CN1920054A - Mammary cancer gene (BRCA1) mutation detecting analysis - Google Patents
Mammary cancer gene (BRCA1) mutation detecting analysis Download PDFInfo
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Abstract
The invention relates the BRCA1 gene, comprising BRCA1 gene mutation, deletion, inserting, transversion ionophresis, checking technology, BRCA1 complex symptom, breast cancer, and treatment field and health field of BRCA1 gene mutation of breast cancer people. The gene mutation is the 11 extron 914T/C (S265S) heterozygosity mutation and base mutation, and any single or combination mutation. The invention also relates the checking method. The invention has important meaning for human, especially Chinese genes.
Description
Technical field
The present invention relates to a kind of biological gene, the present invention relates to Chinese BRCA more precisely
1The electrophoresis of transgenation, disappearance, insertion, transversion, the gene sequencing detection technique, disease before the BRCA1 syndromes, mammary cancer and cancer thereof, the personnel of mammary cancer family are based on the every treatment field and the healthcare field of BRCA1 transgenation.
Background technology
About mastocarcinoma gene 1 (BRCA
1): mammary cancer is the cancer that sickness rate increases fast in the present worldwide.In the mammary cancer universal geography collection of illustrative plates of WHO issue, per 100,000 populations morbidity number is: Australia (83,/10 ten thousand), the U.S., Canada (82,/10 ten thousand), Europe (70,/10 ten thousand).Secondly for south american countries (65,/10 ten thousand), Eastern Europe (49,/10 ten thousand), north African, South Africa, Japan, Israel and Arab are (28,/10 ten thousand), and in non-, the Asia sickness rate is minimum (16-19/10 ten thousand).
Mammary cancer sickness rate in women's cancer holds pride of place, and accounts for more than 1/4 in all women's cancers, and the every 7-9 of occidentals position women will have one mammary cancer takes place in life.Carry the women of mammary cancer BRCA1 Disease-causing gene, the probability that suffers from breast cancer in the time of 70 years old is up to more than 80%; Therefore the molecular pathology of mammary cancer is one of gene of greatest concern in cma gene.Not only clear and definite BRCA
1, BRCA
2Gene is now also relevant for BRCA
3Report with other breast cancer related gene.The most valued BRCA wherein just
1Gene, relative disease that is caused by it and tumour are by WHO called after " BRCA1 syndromes ".The genome website of United States Government has also been set up Breast Cancer Information core Database website specially.This website is registered 1300 BRCA
1The mutational site.The annual subsidy that obtains 8,000 ten thousand U.S. dollars from national health administration of the U.S. maximum commercialization gene sequencing company among the people (Myriad company), the mastocarcinoma gene of finishing public goodization detects 10,000 6 thousand people, obtained a large amount of gene mutation site resource and patent, and at national registrations such as native country, Europe.In the past few years huge profit margin has caused the intergovernmental BRCA1 gene patent war of European and American countries.
Mammary cancer BRCA
1The assignment of genes gene mapping has 24 exons (22 encode exon, variable region 5 ' UTR exon, 1a﹠amp in human chromosomal 17q21; 1b), between the actual chromosomal region that reaches 81kb.Has rare high-density Alu repeatability DNA (41%), BRCA
1The a spot of pseudogene in upstream (BRCA1 φ) causes 1a, 1b and 2 exons polyphone to duplicate and reaches 44.5kb.
At BRCA
1In the gene, the 11st shows the long 3.4kb of son, BRCA
1The coding whole 1863 amino acid in, the 11st exons coding 61% amino acid, so numerous to the research document of exon 11.
The BRCT territory of terminal zinc ring structure territory of BRCA amino acid chain and C-end repeats to guard in the spinal animals, and other protein part may merge with all the other gene product homologies.
Relevant BRCA
1The gene expression research progress is a lot, known BRCA
1There are several shearings to transcribe mode; Most importantly in the disappearance of frameing shift (BRCA1-Δ 11) of the 11st exon, total length and Δ 11 exons can be expressed, but the 100th and 97 К Da Δs 11 isomer proteins lack the signal for locating of nuclear, are positioned at slurry.Another kind of total length 220 К Da albumen mainly are positioned at the nuclear phosphitylation.Slowly arrive highest level from G1 to the S phase.This moment BRCA
1With BRCA
2And Rad51 albumen co each kitchen range point in dispersive nuclear.BRCA during dna damage
1Peroxophosphoric acidization, BRCA
1, BRCA2, Rad51 in each kitchen range point disperse, be repositioned onto the dna replication dna structural region that contains PCNA, form dna repair protein (BASC).It has the various kinds of cell function such as DNA repairs, transcribes, the albumen ubiquitinization of karyomit(e) reorganization.
1, about BRCA
1Transgenation
BRCA
1Sudden change mainly is positioned at the 11st exon, account for full gene mutation rate 2/3, it is divided into ABCD4 district, biologically is divided into frame shift (insertion), frame shift (disappearance), missense, nonsense, shearing, polymorphic, unfiled, 9 kinds of sudden changes such as synonym hypotype, it medically is the mutation type of three kinds of medical significances with its cluster, the first kind is detrimental mutation (deleterior), to frame shift, is cut into the master; Second class is suspicious pathogenic mutation, and they have accounted for the great majority of mutation rate, show as missense mutation, nonsense mutation, and the big base of intron (3bp) changes, and also has the sudden change of part monamino acid disappearance.The 3rd class is the sudden change of a large amount of not bright meaning, based on synonym, polymorphic and non-encoding mutant, silent mutation, and the intron sudden change apart from about the exon 40bp.
Sudden change frequency (prevanlence): i.e. BRCA
1At familial breast cancer, sporadic breast cancer, cancer patient relatives at different levels and irrelevant crowd's frequency distribution.Know best in the data that the BIC of www.genome.gov database announces with Myriad genetic.In breast cancer, ovarian cancer crowd, Jew BRCA1/2 mutation rate raises 〉=30%, American 〉=10%, and Chinese's small sample 〉=3%, Jew in the familial patients with mastocarcinoma 〉=80%, American 〉=60-80% still lacks Chinese related data.
Medical science shows meaning (penetrace) outward: to the medical significance of BRCA1 sudden change, understanding such as penetrance and prognosis are based on Myriad genetic early stage report 10,000 routine mammary cancer and relevant informations in 2002, and research subsequently is classified as follows sudden change:
Hazardous property sudden change, comprise the albumen brachymemma at least distance-C hold 10 more than the amino acid.Special missense and nonsense are inserted the preface sudden change.Wherein 17.2% sudden change belongs to the hazardous property sudden change.Wherein 60% for frameing shift, and 25% is nonsense mutation, and 12.5% is including the subarea, and 2.1% is missense mutation.
Suspicious pathogenic mutation claims not qualitative sudden change again.Comprise missense mutation, the intron region mutation, terminal sudden change accounts for 19.8%.
The non-outer sudden change that shows: also claim not expression mutation, polymorphism sudden change.Do not produce the albumen brachymemma, control group allelotrope changes greater than 1% but does not have clinical impact; Coding region variation neither change aminoacid sequence does not indicate yet shears exon (silence); Sudden change does not damage length and the stability that mRNA transcribes; Account for 60% sudden change and belong to this big class.
On the difference of ethnic group mutation rate, in the 10000 routine mammary cancer of Myriad report, the Aisa people is 112 examples (1.1%) only, 91 philtrums have 11 people to have hazardous property sudden change (12%), add suspicious pathogenic mutation (1.15 times) and the non-outer sudden change (3.5 times) that shows, amount to 56% mutation rate, too big with domestic homogeneous data gap.
The total hazardous property sudden change of the BRCA gene of Myriad report is 17.2%.Wherein there is 20% hazardous property mutation rate in breast cancer tissue, cancer mutation rate low (13%) in the pathology upper conduit, and wetting property breast cancer mutation rate height (24%), male breast carcinoma BRCA has up to 28% mutation rate, and ovarian cancer is up to 34%.With regard to mammary cancer people species diversity, the hazardous property mutation rate of European descent is 16%, and the blood lineage's of Jew is 21%, and African blood lineage accounts for 19%, and Latin/Caribbean blood lineage's is 18%, and U.S. original inhabitants blood lineage is 14%, and the Asia blood lineage is 12%, and visible difference is little.
Domestic research overview: in conjunction with the update search content, comparatively approaching with the data and the Myriad data of direct sequencing report: Xu Guangwei etc. add order-checking with DHPLC and detect the full genome of the 9 routine breast cancer BRCA of family (36 couples of BRCA
1Primer), BRCA the 18th exon of other 32 routine sporadic breast cancer 33 routine normal human blood samples.Familial breast cancer pathogenic mutation rate reaches 11-22%, and sporadic breast cancer and normal people close the 18 exons variation of 33-37% or polymorphic, and he points out treatise data mutation rate that China delivers mostly＜5%, be because the site of detecting not comprehensively, system not.Zhou Dongxian etc. detect totally 11 sudden changes (31.4%) to 35 routine mammary cancer and 10 routine healthy human blood's the 11st exon total length pcr amplifications, 25 polymorphic heterozygosis (71.4%), but analyze wherein disease cause mutation.
On the other hand, Liu Li etc. detect the 2nd, 11,20 exons to 56 routine mammary cancer and the 34 routine ovarian cancers that contain 12 routine family histories with PCR-SSCP and MNA method, do not find transgenation.Wang Shuyu etc. detect the 2nd, the 11st exon to 37 routine mammary cancer (containing 4 routine family histories) with the PCR-SSCP method, and 1 routine phase shift mutation (2.7%) is only found in order-checking then.Wang Xufen etc. detect the 2nd, 11,20 exons to 50 routine mammary cancer with the PCR-SSCP method, do not detect sudden change.Cui waits quietly with method 6 exons of 79 routine breast cancer detections, only detects 1 routine single base (2430T＞C) sudden change.The method that is equal to Lu Yunfei has detected 89 routine mammary cancer, only finds 4 example sudden changes (4.5%).Dong Qiuming detects identical exon 2 example (6%) to 34 routine mammary cancer with method sudden change.Ke Yuxiong detects 86 routine breast cancers the 2nd, 11 and one of 20 exons discovery is polymorphic.Lai Chunning etc. detect 186 routine sporadic breast cancer BRCA1 2.11.20 exons, and checking order then through PCR-SSCP detects 13 example sudden changes.The hazardous property mutation rate is only 2.7%, total mutation rate 7%.The Hu Zhentong method detects the only 3 example sudden changes (7.3%) of 41 examples.As a result their common conclusion be Chinese women mammary gland BRCA1 with abroad compare mutation rate low, there is no need to carry out BRCA
1Sudden change detects.Suter detects 1306 routine Shanghai woman worker BRCA mutation rates only 8% in the U.S. with the PCR method.We carry out BRCA to 46 routine mammary cancer or first degree relative (containing 12 routine familial breast cancer and relatives)
1Order-checking, wherein 14 examples are carried out BRCA
1Genome sequencing.Find 127 mutational sites altogether, 15 kinds of different mutation types, wherein 5 kinds is the hazardous property site, and every routine hereditary breast cancer patient contains the site more than 10, and it is that BIC and NCBI do not appear in the newspapers that 4 novel sites are arranged.46 total philtrums have 19 sudden changes (41%).
2, external BRCA
1Patent application
Gene patent is the product of the 80s and 90s in last century biotechnology rise, and left and right sides human growth genome obtained gene patent power the earliest in 1978.The existing at present Human genome more than 4000 kinds has been declared patent because of its new purposes of constantly finding, wherein 63% belongs to external private company, and 28% belongs to universities and colleges.Enjoy the Human genome patent right maximum be American I ncyte company, covered 2000 kinds of Human genomes approximately; China associating genome company once reported in 1999 has declared 4000 kinds of gene patents." science " magazine was once drawn the patent present situation and was declared detail drawing the end of last year, pointed out 20% Human genome to be declared patent, and wherein medical usage is significantly as senile dementia and cancer related gene, and a gene has been declared the patent of the different purposes of kind more than 20.
There is the problem that runs off in China aspect gene protection at present; Chinese's asthma gene, acquired immune deficiency syndrome (AIDS) gene have been registered patent abroad; some targeted drug gene (EGFR) also has been registered patent abroad, and the patent of relevant BRCA1 gene is the gene patent application incident of influence power maximum in the world.
(1) problem and the shortcoming of prior art existence
BRCA
1Detection technique mainly comprises four aspects at present
1, immunohistochemistry technique
Mainly utilize BRCA
1Antibody, the BRCA of marked tumor and target cell in the histocyte section
1The protein expression situation.Can make the positive, negative sxemiquantitative record.Shortcoming: 1. the susceptibility difference 2. the poor specificity coarse evaluation that 3. belongs to protein level 4. can not observe gene aspect mutation type, mode and coding site.So the assessment transgenation does not have using value.
2, round pcr
The range gene amplification and the gel electrophoresis method of PCR-based, as strand Nucleotide sex change PCR (PCR-SSCP), sex change high performance liquid chromatography (DHPLC), two dimension genescan (TDGS), denaturing gradient gel electrophoresis (DGGI), heteroduple analysis (HA), fluorescence mismatch analysis (FAMA), albumen brachymemma tests (PTT) etc. are to detect BRCA at present
1The mainstream technology of gene, principal feature: 1. use the synthetic primer targetedly of known base sequence, what 2. detect is single base mutation type in the sudden change, 3. detected result only react sampling, examination character short base fragment, write down the result in the mode of running gel.
Shortcoming is 1. to discern electrophoresis strip to have subjective factor, not Jia Shiyi erroneous judgement of the goodness of fit, influenced greatly by experiment condition so the repetition accuracy is low, not enough as template quality, glue quality, temperature, voltage to the accuracy of detection of electrophoresis list base point mutation, produce false negative, 3. detect as being applied to commercialization, then be subjected to having announced in the patent BRCA
1The restriction in mutational site.
3, biochip technology
This is that the external big flux that rises in recent years detects BRCA
1The technology in mutational site can detect that number is many, the site is many, the time is short.But shortcoming is the BRCA that 1. only can be used for known announcement
1The site; based on the point mutation examination; 2. the base length limited is in the exon district of 20-30bp; large fragment deletion shear zone and the then omission of intension; 3. experiment repetition accuracy rate is low; so require 2 repetition rates to reach 70-80% announcements of just transmitting messages when above, the 4. same restriction that when being applied to the commercialization detection, is subjected to intellectual property protection.There are not medical practitioner and pricing policy yet.
4, gene sequencing technology
Genome is checked BRCA by base entirely
1The all kinds transgenation of gene, and demonstrate the form result of each base sequence, belong to the detection technique of the most advanced science of current biology field, can analyze known, the unknown base change state of each race.Shortcoming is gene sequencer and the operation pathology doctor who 1. needs the high quality precision, and 2. by the base order-checking, technology is difficult to the order-checking of the multidigit point of big flux to universal difficulty at present, so document does not in the world almost have BRCA
1Genome total length order-checking data.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, new mastocarcinoma gene mutational site (BRCA is provided
1) and a kind of at detected dna profiling, the BRCA of application ABI
1Complete 44 pairs of primers of genome (RSS000009248-01) carry out pcr amplification, purifying, electrophoretic analysis; Genome sequencing reaction, purifying is after gene sequencer sequencing analysis, data analysis comparison, detect and report and the BRCA of report not
1Exon, intron gene mutation site, mode and type.
(-) inventive principle
1, BRCA
1The morbidity of gene and mammary cancer is closely related, is subjected to the attention of American National Patent Office and EUROPEAN PATENT OFFICE, its clinical medicine diagnosis and treatment, health care, the widespread use that taken the lead in each oncogene of scientific research aspect.
2, use BRCA
1Full genome primer, to Chinese han population protoblast and BRCA1 syndromes, mammary cancer family member and breast cancer tumour somatocyte carry out gene sequencing, detect BRCA
1Transgenation does not have as far as possible to omit comprehensively and detects.
3, utilize BRCA
1Full genome (exon, intron) sequencing result carries out the preceding disease of health examination, mammary cancer and cancer, BRCA
1Syndromes patient's gene test and diagnosis and treatment.
The present invention takes following technical scheme to realize:
The mastocarcinoma gene mutational site, described site is
The first kind: the 11st exon 914T/C (S265S) heterozygous mutant; The 1st intron base mutation, 124C＞T, 385A＞G, 495InsACA/494InsAAC/493InsCAA, heterozygous mutant 124C/T heterozygosis, 385A/G heterozygosis; The 872T/C heterozygous mutant; The 24th exon non-coding region base mutation, 6410G＞T, 7276C＞T;
Second class: the base mutation of the 11st exon: 2201C＞T (S694S), 2430T＞C (L771L), 2731C＞T (P871L), 3232A＞G (E1038G), 3667A＞G (K1183R), 2685 T/C (Y856H), the 13rd exon: 4427T＞C (S1436S), the 16th exon: 4956A＞G (S1613G);
All there is heterozygosity sudden change mode in the above-mentioned site of second class;
And any single or associativity in above-mentioned first, second class mutational site sudden change.
Mastocarcinoma gene (BRCA
1) mutational site check and analysis method, comprise the following steps:
One, invention application and material
1, social crowd's blood, body tissue cell, wax stone and formalin-fixed tissue, culturing cell, all kinds of frozen, the fixing cell of preserving;
2, mammary cancer and all BRCA
1Syndromes patient and the blood relationship personnel's of family thereof tumour cell and blood;
3, BRCA
1Related neoplasms patient's tumor cell and blood preparation;
Two, technological method
1, DNA extraction and quantitative
Flesh tissue: commercialization DNA extraction test kit
Fix and paraffin: commercialization company DNA extraction test kit
Blood and liquid cell: commercialization company DNA extraction test kit
More than all by specification indicating means operations, with gel electrophoresis system, and the nucleic acid quantification instrument detects dna profiling quality and concentration, select dsDNA for measuring target, the 260nm light absorption ratio is greater than (dna content is greater than 2.0ug/ul) more than 0.05, between the light absorption ratio A260/A280 ratio 1.6-1.8,320nm wavelength place light absorption ratio is close to 0 sample as being suitable for template.
2.PCR specific amplification
1. primer
BRCA
1Sequencing primer, OMIM (NCBI): 113705 (sequence is seen disk)
Celera database number: RSS000009248-01 detects BRCA
1The gene order of 100kb total length (exon, intron, shear zone) has comprised zinc finger protein and has included the terminal bases that BRCT and transcriptional activity district are held in subarea and 5 '.
2. pcr amplification condition
PCR system: AmpliTaq Gold PCR Master Mix, 50% glycerine, PrimerMix, template DNA, Distilled water.
PCR condition: put 95 ℃ of pre-sex change 15min in the pcr amplification instrument, 94 ℃ of sex change 50sec, 63 ℃-58 ℃ annealing 1min (0.5 ℃/1min), 72 ℃ are extended 1min, circulate 94 ℃ of sex change 50sec altogether 10 times, 57 ℃ of annealing 1min, 72 ℃ are extended 1min, circulate altogether 30 times, extend 10min in 72 ℃ at last;
The control of PCR product quality: the PCR reaction product requires band single, and concentration 30-100ngPCR enzyme is used HotStarTaq DNA Polymerase, gold medal enzyme and fine other enzyme both at home and abroad;
3. PCR product purification:
Cross column purification: the PCR of Promega company purification kit, and other commercial purification kit.
Alcohol/EDTA purifying
Gel video picture electrophoresis assessment purifying quality behind the product purification, numbering, file (80 ℃), report.
4. sequencing reaction and purifying
Order-checking PCR reaction conditions: 1ul BigDye (2.5X)+1.5ul BigDye SeqBuffer (5X)+3ul primer+1ulPCR purified product+3.5ul ddH
2O.96 ℃, → 60 ℃ of 10sec → (96 ℃ of 10sec → 50 ℃ 5sec → 60 ℃ of 4min) * 25 circulations, 4min → 4 ℃ insulation
Order-checking purifying: with alcohol/NaAc/EDTA method and other commercialization method of purification.Key step is seen the ABI operation instructions.
5. gene sequencing (containing backward sequencing): 96 orifice plate specimen preparations, last sample to 3100 genetic analyzer carries out electrophoresis.Datecollection software carries out data processing and analysis automatically.
6, sequencing result analysis and report
Sequencing result forms written statement, operator and report doctor signature, notes phase, and enclose the primitive sequencer electrophorogram, sequence chart is given clinical or trouble side;
7, records handling registration
It is single that all detected objects are all filled in application for registration, indicates medical history, pathological diagnosis, check HE section;
All DNA template, PCR purified product are all numbered-80 degree very low temperature and are preserved for future reference
Three, processing step
1. BRCA
1Gene sequencing:
Four, processing condition
1, envrionment conditions
Dna profiling extracts pcr amplification and necessary subregion of sequencing reaction or locellus operation, is strictly on guard against crossed contamination, the standard Pollutant Treatment.In three grades of laboratory operation of national molecular biology or management, training, and issue licence.
2, staff condition
The relating operation personnel must be qualified through " National Laboratory's approval system " training seminar's training, has the pathology qualifications of a licensed doctor, and more than Master degree candidate's graduation, clinical pathology diagnosis working had molecular pathology experiment basis and training on operation more than 1 year more than 3 years.
3, hardware condition
DNA spectrophotometric quantitative instrument, automatic gel imager, an order-checking level pcr amplification instrument, electrophoresis apparatus, refrigerated centrifuge, supercentrifuge, DNA analysis instrument (gene sequencer), broadband internet.
4, software condition
Have skilled molecular biosciences and molecular pathology knowledge expertise and consult NCBI and pertinent literature storehouse
Can use 3100-AvantDate Collection 2.0 running software sequenators
Can analyzing DNA sequencing analysis 5.1 software analysis raw data
The automatic icp gene sudden change of DNA seqScape software
The manual icp gene sudden change of DNA star seqman software and other genetic analysis softwares.
Technical result
(1), object
1. breast cancer families: in Jiangsu TCM Hospital's mammary gland surgery, inpatient department and 12 testers (age 41-60 year) that collect 9 breast cancer families of Han nationality in the patient with breast cancer who examines, wherein 2 examples suffer from breast cancer, numbered respectively, its peripheral blood got in signature informed consent postscript.Breast cancer families is collected standard: have at least 1 routine mammary cancer case to be made a definite diagnosis before 50 years old in family's 3 routine mammary cancer, meet the first degree relative or the member of a breast cancer families of above-mentioned primary condition.
2. sporadic breast cancer: the patient with breast cancer in that Jiangsu TCM Hospital's mammary gland surgery is in hospital, gather 3 routine patients' fresh tumor tissues after informed consent.
3, mammary gland precancerous lesion: the mammary gland precancerous lesion patient who is in hospital at Jiangsu TCM Hospital's mammary gland surgery, after informed consent, gather 10 routine patients' new fresh tumor tissues, the right newborn lobulated tumour of 1 example, 5 routine intraductal papillomas become, 2 routine cystic hyperplasia of breast diseases, 2 routine cyclomastopathy.
4, the BRCA1 syndromes: except mammary cancer, ovarian cancer, carcinoma of endometrium, cervical cancer, prostate cancer, liver cancer etc.
3. normal controls: selected condition is informed consent, and the age is more than 35 years old, and is healthy, no mammary cancer family history, and no mammary gland serious disease history collects the peripheral blood (comprising the contrast of 1 normal male) that meets above standard normal control 8 people.
(2), method (see before and state)
(3), result
1, breast cancer families gene sequencing result: 11 have been carried out BRCA among 12 testers
1Full gene sequencing, 1 is detected the 3rd and 11 exon complete sequences.Wherein the 11st exon has 5 all to detect base mutation, be identical base mutation: 2 places same sense mutation: 2201C＞T, 2430T＞C, with 3 places missense mutation: 2731C＞T, 3232A＞G, 3667A＞G (EMBL/Gene Bank/DDBJ gene recording mechanism AY304547), cause the 871st, 1038 and 1183 amino acids to replace (protein recording mechanism AAP70031) respectively.5 individualities are heterozygous mutant in addition, and be the heterozygote of said mutation gene and normal gene: 2201,2430 and 2731 is base C/T heterozygosis, 3232 and 3667 A/G heterozygosis.Other has patient G050434 to find 914T/C (S265S) heterozygous mutant, and the BIC record is not seen in this site.Other has 2685 T/C of 2 patients (Y856H) heterozygous mutant (the two is sister, and elder sister is an infitrating ductal carcinoma, and younger sister is normal health check-up).The 13rd exon has 4 to detect identical base mutation with the 16th exon, is respectively 4427T＞C (S1436S), 4956A＞G (S1613G), and other has 5 individualities is heterozygous mutant 4427T/C heterozygosis, the 4956A/G heterozygosis.The 24th exon non-coding region has 4 to detect identical base mutation, 6410G＞T, and 7276C＞T, other has 5 individualities is heterozygous mutant 6410G/T heterozygosis, the 7276C/T heterozygosis.The 1st intron has 4 to detect identical base mutation, 124C＞T, and 385A＞G, 495InsACA/494InsAAC/493InsCAA, 4 individualities are heterozygous mutant 124C/T heterozygosis, 385A＞G heterozygosis in addition, 495 InsACA.Find the 872T/C heterozygous mutant G050477 number in addition.Tabulation is seen in sudden change in detail.
2, sporadic breast cancer patient sequencing result: 2 have been carried out BRCA among 3 testers
1Full gene sequencing, 1 is detected the 3rd exon.Wherein 2 the 11st, 13,16,24 exons and the 1st intron appearance base mutation identical with above-mentioned breast cancer families tester.
3, mammary gland precancerous lesion patient sequencing result: 5 have been detected the 3rd exon among 10 testers, and 5 are detected the 11st exon partial sequence, and the 3rd exon is not found nucleotide variation, and 2 detect 5 the heterozygosity sudden changes identical with breast cancer families.
4, normal controls: 1 has been carried out full gene sequencing among 8 testers, and 7 have been detected the 11st exon partial sequence, and wherein 2 detect 5 identical heterozygosity sudden changes of full milk gland cancer family.
5, new site
1, patient G050434 finds the 11st exon 914T/C (S265S) heterozygous mutant
2, the 1st intron has 4 to detect identical base mutation, 124C＞T, and 385A＞G, 495InsACA/494InsAAC/493InsCAA, 4 individualities are heterozygous mutant 124C/T heterozygosis, 385A＞G heterozygosis in addition, 495 InsACA.Find the 872T/C heterozygous mutant G050477 number in addition.
3, the 24th exon non-coding region has 4 to detect identical base mutation, 6410CG＞T, 7276C＞T
Sum up:
1, breast cancer families 12 philtrums 12 examples are found 17 site mutations, mutation rate 100%
2,2 examples are found 12 site mutations, mutation rate 66.6% in the 3 routine sporadic breast cancer
3,2 examples are found 5 site mutations, mutation rate 20% in the 10 routine mammary gland precancerous lesions
4,2 examples are found 5 point mutation, mutation rate 25% among the 8 routine normal control crowds.
The foregoing description does not limit the present invention in any form, and all employings are equal to the formed technical scheme of mode of replacing or imitating conversion, all drop within protection scope of the present invention.
Claims (2)
1, mastocarcinoma gene mutational site, described site is
The first kind: the 1st intron base mutation, 124C＞T, 385A＞G, 495InsACA/494InsAAC/493InsCAA, heterozygous mutant 124C/T heterozygosis, 385A/G heterozygosis; The 872T/C heterozygous mutant; The 24th exon non-coding region base mutation, 6410G＞T, 7276C＞T;
Second class: the base mutation of the 11st exon: 2201C＞T (S694S), 2430T＞C (L771L), 2731C＞T (P871L), 3232A＞G (E1038G), 3667A＞G (K1183R), 914T/C (S265S) heterozygous mutant, 2685 T/C heterozygosis (Y856H), the 13rd exon: 4427T＞C (S1436S), the 16th exon: 4956A＞G (S1613G);
All there is heterozygosity sudden change mode in the above-mentioned site of second class;
And any single or associativity in above-mentioned first, second class mutational site sudden change.
2, mastocarcinoma gene 1 (BRCA
1) mutational site check and analysis method, comprise the following steps:
(1) application and material
A, social crowd's blood, body tissue cell, wax stone and formalin-fixed tissue, culturing cell, all kinds of frozen, the fixing cell of preserving;
B, mammary cancer and whole BRCA
1Syndromes patient and the blood relationship personnel's of family thereof tumour cell and blood preparation and above-mentioned materials thereof;
C, BRCA
1Related neoplasms patient's tumor cell and blood preparation;
(2) detection method
A, DNA extraction and quantitative
Flesh tissue: commercialization DNA extraction test kit
Fix and paraffin: commercialization DNA extraction test kit
Blood and liquid cell: commercialization DNA extraction test kit
B. the operation of above all by specification indicating means, with gel electrophoresis system, and the nucleic acid quantification instrument detects dna profiling quality and concentration.Select dsDNA for measuring target
(3), PCR specific amplification
A, primer
BRCA
1Sequencing primer, OMIM (NCBI): 113705
Celera database number: RSS000009248-01 detects BRCA
1The gene order of 100kb total length (exon, intron, shear zone) has comprised zinc finger protein and has included the terminal bases that BRCT and transcriptional activity district are held in subarea and 5 ';
B, pcr amplification condition
PCR system: ABI AmpliTaq Gold PCR Master Mix, Primer Mix template DNA, Distilled water;
PCR condition: put in the pcr amplification instrument pre-sex change 15min, 94 ℃ of sex change 50sec, 63 ℃-58 ℃ annealing 1min, 72 ℃ are extended 1min, circulate 94 ℃ of sex change 50sec, 57 ℃ of annealing 1min altogether 10 times, 72 ℃ are extended 1min, circulate altogether 30 times, extend 10min in 72 ℃ at last;
The control of PCR product quality: the PCR reaction product requires band single, and concentration 30-100ngPCR enzyme is used HotStarTaq DNA Polymerase, gold medal enzyme and fine other enzyme both at home and abroad;
C, PCR product purification:
Cross column purification: commercialization PCR purification kit, and other commercial purification kit;
Alcohol/EDTA purifying
Gel video picture electrophoresis assessment purifying quality behind the product purification, numbering, file (80 ℃), report;
(4), sequencing reaction and purifying
Order-checking PCR reaction conditions: BigDye (2.5X)+BigDye Seq Buffer (5X)+primer+PCR purified product+ddH
2O; 96 ℃, → 60 ℃ of 10sec → (96 ℃ of 10sec → 50 ℃ 5sec → 60 ℃ of 4min) * 25 circulations, 4min → 4 ℃ insulation
Order-checking purifying: with alcohol/NaAc/EDTA method and other commercialization method of purification;
(5), gene sequencing and backward sequencing: 96 orifice plate specimen preparations, last sample to 3100 genetic analyzer (gene sequencer) carries out electrophoresis; Datecollection software carries out data processing and analysis automatically;
(6), sequencing result analysis and report
Sequencing result forms written statement, operator and report doctor signature, notes phase, and enclose primitive sequencer figure and give clinical or trouble side;
(7), records handling registration
It is single that all detected objects are all filled in application for registration, indicates medical history, pathological diagnosis, all DNA template, and the PCR purified product is all numbered, and-80 degree very low temperature are preserved for future reference
(8) processing step, condition
A, BRCA
1Gene sequencing:
B, envrionment conditions
Dna profiling extracts pcr amplification and sequencing reaction must subregion or locellus operation, is strictly on guard against crossed contamination, the standard Pollutant Treatment, and program is by the control of corresponding SOP system.
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| CN 200610085551 CN1920054A (en) | 2006-06-23 | 2006-06-23 | Mammary cancer gene (BRCA1) mutation detecting analysis |
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| CN101200766B (en) * | 2007-12-24 | 2012-06-06 | 北京美康生物技术研究中心 | Multiple PCR reagent kit detecting breast cancer susceptibility gene mutation and preparation method thereof |
| CN104017870A (en) * | 2014-05-28 | 2014-09-03 | 杭州美中疾病基因研究院有限公司 | Breast carcinoma gene BRCA1/BRCA2 mutation detection kit and preparation method thereof |
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