physiological functions of the miRNA.

The dual-luciferase reporter gene experiment is a classical experiment method for detecting the interaction between transcription factors and target gene promoter region DNA, and the core of the dual-luciferase reporter gene experiment is to construct a dual-luciferase reporter gene plasmid inserted with a specific gene promoter region DNA fragment, and the construction of the plasmid is to obtain the promoter region DNA fragment. Therefore, the PCR amplification is utilized to obtain effective promoter region DNA fragments, which have important practical value for developing the function research of related genes. However, in the promoter region of many genes, there are special gene structures such as CpG islands, which often cause primer mismatching or local secondary structure formation, thereby causing PCR amplification failure. Therefore, what method is used to achieve effective PCR amplification and identification of promoter region DNA fragment is a problem that is often faced by related research works.

In addition, Hsa-miR-17 is highly expressed in various tumor tissues such as brain glioma, leukemia and the like and is relevant to prognosis, so Hsa-miR-17 is an important potential oncogene. Meanwhile, Hsa-miR-17 also participates in the chemotherapy response of breast cancer and the targeted therapy drug resistance of non-small cell lung cancer, so that the Hsa-miR-17 is a potential tumor therapy drug target. However, the expression regulation and related functions of Hsa-miR-17 are not completely elucidated, so that the obtained effective DNA fragment of the Hsa-miR-17 promoter region has important practical value for developing the function research of Hsa-miR-17 and has practical significance for promoting the related research of Hsa-miR-17.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the prior art and provide a set of PCR amplification and Sanger sequencing identification primers capable of effectively amplifying Hsa-miR-17 coding gene promoter region and a matched reaction system thereof.

One of the purposes of the invention is to provide a PCR amplification primer capable of effectively amplifying the promoter region of the Hsa-miR-17 coding gene.

The invention also aims to provide a PCR amplification system capable of effectively amplifying the promoter region of the Hsa-miR-17 coding gene.

The invention further aims to provide a primer system for identifying the sequence of the promoter region of the Hsa-miR-17 encoding gene by forward and reverse Sanger sequencing.

Another purpose of the invention is to provide a promoter region sequence of the Hsa-miR-17 gene.

The above purpose of the invention is realized by the following technical scheme:

the invention provides a PCR amplification primer capable of effectively amplifying an Hsa-miR-17 coding gene promoter region, wherein upstream and downstream primers are shown in SEQ ID NO.1 and 2.

The invention also provides a PCR amplification system capable of effectively amplifying the promoter region of the Hsa-miR-17 coding gene, and DMSO with the final concentration of 3-5% in volume ratio is added into the conventional PCR amplification system.

Preferably, the PCR amplification system is: green Mix 7.5. mu.l, 10. mu.M upstream and downstream primers 1. mu.l each, 3-5% DMSO H₂O 4.5μl，DNA 1μl。

The invention also provides a primer system capable of identifying the sequence of the Hsa-miR-17 coding gene promoter region by forward and reverse Sanger sequencing, which comprises four forward sequencing primers and four reverse sequencing primers, wherein the sequences are respectively shown in SEQ ID NO. 3-10.

In conclusion, the invention provides a set of primers for PCR amplification and Sanger sequencing identification capable of effectively amplifying Hsa-miR-17 coding gene promoter region and a matched reaction system thereof, and the primers mainly comprise the following three characteristics:

(1) a special 5' tailed PCR primer; (2) adding a special PCR reaction system with a final concentration of 3-5% DMSO into a conventional PCR amplification system; (3) a set of Sanger sequencing primer system capable of identifying the correctness of the sequence of the amplified target fragment in forward and reverse directions twice.

Firstly, the invention uses a special 5' tailing PCR amplification primer, which comprises the following specific steps:

upstream primer (SEQ ID NO. 1):

5′-GAACCAGATCTTGGAATTCTTCGAAGGCTACGCGGAGAATC-3' (the underlined part is the tailed primer sequence),

downstream primer (SEQ ID NO. 2):

5′-GCTCGGTACCAAGCGCTACCGGTTACCTGCACTGTAAGCACTTT-3' (the underlined part is the tailed primer sequence).

Secondly, DMSO with the final concentration of 3-5% is added into a conventional PCR amplification system.

Successful amplification of the Hsa-miR-17 coding gene promoter region can be realized by jointly using a 5' tailed PCR primer and a PCR amplification system containing DMSO with a final concentration of 3-5%.

Finally, the invention realizes the complete sequence determination and identification of the successfully amplified Hsa-miR-17 coding gene promoter region fragment by utilizing a forward and reverse Sanger sequencing identification primer system.

The forward and reverse Sanger sequencing and identifying primer system comprises the following components:

forward sequencing primer:

forward sequencing primer 1 (SEQ ID NO. 3): 5'-TGCCATCAGGACCACA-3', respectively;

forward sequencing primer 2 (SEQ ID NO. 4): 5'-TAGATTTGGACGGTGGTA-3', respectively;

forward sequencing primer 3 (SEQ ID NO. 5): 5'-TGGCTTATGCAGTTTACG-3', respectively;

forward sequencing primer 4 (SEQ ID NO. 6): 5'-CCCATTAGGGATTATGCT-3', respectively;

reverse sequencing primer:

reverse sequencing primer 1 (SEQ ID NO. 7): 5'-TGCTACAAGTGCCTTCAC-3', respectively;

reverse sequencing primer 2 (SEQ ID NO. 8): 5'-CTGGAAGTGGTGGCTCT-3', respectively;

reverse sequencing primer 3 (SEQ ID NO. 9): 5'-TGCATAAGCCAGTTTCC-3', respectively;

reverse sequencing primer 4 (SEQ ID NO. 10): 5'-AGAGTAAGAGTCGAGCAATAC-3' are provided.

Therefore, the invention also provides a method for effectively amplifying the promoter region of the Hsa-miR-17 coding gene, which comprises the following steps:

s1, using sample DNA as a template, and carrying out PCR amplification by using the PCR amplification primer according to claim 1, wherein an amplification system contains DMSO with the volume concentration of 3-5%;

s2, obtaining an amplification product with a 1608bp target band by using amplification product electrophoresis;

s3, purifying the amplification product obtained in the step S2, and performing sequencing reaction on the purified PCR product to obtain a promoter region sequence of the Hsa-miR-17 encoding gene.

Wherein, preferably, the amplification system of the PCR amplification in step S1 is: green Mix 7.5. mu.l, 10. mu.M upstream and downstream primers 1. mu.l each, 3-5% DMSO H₂O 4.5μl，DNA 1μl。

Preferably, the reaction conditions for PCR amplification in step S1 are: 3min at 94 ℃; 30s at 94 ℃, 30s at 53-62 ℃ and 45s at 72 ℃ for 40 cycles; 7 min at 72 ℃.

Preferably, the purification method of step S3: and adding 2. mu.l of Beijing Xinnuo SAP enzyme mixture into 5. mu.l of PCR product, mixing uniformly, and keeping the temperature at 37 ℃ for 1h and 80 ℃ for 15 min.

Preferably, the reaction system of the sequencing reaction described in step S3: PCR product 3. mu.l, Bigdye 3.11. mu.l, sequencing primer (5 pM/. mu.l) 2. mu.l were purified.

Preferably, the reaction conditions of the sequencing reaction described in step S3 are: 1 min at 96 ℃; at 96 deg.C for 10s, at 50 deg.C for 5s, at 60 deg.C for 4 min, for 25 cycles; keeping the temperature constant at 12 ℃; then purifying sequencing reaction products by sodium acetate and ethanol, and performing machine sequencing.

A kit capable of effectively amplifying the promoter region of the Hsa-miR-17 encoding gene is also within the protection scope of the invention, and the kit comprises the primers shown in SEQ ID NO. 1-10.

Preferably, the kit further comprises DMSO.

Preferably, the kit further comprises reagents required for conventional PCR amplification.

The method of use of the kit is as described above.

In addition, the promoter region sequence of the Hsa-miR-17 gene obtained by the invention also falls within the protection scope of the invention, and the specific sequence is shown in SEQ ID NO. 11.

The invention has the following beneficial effects:

compared with the conventional PCR reaction system which can not effectively amplify the promoter region fragment of the Hsa-miR-17 encoding gene, the primer system and the reaction system can realize effective amplification of the target fragment and can identify the sequence correctness of the whole amplified fragment. Therefore, the technical scheme of the invention has practical significance for promoting related research of Hsa-miR-17 expression regulation, and the PCR system primer design thought and the system optimization strategy provided by the invention can also be applied to amplification of other DNA fragments with high and low GC contents, and have good application value.

Drawings

FIG. 1 shows the sequence characteristics of the promoter region of the Hsa-miR-17 gene.

FIG. 2 is an electrophoresis identification chart of PCR amplification products of Hsa-miR-17 gene promoter region; m is a Beijing Tiangen 1KB DNA Marker; NTC is negative control; 1 to 6 represent annealing temperatures of 47 deg.C, 50 deg.C, 53 deg.C, 56 deg.C, 59 deg.C and 62 deg.C, respectively.

FIG. 3 is a sequencing diagram of PCR amplification products of Hsa-miR-17 gene promoter region.

FIG. 4 is a result diagram showing the specificity of a PCR amplification system in the promoter region of the Hsa-miR-17 gene; note: m is a Beijing Tiangen 1KB DNA Marker; NTC is negative control; PC is a positive control; 1-3 represent 3 nude mouse tissue DNA samples.

FIG. 5 is an electrophoretic identification chart of PCR amplification products of Hsa-miR-17 gene promoter region of 10 cell samples; note: m is a Beijing Tiangen 1KB DNA Marker; NTC was a negative control.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 acquisition of sequences around the promoter region of Hsa-miR-17 Gene, characterization, and primer design

1. Searching and obtaining a DNA sequence of an Hsa-miR-17 gene upstream 2000bp promoter region on a UCSC website (http:// genome. UCSC. edu /); after downloading the sequence, sequence characteristics were analyzed using oligo7 software; the analysis of oligo7 software shows that the average GC content of the DNA sequence of the upstream 2000bp promoter region of the Hsa-miR-17 gene is 48%, but the GC contents of the first 500bp and the second 1500bp sequences are 80.8% and 37.1% respectively, which indicates that the target sequence is a chimeric sequence of DNA fragments with high and low GC contents and has specificity. Melting temperature analysis of the target sequence was also characterized by high front and low back, as shown in FIG. 1.

2. PCR upstream and downstream primers are respectively designed behind a high-GC-content DNA fragment junction region, a low-GC-content DNA fragment junction region and a downstream initial transcription site, and a pair of tailed primers with 5' ends added with a linker sequence are additionally designed on the basis of covering the whole low-GC-content promoter region; and the sequencing identification primer comprises a forward primer system and a reverse primer system, each primer system comprises 4 primers, each primer is uniformly distributed at the front part, the middle part and the rear part of the target sequence, and the sequencing primer system can be used for detecting the whole section of the target sequence through sequence splicing analysis.

The primers are detailed in tables 1 and 2. BLAST analysis showed good primer specificity.

TABLE 1 Hsa-miR-17 Gene promoter region specificity PCR amplification primers

TABLE 2 Sanger sequencing primers for the Hsa-miR-17 gene promoter region

Example 2 construction of PCR amplification System for Hsa-miR-17 Gene promoter region

1. Extracting non-small cell lung cancer cell strain HCC827 genome DNA, and measuring the nucleic acid concentration by a microspectrophotometer. Using HCC827 cell genome DNA as a sample, respectively carrying out PCR amplification by using a conventional amplification primer and a 5' tail amplification primer in the conditions of different annealing temperatures and dimethyl sulfoxide (DMSO) concentrations, and identifying PCR amplification products by 1.5% agarose gel electrophoresis.

Reaction system: green Mix 7.5. mu.l, 10. mu.M upstream and downstream primers 1. mu.l each, H₂O or DMSO H₂O (9.9%,16.5%) 4.5. mu.l, DNA 1. mu.l. The final concentration of DMSO in the system is 0%, 3%, 5% by volume

Reaction conditions are as follows: 3min at 94 ℃; 30s at 94 ℃ (47, 50,53,56,59, 62) DEG C, 30s at 72 ℃ and 45s at 72 ℃, and 40 cycles are carried out; 7 min at 72 ℃.

2. As a result: the conventional PCR primers can not successfully amplify target fragments under various conditions; the tailed primers did not successfully amplify the target fragment at each annealing temperature without DMSO addition; the tailed primer can successfully amplify the target fragment when the final DMSO concentration is 3% and the annealing temperature is 53-62 ℃ and when the final DMSO concentration is 5% and the annealing temperature is 53-59 ℃, and the electrophoresis of the product can see a 1608bp clear single target band (see figure 2).

Meanwhile, the result also shows that DMSO with the final concentration volume ratio of 3-5% is added into an amplification reaction system to be combined with a 5' tailing primer, so that the successful amplification of the Hsa-miR-17 coding gene promoter region can be realized.

In conclusion, experiments prove that the PCR reaction is unsuccessful without adding DMSO in the amplification system; without the use of tailed primers, the PCR reaction was unsuccessful even after DMSO addition. Therefore, the use of tailed primers in combination with the addition of DMSO is critical to the successful amplification of this PCR system.

Example 3 sequencing identification of PCR amplification products of Hsa-miR-17 Gene promoter region

The PCR product from the reaction tube of example 2 with final DMSO concentration of 5% and annealing temperature of 59 ℃ was selected for product sequencing. And (3) purifying PCR amplification products of corresponding reaction tubes: and adding 2. mu.l of Beijing Xinnuo SAP enzyme mixture into 5. mu.l of PCR product, mixing uniformly, and keeping the temperature at 37 ℃ for 1h and 80 ℃ for 15 min. And (3) carrying out sequencing reaction on the purified PCR product: reaction system: PCR product 3. mu.l, Bigdye 3.11. mu.l, sequencing primer (5 pM/. mu.l) 2. mu.l were purified. Reaction conditions are as follows: 1 min at 96 ℃; at 96 deg.C for 10s, at 50 deg.C for 5s, at 60 deg.C for 4 min, for 25 cycles; keeping the temperature constant at 12 ℃. Then purifying sequencing reaction products by sodium acetate and ethanol, and performing machine sequencing.

The PCR product sequence obtained by sequencing completely conforms to the target sequence, which indicates that the 5' tailed PCR amplification primer successfully realizes the PCR amplification of the DNA fragment of the Hsa-miR-17 gene promoter region under the condition of using a specific matched PCR reaction system (a conventional PCR reaction system with the final DMSO concentration of 5%) (see figure 3).

Example 4 Performance evaluation of PCR amplification System of Hsa-miR-17 Gene promoter region

Water, HCC827 cell DNA and 3 cases of nude mouse tissue DNA are respectively used as a negative control, a positive control and an experimental sample, and the specificity experiment is carried out by utilizing the Hsa-miR-17 gene promoter region PCR amplification system established by the invention. And (3) additionally performing a detection lower limit and repeatability evaluation experiment of the system: 40 ng/. mu.l HCC827 cell DNA was diluted 2-fold to 5 concentrations to form a sample series, which was amplified using an established PCR system, and each sample was set to 3 duplicate tubes.

The results of the specificity experiment show that: no target amplification band is seen in the negative control and the positive control, no target amplification band is seen in the amplification of 3 nude mouse tissue samples, and no non-specific amplification band is seen in all the samples, indicating that the specificity of the PCR system is good. See figure 4 for details.

The lower detection limit and the repeatability experiment result show that: the established PCR amplification system of the Hsa-miR-17 gene promoter region can see a single and clear target amplification band in the electrophoresis identification of PCR products of each reaction tube (including a repeat tube) within the range of 10-40 ng/mu l of the sample genome DNA concentration; while 3 repeat tubes of each sample gradient within the range of 1.25-5 ng/mu l can not see all or no single and clear target amplification bands; the results show that the PCR system has good repeatability and the detection sensitivity can reach 10 ng/mu l.

Example 5 cell sample PCR amplification of Hsa-miR-17 Gene promoter region

10 cells (lung cancer HCC827, H1650 and MSTO211H cells are purchased from Shanghai cell institute of Chinese academy of sciences, G6, B2, C10, C8 and E5 are single clone daughter cells of HCC827 cells, lung cancer H460 cells, brain glioma U251 and T98G cells are stored cells in the laboratory) are amplified by using an established Hsa-miR-17 gene promoter region PCR system, the PCR products are identified by 1.5% agarose gel electrophoresis, and the PCR amplification products of 1 sample are randomly extracted for Sanger sequencing identification.

The result shows that 10 cases of cell sample genome DNA are successfully amplified by utilizing the established Hsa-miR-17 gene promoter region PCR amplification system, and a single clear target amplification band can be seen in the product electrophoretic identification, which is shown in figure 5; all PCR products are sequenced, and the sequencing result is completely consistent with the target amplification sequence.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> Guangzhou medical university affiliated tumor hospital

<120> Hsa-miR-17 gene promoter region, PCR amplification identification primer group and reaction system thereof

<130>

<160> 11

<170> PatentIn version 3.3

<210> 1

<211> 41

<212> DNA

<213> PCR amplification upstream primer

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gaaccagatc ttggaattct tcgaaggcta cgcggagaat c 41

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<212> DNA

<213> downstream primer for PCR amplification

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gctcggtacc aagcgctacc ggttacctgc actgtaagca cttt 44

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<212> DNA

<213> Forward sequencing primer 1

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tgccatcagg accaca 16

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<212> DNA

<213> Forward sequencing primer 2

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tagatttgga cggtggta 18

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<212> DNA

<213> Forward sequencing primer 3

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tggcttatgc agtttacg 18

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<213> Forward sequencing primer 4

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cccattaggg attatgct 18

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<212> DNA

<213> reverse sequencing primer 1

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tgctacaagt gccttcac 18

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<213> reverse sequencing primer 2

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ctggaagtgg tggctct 17

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<213> reverse sequencing primer 3

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tgcataagcc agtttcc 17

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<213> reverse sequencing primer 4

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agagtaagag tcgagcaata c 21

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<213> Hsa-miR-17 gene promoter region sequence

<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 (3)

1. A PCR amplification system capable of effectively amplifying Hsa-miR-17 coding gene promoter region is characterized in that the amplification system is as follows: 7.5 mul of Green Mix, 1 mul of 10 mul of upstream and downstream primers respectively, 4.5 mul of 3-5% DMSO (dimethyl sulfoxide) in a final concentration volume ratio, and 1 mul of DNA (deoxyribonucleic acid); the upstream and downstream primers are shown as SEQ ID NO.1 and SEQ ID NO. 2.

2. A method for effectively amplifying Hsa-miR-17 coding gene promoter region for non-therapeutic diagnosis purposes is characterized in that,

the method comprises the following steps:

s1, carrying out PCR amplification by using the PCR amplification system of claim 1 by using a sample DNA as a template;

s2, obtaining an amplification product with a 1608bp target band by using amplification product electrophoresis;

s3, purifying the amplification product obtained in the step S2, and performing sequencing reaction on the purified PCR product to obtain a promoter region sequence of the Hsa-miR-17 encoding gene.

3. A kit capable of effectively amplifying a promoter region of an Hsa-miR-17 encoding gene, which is characterized by comprising the PCR amplification system of claim 1.

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