CN110055258A - A kind of site breast cancer related gene ERBB2 g.39717320G > A mutant and its application - Google Patents
A kind of site breast cancer related gene ERBB2 g.39717320G > A mutant and its application Download PDFInfo
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Abstract
The invention discloses a kind of site breast cancer related gene ERBB2 g.39717320G > A mutant and its application, the invention belongs to genetic engineering and medical oncology fields.Saltant type ERBB2 gene order therein and wild type ERBB2 gene order have g.39717320G > A site mutation, detect the specific primer of the mutant and the kit comprising the specific primer.A kind of site breast cancer related gene ERBB2 g.39717320G > A mutant and its application, being capable of quickly and easily auxiliary judgment breast cancer.
Description
Technical field
The present invention relates to a kind of medical biotechnology more particularly to a kind of sites breast cancer related gene ERBB2
G.39717320G > A mutant and its application.
Background technique
Breast cancer belongs to a kind of systemic disease by cognition, and the cause of disease of breast cancer not yet clearly discloses so far, with swollen
The development of tumor molecular genetics, oncocytogenetics and molecular epidemiology, it is now recognized that environmental factor and inherent cause are total
Same-action influences the generation of breast cancer, and in inherited cancer syndrome, the germ line mutation of cancer associated gene determines the family
Genetic predispostion；And in distributed cancers, Major Risk Factors are environmental factors, the genetic polymorphism of gene related to this
Property determine individual to the neurological susceptibilities of these factors.
Some researches show that the women for carrying breast cancer inheritance susceptible gene, the onset risk of breast cancer will be greatly improved,
By taking BRCA1 and BRCA2 gene as an example, the risk that carrier suffers from breast cancer throughout one's life is up to 80%, therefore is directed to these and breast cancer
The secondary prevention (early discovery, early diagnosis, early treatment) of breast cancer can be improved in the detection of relevant tumor susceptibility gene.Wherein, breast cancer
Related gene ERBB2 is also known as neu or HER-2 gene, is that a kind of cell carrys out proto-oncogene, in kinds of tumors its oncogene and
Its protein product (p185) has overexpression and amplification.It is more first to the pathological study of ERBB2 oncogene protein product p185
Breast cancer is seen, is acted on also more clear.At present it is believed that the positive expression of ERBB2 protein product can be used as judgement cream
One individual index of gland cancer prognosis.
Summary of the invention
Goal of the invention: in order to overcome the deficiencies in the prior art, the present invention provides a kind of breast cancer related gene
The site ERBB2 g.39717320G > A mutant, detect the specific primer of the mutant and the examination containing specific primer
Agent box, so as to quickly and easily auxiliary judgment breast cancer.
Technical solution: to achieve the above object, the site a kind of breast cancer related gene ERBB2 of the invention
G.39717320G > A mutant, wild type ERBB2 gene order is as shown in SEQ ID NO:1, saltant type ERBB2 base
Because as shown in SEQ ID NO:2, saltant type ERBB2 gene order and wild type ERBB2 gene order have g.39717320G >
A site mutation.
Further, a kind of for detecting a kind of above-mentioned site breast cancer related gene ERBB2 g.39717320G > A
The specific primer of mutant, the sequence of the upstream primer of the specific primer is as shown in SEQID NO:3, the specificity
The sequence of the downstream primer of primer is as shown in SEQID NO:4；For detecting ERBB2 gene, g.39717320G the site > A is prominent
Become；G.39717320G > A site mutation refers to mutational site in the 92nd alkali of SEQID NO:1 sequence to the ERBB2 gene
Base G sports A.
Further, a kind of Computer-aided Diagnosis of Breast Cancer kit, including for detecting the site gene ERBB2
G.39717320G the specific primer of > A mutant.
Beneficial effects of the present invention are as described below: g.39717320G > A is prominent in a kind of site breast cancer related gene ERBB2
Variant, the specific primer for detecting the mutant and the kit containing specific primer, may make the diagnosis of breast cancer more
Add convenient and easy, grasps conditions of patients quick and precisely for clinician, lay the foundation for clinical therapeutic efficacy evaluation, and be discovery
New small molecule drug targets with potential treatment value provide help.
Detailed description of the invention
Attached drawing 1 is that g.39717320G > A mutant detects gel electrophoresis figure in the site breast cancer related gene ERBB2；
Attached drawing 2 is the gene sequencing figure of the site breast cancer related gene ERBB2 g.39717320G > A mutant.
Specific embodiment
The present invention will be further explained with reference to the accompanying drawing.
A kind of site breast cancer related gene ERBB2 as described in attached drawing 1 to 2 g.39717320G > A mutant, it is wild
Raw type ERBB2 gene order is as shown in SEQ ID NO:1, and saltant type ERBB2 gene is as shown in SEQ ID NO:2, saltant type
ERBB2 gene order and wild type ERBB2 gene order have g.39717320G > A site mutation.
In the present embodiment, by having the breast cancer patients of Family history of cancer and 300 normal control members to 120
G.39717320G the mutational site > A carries out screening to ERBB2 gene, finds 8 patients with ERBB2 gene
G.39717320G > A is mutated (6.7%).The variation is a missense variation.This variation is only 3 in 300 control groups
It carries (1.0%), illustrates that this variant sites increases the risk of mammary gland.Variation occurrence frequency in patient with breast cancer is aobvious
It writes and is higher than normal control, illustrate that the mutational site and breast cancer are closely related.
The mutational site relevant to Computer-aided Diagnosis of Breast Cancer of above research institute's discovery is g.39717320G > A, the mutation
39717320 positions of No. 17 chromosome are betided, number of the gene in NCBl reference database GRCh38.p12 is
NC_000017.11 (39688084~39728662), be listed in the database here include this site wild type part
Base sequence for reference, as shown in SEQID NO:1, the corresponding sequence of ERBB21 gene mutation as shown in SEQID NO:2,
Wherein mutational site sports A by bases G in the 92nd of SEQID NO:1 sequence.
SEQID NO:1
GAAGCACAAAGGGGACCCAACTAAGGGCCTGATCCTACTGCCCTGGGGGTGTCAGTGCC AGCCCCCC
ACAAATCTTTTCTGCCCCCCCCAGGAGGCTGACCAGTGTGTGGCCTGTGCCCACT ATAAGGACCCTCCCTTCTGC
GTGGCCCGCTGCCCCAGCGGTGTGAAACCTGACCTCTCCTACA TGCCCATCTGGAAGTTTCCAGATGAGGAGGGC
GCATGCCAGCCTTGCCCCATCAACTGCACCC ACTCGTGAGTCCAACGG
SEQID NO:2
GAAGCACAAAGGGGACCCAACTAAGGGCCTGATCCTACTGCCCTGGGGGTGTCAGTGCC AGCCCCCC
ACAAATCTTTTCTGCCCCCCCCAGAAGGCTGACCAGTGTGTGGCCTGTGCCCACT ATAAGGACCCTCCCTTCTGC
GTGGCCCGCTGCCCCAGCGGTGTGAAACCTGACCTCTCCTACA TGCCCATCTGGAAGTTTCCAGATGAGGAGGGC
GCATGCCAGCCTTGCCCCATCAACTGCACCC ACTCGTGAGTCCAACGG
The mutational site is that this research is found in Chinese Han women patient with breast cancer for the first time, database displaying occidentals
The scanning in the group region does not find the mutational site yet.By being included in prospective cohort study, it was demonstrated that carry the trouble of the mutation
Person has been eventually developed to breast cancer, it was demonstrated that the site is the pathogenic factor of breast cancer.
The influence factor that the present invention develops disease by the control age studies mutational site in Computer-aided Diagnosis of Breast Cancer
Application prospect illustrates influence of the mutational site for breast cancer progression, discloses its diagnostic value.
A kind of specificity for a kind of site breast cancer related gene ERBB2 of right to examin g.39717320G > A mutant
Primer, the sequence of the upstream primer of the specific primer is as shown in SEQID NO:3, the downstream primer of the specific primer
Sequence as shown in SEQID NO:4；For detecting ERBB2 gene g.39717320G > A site mutation；The ERBB2 gene
G.39717320G > A site mutation refers to that mutational site sports A in the 92nd bit base G of SEQID NO:1 sequence.
A kind of Computer-aided Diagnosis of Breast Cancer kit, including described for detecting the site gene ERBB2 g.39717320G >
The specific primer of A mutant.
Detection method in the present embodiment is the specific sequence site using mankind ERBB2 gene different genotype
Change, design the primer sequence for mutational site, carries out polymerase chain reaction to complete.
The present invention uses Sanger sequencing approach, with PCR amplification primer, carries out abrupt climatic change to ERBB2 gene；Wherein
The sequence of the upstream primer of PCR amplification primer is for example shown in SEQID NO:3, and the sequence of downstream primer is as shown in SEQID NO:4.
Specifically, g.39717320G > A mutated gene detects, is auxiliary for breast cancer for the preparation of DNA profiling, ERBB2 gene
Help being produced as follows for diagnosis mutational site kit described.
The preparation of DNA profiling extracts Whole Blood Genomic DNA using the kit purchased, the specific steps are as follows:
(1) sterile 2.0mL centrifuge tube one is taken, 1mL cell pyrolysis liquid is added；
(2) whole blood sample so anticoagulant through EDTA is gently shaken, until thoroughly mixing；Then the addition of 500 μ L sample of blood is drawn
In the above-mentioned centrifuge tube containing cell pyrolysis liquid, centrifuge tube 5~6 times mixings are gently toppled over；
(3) it is incubated at room temperature 10 minutes, during which overturns 2~3 mixings of centrifuge tube；
(4) 12000rpm room temperature is centrifuged 5 minutes；
(5) slowly supernatant is moved as far as possible with pipettor and is abandoned completely, paid attention to the whiteness of two-phase intersection not
It is sucked out；
(6) it is acutely mixed using turbula shaker (Votex), until (10~15 seconds) are resuspended in leucocyte；
(7) 300 μ L of karyorhexis liquid is added into resuspension cell solution.It is white thin that solution 5~6 times cracking are put with liquid transfer gun head suction
Born of the same parents.Solution should become very sticky at this time.If visible cell agglomerate after mixing, solution is placed in 37 DEG C and is incubated for until agglomerate disappears
It dissipates, if still visible cell agglomerate after being incubated for 1 hour, separately adding 100 μ L of karyorhexis liquid to lay equal stress on is reset in 37 DEG C of incubations；
(8) 100 μ L of albumen precipitation liquid is added into karyorhexis object, is acutely shaken with turbula shaker 10~20 seconds；
(9) 12000rpm room temperature is centrifuged 5 minutes；
(10) its supernatant is gone into being added in the 2.0mL centrifuge tube of 300 μ L room temperature isopropanols of reference numeral；
(11) it gently overturns to mix solution, until white linear DNA forms precipitating；
(12) 12000rpm room temperature is centrifuged 1 minute；
(13) liquid, addition and isometric 70% ethyl alcohol of room temperature of sample size are discarded supernatant, gently overturns centrifuge tube for several times；
(14) slowly ethanol is moved as far as possible with pipettor and is abandoned completely.Centrifuge tube is placed in 50 DEG C and toasts 5~10 points
Clock allows remaining ethanol volatilization clean as far as possible；
(15) the DNA lysate of 50~100 μ L is added into centrifuge tube, mixes gently；
(16) DNA extraction effect is assessed with 1% agarose gel electrophoresis, Nanodrop nucleic acid instrument detection level is quantitative
To 20~50ng/ μ L, -20 DEG C of preservations.
ERBB2 gene g.39717320G > A mutated gene detection technique scheme:
Instrument: Veriti96 type PCR instrument, is coagulated at BIO-RAD Gel Doc XR+ type gel imager (Bio Rad Laboratories)
Gel electrophoresis instrument (6 1 company of Beijing).
Reagent: QIAamp DNA extraction kit (German Qiagen company)；DNA Isolation Kit extracts kit
(Beijing PELFREEZ company)；PCR buffer, dNTP, Taq enzyme (American AB I company)；Primer is by the limited public affairs of the raw work biology in Shanghai
Department's synthesis.
(1) design of primers: the ERBB2 gene (sequence recorded according to American National Bioinformatics Institute (NCBI) GenBank
Number: NC_000017.11), by Oligo6.0 primer software Design primers, finally determine 1 pair of specific oligonucleotide primer sequence
It is classified as F:5 '-ATCTTTTCTGCCCCCCCCAGA-3 ' (SEQID NO:3) and R:5 '-CTGCAGAAAAGACCGTTGGAC-3 '
(SEQID NO:4), amplified production fragment length are 206bp；
(2) total volume: 50 μ L, the 10 μ L containing 5 × buffer of PCR, 5.0 μ L of DNA profiling, Taq polymerase (1U/ μ L) is reacted
1.0 μ L, MgCl2 final concentration 2.0mmol/L, dNTP final concentration 200nmol/L, and the upper and lower primer final concentration of specificity
200nmol/L, and add disinfection distilled water to 50 μ L of total volume；
Reaction condition: 95 DEG C initial denaturation 5 minutes, then successively 95 DEG C be denaturalized 30 seconds, then 56 DEG C anneal 30 seconds, 72 DEG C
Extend 30 second minute, totally 35 circulations；
(3) g.39717320G > A mutated gene detects ERBB2 gene: with 1.5% Ago-Gel to by step (2)
The resulting object of expanding production that increases carries out electrophoresis, to detect whether it has purpose segment.Result is observed by gel imager and is taken pictures,
PCR product is shown as single band after electrophoresis, and no miscellaneous band then prompts PCR product single, no non-specific amplification, if band position
It is then purpose segment setting in appropriately sized position.As shown in Fig. 1, M:50bp ladder molecular weight markers object, 1: blank pair
According to 2: wild type control, 3:ERBB2 gene g.39717320G > A sudden change sample；
(4) amplified production purifies: this research uses the Agarose Gel DNA Purification of Takara company
PCR product after agarose gel electrophoresis is carried out purification and recovery by Kit kit, prepares sequencing；
(5) Sanger sequencing judges with result: PCR product carries out in the full-automatic DNA sequencer of ABI3730 type after purification
Sequencing.By sequencing result and ERBB2 wild-type reference sequence (NCBI Reference Sequence:NC_000017.11) into
Row compares, and is reported according to practical catastrophe result.Detection gained gene mutation figure is as shown in Fig. 2, arrow institute in figure
It is shown as ERBB2 gene and shows g.39717320G > A mutation.
The production of mutational site kit and operating process are that scanning detection technology is sequenced in based on PCR amplification and Sanger.
Kit contains a collection of specific primer on the mutated site (including following primer: the g.39717320G primer sequence in the mutational site > A
It is classified as SEQID NO:3 and SEQID NO:4), which can also include that PCR reacts common reagent, such as Taq enzyme, dNTP
Mixed liquor, MgCl2 solution, deionized water etc.；These common agents be all it is well known to those skilled in the art, in addition it can contain
There are standard items and/or reference substance (such as determining standard items and the blank control of genotype).The value of this kit is only to need
It wants peripheral blood without other tissue samples, detects mutational site with special primer pair by most simplifying, then pass through mutation
Auxiliary judgment breast cancer is composed in site, not only stable, easy to detect and accurate, greatly improves the sensibility of medical diagnosis on disease and special
Property, therefore this kit is put into and is practiced, it can help to instruct diagnosis and more effective individualized treatment.
Relevant mutational site Oligonucleolide primers sequence, as shown in table 1；
Table 1
Wherein * F=forward primer sequence；R=reverse primer sequences.
The above is only a preferred embodiment of the present invention, it should be pointed out that: for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>the 5th the People's Hospital of Wuxi City
<120>a kind of site breast cancer related gene ERBB2 is g.39717320G>A mutant and its application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 2
<211> 265
<212> DNA
<213> Homo sapiens
<400> 2
gaagcacaaa ggggacccaa ctaagggcct gatcctactg ccctgggggt gtcagtgcca 60
gccccccaca aatcttttct gcccccccca ggaggctgac cagtgtgtgg cctgtgccca 120
ctataaggac cctcccttct gcgtggcccg ctgccccagc ggtgtgaaac ctgacctctc 180
ctacatgccc atctggaagt ttccagatga ggagggcgca tgccagcctt gccccatcaa 240
ctgcacccac tcgtgagtcc aacgg 265
<210> 3
<211> 265
<212> DNA
<213> Homo sapiens
<400> 3
gaagcacaaa ggggacccaa ctaagggcct gatcctactg ccctgggggt gtcagtgcca 60
gccccccaca aatcttttct gcccccccca gaaggctgac cagtgtgtgg cctgtgccca 120
ctataaggac cctcccttct gcgtggcccg ctgccccagc ggtgtgaaac ctgacctctc 180
ctacatgccc atctggaagt ttccagatga ggagggcgca tgccagcctt gccccatcaa 240
ctgcacccac tcgtgagtcc aacgg 265
<210> 4
<211> 21
<212> DNA
<213> Homo sapiens
<400> 4
atcttttctg ccccccccag a 21
<210> 5
<211> 21
<212> DNA
<213> Homo sapiens
<400> 5
ctgcagaaaa gaccgttgga c 21
Claims (3)
1. a kind of site breast cancer related gene ERBB2 g.39717320G > A mutant, it is characterised in that: its wild type ERBB2
Gene order is as shown in SEQ ID NO:1, and saltant type ERBB2 gene is as shown in SEQ ID NO:2, saltant type ERBB2 gene
Sequence and wild type ERBB2 gene order have g.39717320G > A site mutation.
2. it is a kind of for detect the site a kind of breast cancer related gene ERBB2 described in claim 1 g.39717320G > A mutation
The specific primer of body, it is characterised in that: the sequence of the upstream primer of the specific primer is described as shown in SEQID NO:3
The sequence of the downstream primer of specific primer is as shown in SEQID NO:4；For detect ERBB2 gene g.39717320G > site A
Mutation；The ERBB2 gene g.39717320G > A site mutation refers to mutational site in the 92nd alkali of SEQID NO:1 sequence
Base G sports A.
3. a kind of Computer-aided Diagnosis of Breast Cancer kit, it is characterised in that: including as claimed in claim 2 for detecting gene
The site ERBB2 g.39717320G > specific primer of A mutant.
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Cited By (1)
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| CN111139301A (en) * | 2020-03-10 | 2020-05-12 | 无锡市第五人民医院 | Breast cancer related gene ERBB2 site g.39397319C > A mutant and application thereof |
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| CN111139301A (en) * | 2020-03-10 | 2020-05-12 | 无锡市第五人民医院 | Breast cancer related gene ERBB2 site g.39397319C > A mutant and application thereof |
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