CN1661048A - Relativity between proto-oncogene of leukaemia in myeloblast class essential hypertension - Google Patents

Relativity between proto-oncogene of leukaemia in myeloblast class essential hypertension Download PDF

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CN1661048A
CN1661048A CN 200410016467 CN200410016467A CN1661048A CN 1661048 A CN1661048 A CN 1661048A CN 200410016467 CN200410016467 CN 200410016467 CN 200410016467 A CN200410016467 A CN 200410016467A CN 1661048 A CN1661048 A CN 1661048A
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myb
seq
gene
primer
hypertension
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金力
黄薇
姜正文
王颖
张晨辉
李艳平
王志敏
肖君华
卢大儒
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Fudan University
Shanghai Human Genome Research Center
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Fudan University
Shanghai Human Genome Research Center
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Abstract

A process for testing the susceptibility of primary hypertension includes detecting if there are variations in premyelocytic leukemia proto-oncogene MYB, transcript and/or protein of an individual, and determining its high susceptability if there are. Its reagent kit is also disclosed.

Description

The dependency of myeloblastosis proto-oncogene and essential hypertension
Technical field
The present invention relates to molecular biology and medical field.Relate more specifically to the myeloblastosis proto-oncogene (myeloblastosis oncogene, single nucleotide polymorphism MYB) (single nucleotidepolymorphism, SNP) and with the dependency of essential hypertension.The invention still further relates to the method and the test kit that detect these SNP.
Background technology
Hypertension is meant one group of clinical symptom grouping that systolic pressure or diastolic pressure raise.The rising of blood pressure and coronary heart disease, renal tubal dysfunction, hypertensive heart disease and the concurrent cerebral apoplexy of hypertension have an apparent causal connection.The up-to-date Case definition of hypertension is systolic pressure 〉=19kpa (140mmHg) or diastolic pressure 〉=12kpa (90mmHg), and meeting wherein, a person can be diagnosed as hypertension.
Most hyperpietic only has slight subjective symptoms in early days in elevation of blood pressure, as headache, dizziness, insomnia, tinnitus, fidgety, the difficult concentrated fatigue etc. that also occurs easily of working and learning energy.Along with the development of the state of an illness, when particularly developing complications, symptom increases and obviously gradually, as numbness of the fingers and stiff, lower limb pain appears when walking more, or the nervous sense of nape portion sore muscle appears.Show when, shortness of breath nervous, uncomfortable in chest, precordialgia that heart gets involved, frequent micturition at night, diuresis occur, show when urine is light that kidney is got involved, the kidney arteriole hardens when occurring.If obnubilation appears suddenly in the hyperpietic, breathe promptings such as dull irregular, gatism hematencephalon may take place, if engender a side limb activity inconvenience, numbness even benumb, should suspect the formation whether cerebral thrombosis is arranged.
The early stage Non Apparent Abnormality sign of hypertension occurs.When appearring in vitals such as brain, the heart, kidney, the mild damage can have unusual sign to occur.The performance of common heart abnormality has that apex beat moves to left, pareordia is praised the sample pulsatory feeling, auscultation apical region of heart first heart sound strengthens, second sound of aortic area strengthens and systolic murmur and diastolic murmur are arranged, show arteriosclerosis and left ventricular hypertrophy take place, if listen in the apical region of heart and the galloping horse sample rhythm of the heart may show the appearance of central force depletion.Also common in addition ear-lobe folding line, capillary pulsation, radial artery occur and pulsus durus or acrotism and lower limb intermittent claudication etc. occur.
In addition, because the development of some risk factor or hypertension itself can cause the remarkable or hurried rising of some hyperpietic's blood pressures, simultaneously with vitals functional lesions such as brain, the heart, kidney, retinas, a series of clinical special signs appear in serious threat to life, are called hypertensive emergency.The sickness rate of hypertensive emergency accounts for 5% of Hypertensive Population, and common have hypertensive encephalopathy, hematencephalon, acute left heart failure, the acute withdrawal syndrome of clonidine, Acute Myocardial Infarction, a radical type malignant hypertension etc.
About 5% left and right sides no conscious sympton among the hyperpietic does not know when blood pressure raises, and does not more know to have produced when the complication of blood vessel and organ injury yet, some patient even just know after cardiovascular accident has taken place and oneself suffer from hypertension.So, find out the genetic cause of hypertension incidence, hypertensive morbidity can be effectively controlled in preventing and detecting early, and disease is reduced to minimum to the injury of human body.
(essential hypertension EH) also is essential hypertension to essential hypertension, is a kind of independently disease, and the cause of disease of oneself, rule and the clinical manifestation that the generation development lapses to are arranged.Mainly show as the rising of arteriotony clinically.Account for more than 90% of crowd hyperpietic, at present pathogeny is not clear fully as yet, mainly just can be diagnosed as essential hypertension (essential hypertension) after having got rid of the hypertension that other diseases causes.The rising of arteriotony mainly is because of due to periphery arteriole resistance increases, and in various degree Q volume of blood and kinemic increase are arranged simultaneously.Often cause late period internal organs such as the heart, brain, kidney to be got involved severe complications such as hypertensive heart disease, heart failure, renal tubal dysfunction, hematencephalon take place.The treatment of essential hypertension mainly is to bring high blood pressure down to prevent the generation of complication simultaneously.The primary hypertension patient cause of death is cerebrovascular accident, cardiovascular accident and renal insufficiency, and for seeing, in heart failure and uremia is taken second place more with cerebrovascular accident in China, and American-European countries sees with heart failure more, and cerebrovascular accident and uremia are taken second place.
Essential hypertension is as modal cardiovascular disorder, and its sickness rate rises year by year, and can cause the serious heart, brain, kidney complication, is the primary hazard factor of cerebral apoplexy and coronary heart disease.EH is a kind ofly interacted and the polygenic disease of morbidity by inherited genetic factors and environmental factors, and the genetic mechanism of seeking the EH genes involved and then illustrating hypertension incidence has become the focus of present research.
Though have many researchs about range gene polymorphism and essential hypertension, do not confirm the report of myb gene and essential hypertension dependency, more do not confirm the SNP of myb gene and the report of essential hypertension dependency.
In sum, for the final treatment hypertension that realizes, this area presses for seeks the essential hypertension tumor susceptibility gene, and develops method, test kit that detects essential hypertension and relevant medicine.
Summary of the invention
Purpose of the present invention just provides a kind of diagnosis (especially early diagnosis) hypertensive method and detection kit.
Another object of the present invention provides the hypertensive method of a kind of new treatment.
A further object of the present invention provides the hypertensive pharmaceutical composition of a kind of treatment.
In a first aspect of the present invention, a kind of method that the hypertension susceptibility of individuality is diagnosed is provided, it comprises step:
Detect this individual myb gene, transcript and/or albumen, and compare with normal myb gene, transcript and/or albumen,
There are differences and just show that this individuality suffers from hypertensive possibility and be higher than normal population.
In another preference, what detect in the described method is gene or the transcript of MYB, and with normal MYB nucleotide sequence comparing difference.
In another preference, described difference is following single nucleotide polymorphism:
24071 A → G;
Wherein, the nucleotide position numbering is based on SEQ ID NO:1.
In a second aspect of the present invention, provide a kind of test sample whether to have the method for the single nucleotide polymorphism of myeloblastosis proto-oncogene MYB, comprise step:
(a) with the myb gene of myb gene primer amplified sample, obtain amplified production; With
(b) detect whether there is following single nucleotide polymorphism in the amplified production:
24071 A → G;
Wherein, the nucleotide position numbering is based on SEQ ID NO:1.
In another preference, described gene-specific primer has the sequence of SEQ ID NO:2 and 3.
In another preference, the length of described amplified production is 100-3000bp, and contains among the SEQ IDNO:1 the 24071st.
In a third aspect of the present invention, provide a kind of detection hypertensive test kit, it comprises the primer of specific amplification myb gene or transcript, more preferably, it is 100-3000bp that described primer amplification goes out length, and contains among the SEQ ID NO:1 the 24071st amplified production.
In another preference, described test kit also contains the reagent that is selected from down group:
(a) with SEQ ID NO:1 in the 24071st sudden change bonded probe;
(b) the 24071st sudden change restriction enzyme among the identification SEQ ID NO:1.
In another preference, described sudden change is selected from following single nucleotide polymorphism:
24071 A → G;
Wherein, the nucleotide position numbering is based on SEQ ID NO:1.
In a fourth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains the proteic inhibitor of MYB or the antagonist and the pharmaceutically acceptable carrier of safe and effective amount.
Embodiment
The inventor is through deeply and extensive studies, and the SNP of a large amount of candidate genes is measured and analyzes.Find first and proved that the part SNP of myb gene and hypertension are closely related, and found its new function: the change of MYB will cause hypertension, wherein the association study result shows, (24071 A → G) there is significant difference (P＜0.05) in the distribution in case and control group, therefore can be used as to detect hypertensive specificity SNP at the SNP of the 24071st of MYB.Finished the present invention on this basis.
Myb gene
(myeloblastosis oncogene, detailed sequence MYB) can be the nucleotide sequence (can referring to network address http://www.ncbi.nlm.nih.gov/) of U22376 referring to accession number to the myeloblastosis proto-oncogene, shown in SEQ ID NO:1.
The c-myb gene belongs to more clearly proto-oncogene on the 26S Proteasome Structure and Function, and it is that a kind of DNA is conjugated protein.The expression of c-myb is that growth is dependent, and the resting cell expression level is low, and when cell proliferation was vigorous, its genetic expression increased sharply, G1 peak late period (Bwown KE et al, J Biol Chem.1992 Mar 5; 267 (7): 4625-30.).C-myb it is found that except closely related with the propagation of tumour cell the expression enhancing as transcription factor MYB is to cause one of reason of smooth muscle cell (VSMC) propagation.
Vascular smooth muscle cell (VSMC) propagation and phenotypic alternation be atherosclerosis and other obliterative vascular diseases main pathological basis (Miano JM, et al.Am J Pathol, 1990,137:7612765.)., the smooth muscle cell proliferation of MYB and blood vessel concerns that this points out it may be relevant with blood pressure regulation because existing.Human foley's tube injury rats aortas such as Simons M in 1992, use antisense and just myb oligonucleotide (ODNs) to import injury region respectively simultaneously, find that antisense myb oligonucleotide (ODNs) can suppress the propagation of carotid artery smooth muscle cell (carotid artery smooth muscle cells) significantly, film in normal there are not obvious effect (Simons M, et al.Circ Res.1992 Apr; 70 (4): 835-43.).
Intracellular Ca 2+The variation of concentration is relevant with the cell cycle, and G1 is by Ca in the cell to the transformation of S phase 2+The rising activated of concentration, and transcription factor c-Myb by with cytolemma Ca 2+Ca in the cell is regulated in the combination of promoter region ATPase-1 (PMCA1)) 2+Level causes the transformation of G1/S.Therefore at the medicine of c-Myb target spot, can regulate the propagation of arterial smooth muscle cell, reduce sanguimotor Peripheral resistance, may reach treatment atherosclerosis and hypertensive purpose by the expression of inhibition MYB.(Afroze?T，et?al?Curr?Drug?Targets?Cardiovasc?Haematol?Disord.2001?Jun；1(1)：23-40)。
The inventor checks order to the almost whole zone in the myb gene, has found many SNP, and wherein most of SNP and hypertension susceptibility are also uncorrelated, yet association study shows 24071 A → G but are and the very high SNP of hypertension susceptibility cognation.This SNP is arranged in No. 12 intron of MYB, points out influentially to the transcriptional level of MYB, and then causes influencing the expression of MYB and the propagation of smooth muscle cell, and the hypertension susceptibility that finally causes the carrier is apparently higher than normal population.
Based on new discovery of the present invention, MYB albumen or polypeptide have many-sided new purposes.These purposes include, but is not limited to: be used to screen the material that promotes the MYB protein function, as antibody, polypeptide or other part.The peptide molecule that can suppress people MYB protein function that can be used for seeking therapeutic value with the recombinant human MYB protein screening peptide library of expressing.
On the other hand, the present invention also comprises people MYB DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people's myb gene product or fragment.Preferably, refer to that those can combine with people's myb gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people MYB, comprise that also those do not influence the antibody of people MYB protein function.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) 2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule; Or chimeric antibody.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, people's myb gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human MYB albumen or its have antigenic segmental cell and can be used to immune animal and produce antibody.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.
Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare.Antibody of the present invention comprises the antibody that can block people MYB protein function and the antibody that does not influence people MYB protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people's myb gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people's myb gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).
The proteic antibody of anti-people MYB can be used in the immunohistochemistry technology, detect people MYB in the biopsy specimen proteic what and/or whether suddenly change.A kind of preferred anti-MYB antibody is the antibody of the normal MYB of nonrecognition MYB but identification suddenlys change, perhaps discerns normal MYB but the antibody of nonrecognition sudden change MYB.Utilize these antibody, the hypertension susceptibility that can carry out protein level easily detects.
Utilize MYB albumen of the present invention,, can filter out with MYB albumen interactional material takes place, as inhibitor, agonist or antagonist etc. by various conventional screening methods.
MYB albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intravenously or subcutaneous administration.
The present invention also provides a kind of pharmaceutical composition, and it contains the proteic inhibitor of MYB of the present invention and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 0.1 microgram/kg body weight-Yue 10 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that the proteic antagonist of the MYB of safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 0.1 microgram/kg body weight, and in most of the cases be no more than about 10 mg/kg body weight, preferably this dosage is about 0.1 microgram/kg body weight-Yue 100 micrograms/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
The proteic polynucleotide of people MYB also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of MYB of the proteic nothing expression of MYB or unusual/non-activity.The method that structure carries the recombinant viral vector of myb gene is found in existing document (Sambrook, et al.).The recombinant human myb gene can be packaged in the liposome in addition, and then is transferred in the cell.
Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people MYB protein level.These tests are known in the art, and comprise ELISA etc.
Whether having the proteic method of MYB in a kind of detection test sample is to utilize the proteic specific antibody of MYB to detect, and it comprises: sample is contacted with the MYB protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample MYB albumen.
The proteic polynucleotide of MYB can be used for the diagnosis and the treatment of MYB protein related diseases.Aspect diagnosis, the proteic polynucleotide of MYB can be used for detecting the proteic expression of MYB MYB abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of MYB as the MYB dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of MYB albumen and also can detect the proteic transcription product of MYB.
The sudden change that detects myb gene also can be used for diagnosing hypertension.Detection can be at cDNA, also can be at genomic dna.The form of MYB protein mutation comprises that the point mutation compared with normal wild type MYB dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.
The method of the detection SNP of the present invention of most convenient is by the myb gene with myb gene primer amplified sample, obtains amplified production; Detect then and whether have following single nucleotide polymorphism in the amplified production: 24071 A → G, wherein, nucleotide position is numbered based on SEQ ID NO:1.
Should understand, after the present invention has disclosed the SNP and hypertensive dependency of myb gene first, but those skilled in the art can design the amplified production that specific amplification goes out to contain this SNP position easily, determine whether to exist 24071 A → G by methods such as order-checkings then.Usually, the length of primer is 15-50bp, preferably is 20-30bp.Though the complete complementation of primer and template sequence is preferred, one skilled in the art will appreciate that at primer and template to have under the situation of certain not complementary (especially 5 of primer ' end) also can increase specifically (promptly only amplifying required fragment).The method that contains the test kit of these primers and use these primers is all within the scope of the invention, as long as the amplified production that this primer amplification goes out contains the correspondence position of SNP of the present invention.A kind of preferred primer is to having the sequence of SEQ ID NO:2 and 3.
Though the length of amplified production is not particularly limited, the length of amplified production is 100-3000bp usually, preferably is 150-2000bp, more preferably is 200-1000bp.These amplified productions all should contain among the SEQ ID NO:1 the 24071st.
Because SNP of the present invention and hypertension have very high cognation, therefore not only can be used in early days diagnosing primary hypertension more exactly, and can make the carrier just take reasonable precautions (as on diet, taking corresponding control) against a rainy day at premorbid not, thereby improve carrier's lifetime and life quality, therefore have earth shaking using value and social benefit.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
1.1 research object
Because blood pressure is the quantitative character that is subjected to multiple such environmental effects that polygene participates in, the inevitable problem that when analyzing genes involved, will consider genetic heterogeneity.Select the relatively single isolated crowd of genetic background will help to reduce the influence of genetic heterogeneity, simultaneously because these colonies have comparison fixed living environment and more consistent living habit make Effect of Environmental reduce greatly as the material of research multigenic disease.Select such colony,, still therefrom choose family and carry out pedigree analysis, all help to improve the detection sensitivity in hypertension susceptible gene site no matter be to be used for the linkage disequilibrium analysis.Therefore selected in the present embodiment isolated population is as research object (isolated population has the relative uniformity of genetic background, the advantage that founder's number is fewer).
On the basis of informed consent random collecting 324 ages at the Han nationality's individuality more than 50 years old, all come from Dabie Mountain, Yuexi County, Anhui Province and ring the intestines town.80% individuality has only 6 surnames, and founder's number should be fewer, and other individuality of identity comes from same founder.The hypertension sample of selecting for use requires systolic pressure to be not less than 140mmHg, and diastolic pressure is not less than 90mmHg, measures blood pressure and averages for twice.
Select 11% being positioned at the top that blood pressure distributes (37 examples are individual wherein; SBP＞178mmHg) and 7% (22 individualities that are positioned at the lowermost end that blood pressure distributes are arranged; SBP＜104mmHg) is totally 59 individualities, carries out the detection of SNP by the method for direct order-checking.
1.2 experimental technique and result
1.2.1 DNA extraction
Extract DNA with conventional phenol chloroform method from people's blood, concentration correction is used for conventional pcr amplification to 20ng/ul.
1.2.2 the design of PCR and sequencing primer
According to the genome sequence of MYB among the GenBank, design and synthetic following primer.Concrete primer is as shown in table 1 below.
Table 1 primer sequence table
The primer title Sequence (5 '-3 ') SEQ?ID?NO：
Adopted primer is arranged ttttaatggcaccagcatca ?????2
Antisense primer catcatgctttggggaatct ?????3
1.2.3 the pcr amplification of myb gene
With the dna profiling that extracts, use the Taq enzyme, on GeneAmp 9700 PCR instrument, carry out pcr amplification with the Touchdown program.Reaction conditions is: 94 ℃ of pre-sex change 2 minutes, and 94 ℃ of sex change 30 seconds, 63 ℃ of annealing 40 seconds, 72 ℃ were extended totally 10 circulations, 0.5 ℃ of each cycle annealing lapse of temperature 40 seconds; Later 94 ℃ of sex change 30 seconds were annealed 40 seconds for 58 ℃, and 72 ℃ were extended totally 30 circulations 40 seconds; Last 72 ℃ were extended 7 minutes.Pcr amplification product is verified through agarose gel electrophoresis.
As a result, obtain the amplified production of 334bp.
1.2.4 the discovery of SNP and detection
The PCR product is used ABI-PRISM behind the Resin resin purification TM377 dna sequencing instrument (appliedbiosystems of u.s.a. applied biosystem company (ABI)) carry out the two-way order-checking of the terminal cessation method of fluorescent mark, and the interpretation and the SNP that carry out sequence with Polyphred software (the http://droog.mbt.washington.edu/Polyphred.html of Washington, DC university) confirm.
As a result, find to exist following SNP:24071 position A → G.
1.2.5 SNP gene type and association analysis
Carry out the SNP gene type with direct unidirectional sequencing.Promptly in hypertensive patient and normal arterial pressure control group, carry out somatotype and association analysis.
Carry out chi square test after allelic frequency statistics is come out,, determine whether that tentatively allelic frequency and blood pressure level are chain by blood pressure the highest 11% and 7% minimum individual data items are compared.
The frequency of G type SNP in the hypertension individuality that found that among the SEQ ID NO:1 24071 is 38.2%, and the frequency in the ypotension crowd is 18.1%.Difference on the frequency between the two is 20.1%, has confirmed among the SEQ IDNO:1 24071 A/G type SNP and the hypertensive dependency that exists.
Embodiment 2
Essential hypertension susceptibility detection kit
As described in embodiment 1, sudden change and the high blood pressure disease of 24071 A → G are closely related among the SEQ ID NO:1.Therefore, can be that template increases and detects at DNA based on this sudden change design myb gene Auele Specific Primer with patient.
Prepare a test kit (100 person-times), it contains:
Title Sequence Concentration
Adopted primer is arranged SEQ?ID?NO：2 100pmol
Antisense primer SEQ?ID?NO：3 100pmol
The PCR reaction solution Contain Taq enzyme dNTP magnesium ion PCR reaction buffer
Extract male sex's patients'blood 3ml to be detected, use ordinary method (or using specific test kit) from blood, to extract DNA.PCR primer in the hypertension detection kit is diluted to 1 ì mol/ ì l, is that template is carried out the PCR reaction with the primer that is provided with the DNA that is extracted.Behind the PCR product purification, use ABI-PRISM TM377 dna sequencing instrument carry out the two-way order-checking of the terminal cessation method of fluorescent mark, and the interpretation and the SNP that carry out sequence with Polyphred software confirm.
Perhaps, amplified production and normal control are carried out stratographic analysis with sex change high performance liquid chromatograph (DHPLC), also can detect 24071 A → G.
Detected result, the hypertension susceptibility that contains the detected object of 24071 A → G is higher than normal population.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Fudan University
Research Center of Shanghai Human Genome
＜120〉dependency of myeloblastosis proto-oncogene and essential hypertension
<130>040144
<160>3
<170>PatentIn?version?3.1
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＜213〉homo sapiens (Homo sapiens)
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gaattcttta?ttggactatg?catattccca?aacatattat?tgtcatacaa?ctgagggaaa??????60
aatgtaaaac?tatatctggg?aaatgaaaaa?ttataaattc?tcaaaatgat?acataggaaa?????120
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caagaactta?ggagtagtta?cacacctcct?aaaatcttga?aaaagaggct?atgaactcac?????240
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attttaaaga?aagtctaaaa?caccagatag?tcttgaaacc?tcgcatgctt?caccgtttaa?????660
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gctaagtttg?aactgctggg?gcacatggga?cacccacaaa?cccagtcagc?agaagtctca?????780
aactcctaaa?agattaaagg?aaaaatacat?tagtgtgtac?agagaatgaa?aaaaaaatcc?????840
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gtgctggggt?gagagtgaag?gacatgtgac?aatacgacgg?tggcggctgc?aggatccggg????1320
aggggagtga?aagctccgct?ggggcgctcc?attagtgagc?ggtgatggtt?gccgcccact????1380
tgtattgaag?cgtcctttgt?cactaacaag?ttaaattaga?gatgttattt?atttaagaag????1440
aaggaaaaaa?aaccctagcc?aaacagccta?tgaatacata?tgctcacatc?ccctactcct????1500
ccaactccta?atttccccgt?ctccagaggg?cacagttgta?aaccttgacg?aaaatccaat????1560
cttctgtgcg?ggaatttccc?cccaccgctt?gccgcccccg?cgacagtgag?tgggagctgg????1620
aggagctctg?gtcccgctgc?ccgggagcac?gcggagccgg?gcgaccgcgg?tgcggcagcc????1680
agggaggagg?ggaggcggcg?ggactgggcg?cgggcgggcc?gccgccgcga?cccgggagcg????1740
gggtttgctc?aggaaaaggc?gccgtcgcgg?cccccggcca?cccctccctg?gccccgggct????1800
ccctgcccgc?gcgcctcccg?ggcctccggg?cggctagggc?accgcgcggg?cgagcgccga????1860
atgggagcgg?cgacccggcc?agcccggcag?cccgcgggcg?gcagccaggg?cgaccgcgga????1920
ggcggcgggc?agggcgcgtg?cgcactgcag?gggcgccaga?tttggcggga?gggggagtgt????1980
ccaaagctct?ttgtttgatg?gcatctctgt?ttacagagtt?tacactttaa?tatcaacctg????2040
tttcctcctc?ctccttctcc?tcctcctccg?tgacctcctc?ctcctctttc?tcctgagaaa????2100
cttcgcccca?gcggtgcgga?gcgccctgcg?cagccgggga?gggacgcagg?caggcggcgg????2160
gcagcgggag?gcggcagccc?ggtcggtccc?cgcggctctc?gcggagcccc?gccgcccgcc????2220
gcgccatggc?ccgaagaccc?cggcacaggt?aacggggagc?cgggcgggcg?gccgagggcg????2280
ggggcggggg?tcgcggggcg?ccaggctccc?gggagcaggt?gggaattcgt?tccgggatca????2340
tctgaggggc?tgtcagaccc?tccgaggacc?tggagcccct?gcctcggcag?cagaagccgc????2400
tccagagact?gatgaatgga?agagtctttg?cgggaaatgt?atccggacgt?tgggaagcac????2460
gccctgcggc?tcattttgca?agttgcatgg?ggatacattt?ttctttaggg?ggaaaaagaa????2520
agcacgttcc?agccgagggc?agagcggtgc?ccaccagccc?cggggagccc?accaggtgcc????2580
tggcttgatg?ccgcgggtgt?cggcgaggga?tccgcagcgt?aattgctgga?tgcattgaga????2640
tatgtttgga?cttggaagtg?cagagcatat?ggcagataca?gggaaaatgc?tggattgttt????2700
agtagctgca?ggtagttcat?ttaaatttca?ttcattcttt?atggaggcga?ggaccagtgt????2760
cagctgacag?gcggctgaaa?agcagatttt?agggggataa?aggggtctct?acttttgcaa????2820
attgtcttaa?gacctggttt?tggaggttcg?aatcattttt?ctctgaaagg?cttttacttc????2880
cactgcaaac?attgcacggc?acccacacgc?cgccgtcgca?catgtcactg?cacttaggga????2940
ggctcgccgg?gaagatgggt?gcagtccctt?cacatttttg?gccggacaga?tgggaagagg????3000
gagaggagga?ggaggaggga?aatcctcgtc?cgaactgtca?gttgctcgtt?ccccagtctc????3060
caccggcctc?tggactttcc?cagcagcaca?cgccggggct?ccctgcgagc?ggcgggtgca????3120
gggctagctc?ggcagccgtc?gcagtcgcca?ctcggctcaa?ggggacagag?gccggcagca????3180
cccaaggccg?ctgcgcgcgc?aaccgggacg?cgatccccgc?gggctctgcc?cccaggccaa????3240
aggtccccgc?ctggggcgca?cgcgtggatg?cggctgaggt?cgccgcgcct?tctcgcaccc????3300
tccttcagcc?gccttaggtc?gccccggccg?aggcgcggtg?accggacaga?tcctcgcgaa????3360
ggagttctaa?ggcgaagtcc?atggaggcaa?agcaaatcca?ttcgcctttc?cttagattgt????3420
ttgattttct?cctactggtt?tgttgttgag?cctgcggcgc?tggtccccgc?gcggcgctgg????3480
gaagttgcaa?agagcgtggg?tggagaccct?gcagcagcac?ccgcgggctg?ggctgggcgg????3540
ggagcagcgc?cggggcttgg?ttgggctctg?gggacgagag?ggcgacttgg?gggagctccg????3600
ggctccctat?gctcctccgg?agcctggccg?cgcaggacgg?tgtgctgccc?ctcgacgctc????3660
cacttttcgc?ctttaggact?tcacaggaca?gctaccgggg?tcatctgttt?tttttttttt????3720
ttttttcgag?agctgaaact?ggcagcagga?ggagggagtc?gggcaggggt?gctgggagga????3780
ggcgcggcac?ggctccgccg?ccccgaggtc?tcggcccccg?gggcggggcc?gcgcaggttc????3840
cctgaggctc?caggtagccc?agaggggagg?cggctgggag?gggcgggagc?cttgaacctg????3900
cctcagggga?agagaaaccc?gactgcgggc?tcggagccgt?ttcttgcaag?ggcgagcgtc????3960
aactcggcag?ggattatatt?aaccaggtca?gcgaaatgag?tttgaacccc?tccgaatcac????4020
agtagcggcc?acaaggcact?tcctcccgct?cccctccctc?ttcctaaccc?ccagcacctc????4080
ttttcttttt?cgtttctttg?cacggtttgt?ttccttgaca?gaatgtaaca?ttcatagttg????4140
tactcttgta?accacaccgg?gtctagcagg?tggggagtcg?agtgcatcaa?ccctgaaatc????4200
agcctttgaa?aatcctctga?ctgttgggtc?tgagatcttg?tcagagcaga?taaaggaaaa????4260
gttatttcag?gttccttagt?agaaattaat?catgcttcta?ggacgactta?gagcaagcaa????4320
atcgcatgca?aagttcaaag?gcgagcctgg?gtggccagcc?tggcagggaa?actcaggagc????4380
tgggcgcgtg?agcagccccg?gactcgccct?gctgcgtcct?ggccccttca?ggtcctgcag????4440
aagtcactct?ccaccttgcc?cccaagacct?tagttttgca?catctttgcc?tctgtgctta????4500
gcttttaaga?ggcacggaag?gacatggcat?ggtcatgtat?tagcgctgga?aagtacctta????4560
gaaataggat?ccaagcaagc?ccttattgta?aaggcgagga?actgaagtct?tgagagagag????4620
gaagtgattt?gtccaagtca?tacagctagg?tggaggctag?actagaacct?ttgcctccag????4680
aaccctagtc?ctgttgattc?ccgtgctctc?acctgacgtt?ggctgccccc?tgcgggcggc????4740
tgaataggtt?gcttgttcct?gggttgttaa?accgctgcgg?agagagaggt?gctcaatgac????4800
tactctgaaa?aatgtgctca?gttctgaaaa?tgtgacctct?agatccagcc?aaacctgaaa????4860
agttaattcg?acttgaatct?atgactgcag?tttcagattc?ctctggtttt?cttggtgtct????4920
ctcacatttc?acaagggact?gcatttgggg?tctttccaag?gagttcccta?tgaatctttc????4980
aaaagatgca?gacttacacc?tctgggcttt?atagcttttt?tttttttttt?tttttttgct????5040
ttatcatatc?gtgttttaca?tttttgtagt?tcttgttcag?aagttgttta?gtgcctcctg????5100
ttgacagtgg?aaacagaact?aggctattgt?ttcaaaacac?agacactaaa?tgaaaggaat????5160
tattacagat?tataaatata?tatttcctgg?gattttattt?ggtggtggtg?gtattttaaa????5220
tttggattac?atcatgtcgc?cattcaacct?ggactatcag?attgttgctt?aaaataatag????5280
ctttaaatta?tgagaacccc?caattcagga?tttttggtaa?gcacttcagc?ttcacttgta????5340
gggggctggt?aatatgcata?ccagtgagtt?agtttgggaa?gacaaaacct?gtaatgaata????5400
taacttttct?ttttaaaaac?aatgatttac?attatgatat?ggctttatat?cattgttttt????5460
attggtctta?gtttaatggg?tttcccaaca?ttgcaatgac?aaattttagt?gaagtttcta????5520
agatagtatt?tttcctattt?taagttttat?aagaaaaact?ataaaataaa?ccgtgtctgt????5580
aaaattattg?tcatctgatc?agttgattag?attaacaaac?attatgatta?tgattaacaa????5640
acagtaagca?tatagttact?tcttcaaaag?tcataaaatt?tcctaggtta?ggttaactaa????5700
ataaaagaaa?aagctttcga?aaatagtaat?gggttctttt?aaatccatgg?cttttttctg????5760
gggaaatccc?ttctggatta?ggttacattt?tataatattt?caaacaccat?tccatcagag????5820
acagaaaaaa?caagagtaag?taagactctg?actaacaagt?ggcctaatta?ttcacttagt????5880
tactctagaa?actaagtatt?gtaaacatgg?gcacaagttg?gatcaaccag?gcctggagtt????5940
gtgagcaatt?tggtattaat?tttatttaca?aaacattaaa?gcttgatcac?tcaatgttct????6000
tatctttgct?ttggttttaa?aatcctttcc?tcttagattc?tcctaatcct?ctagacttta????6060
tgggatcact?ataatcctgt?tttgcgctgt?actacttctt?gatttttttc?ttcttttaat????6120
aaaacaaaaa?ccccattgga?atagcatagt?tgaattgttt?attatgtttg?agaaatatta????6180
tttaaacgat?gtgacagatg?ccaaagattt?tgagtgtgca?cttatataaa?ggacatgggt????6240
tcttcttcct?tttcttatcc?ttaaccttaa?gttttcaact?taaaccttca?ctggttggaa????6300
ggtggccaaa?tgtataactt?gtccctggtc?taatagtaac?agcaggttca?gacatgcagg????6360
ggaataggaa?ggtgccaggt?ccttggccgt?gtctgtggat?acccataaca?gcagaaccag????6420
tttacaatac?tagagcaaca?gaatgcagca?aacaatcttg?ttgtgcaagt?tttcaaagtt????6480
ttgtcttcat?aacctttgaa?aagattgttg?aggagttttg?tgtaagtttt?gtaatccagt????6540
agtagtctaa?atcctcttgt?ttcagcccac?gtctacccat?tcttatttct?gcagcatata????6600
tagcagtgac?gaggatgatg?aggactttga?gatgtgtgac?catgactatg?atgggctgct????6660
tcccaagtct?ggaaagcgtc?acttggggaa?aacaaggtgg?acccgggaag?aggtaactaa????6720
tcattttatc?aagacgcaga?aaatagatag?agtgtataat?ttataaaaaa?caaaatttca????6780
actaaactac?aggtgctcaa?gctcattagc?aggctccttg?agatcttatc?ctagcccgca????6840
tgtgcagcgt?gccctatgcc?ccccacttca?ttatattatc?catttccaca?ctatttgcag????6900
ttctttgaat?gtcctcttct?cattttaggc?ttcatctgtg?tttccttttg?cctgttgagt????6960
tctcatcctt?ttgtcccacc?tcttcttcct?acttttacat?tacccatgaa?ggcatcacct????7020
cttctgtgaa?gccatccctg?gcttcgccgg?acaaggattg?gatgcatcct?cctgtgatcc????7080
catgcatggg?tgcatgttct?actttattgt?tgctgggaca?tatatacctc?ctccagctga????7140
ctgcaagtct?gttgagggca?gggctcttgt?ctattcatct?ttgtgtttcc?tgaattcaca????7200
aatgcttgga?cccaaaatag?gatattaata?aatgttctct?caataagtga?atgacttgct????7260
actcccaaaa?acaaaacaaa?caaaagaata?aaacaaaaca?ctcagagaac?tcatcttttt????7320
acagtgtatt?gatttaatat?ccttgatata?cttaacagag?ccatctatat?gaaggtatgt????7380
tttaaaatca?tccgataatg?gatggagaca?catctgcttc?tcctgtttct?ttttcggctg????7440
gcttatcaaa?agtttgattt?tgtaggaatg?gaaaaatatt?ttagaagcaa?aattattaat????7500
atgtggagaa?aagtttctct?aatgttctta?aaatttttgc?aactaatttt?agatgattca????7560
tgttacaata?aatggtattg?gctcttcttc?agtttcactg?ttctctgctt?catttaaacc????7620
agagctcctt?ttaacaggca?ggtaggattc?ccagagaggc?ccataggaaa?ttcatatttt????7680
acctattaag?attatccact?tggataatcc?ttatgatacg?gaaattctgt?tgtcaacatt????7740
ggtttcctaa?ttcttgtgtc?tgtattgtac?cagggacctc?cactgtgtgt?gaatgttacc????7800
tttgcagaaa?cataagaagg?aatgcattta?tatacttatg?aactggtagg?attgtgaaca????7860
gcatataaac?taaaagaact?gattagctgg?aaatgtttaa?tgtcttttat?gttctaacac????7920
atctggccta?tctaaatcta?actgattttc?ttatatagct?ttcaaattta?attttaaaaa????7980
tggtctttat?ttcttttata?aaaagttaaa?ctaactggaa?atattctatt?tcttgctcaa????8040
gatcctattt?attaccatag?gtaaatgtgt?actgaatcta?gcactggaat?gcattttttc????8100
ctaacagagt?aaagcagtta?tttataataa?acactcaata?tatgccaggc?attatgataa????8160
atgttttgca?aaattgcctc?atgtaatccc?caaataaccc?tgttaatatc?agcctcatgt????8220
taataaattg?ccccatgtaa?tccccaaata?accctgttaa?tttccatttt?atagatggca????8280
atactaaggc?ccagaaaggt?aaaatggtat?acccgcttta?cacaattaat?agttgagtca????8340
agatgccaac?tctggtctga?ttctaaataa?atctatactc?ccaagtccgt?atattacatt????8400
aatatctcct?gtattcggat?actttagaga?gactttttat?tctagattcg?ttccctcatt????8460
cactttaact?tgaacagagt?tatatagtaa?ataactgata?tcctaggatt?acctttaaat????8520
gtcttaggat?gaaaaactga?agaagctggt?ggaacagaat?ggaacagatg?actggaaagt????8580
tattgccaat?tatctcccgg?taagttagta?agtttttctt?ataacgaaag?gaaagcttct????8640
cccaaagatt?tctataaaat?attctaggag?gagttattaa?gatagtgtat?aatttactca????8700
catttaagaa?gtgggagaaa?tgccctaccc?ataacttata?ttttatatta?taatgcatat????8760
aattaaatta?ttagaattta?gattattaga?atcgattatt?ataatttaga?ttatcaaatt????8820
ttaaaaacag?ttgcttcttg?ttttatcttt?tcatttatag?gataattagt?attttgatga????8880
tttgttacat?ggtagttgat?taaacttgat?atttcctcca?agtcagtttt?tcctgaattc????8940
cctatttttg?tcaatagcaa?tacttttggc?accaaggttt?aaaattctga?agacctttct????9000
tgacttatca?cattccccca?acccctacat?ctaagcagtt?actaaggacc?ctggaattcc????9060
tttatagtca?gtcccgtcct?tttcattctc?atggctgtgc?tgaaggttgg?gctcctgcct????9120
ctcatttgga?ctatcatata?acttcctaac?tggcttcccc?acttgtagtt?ttccattttt????9180
ctttcttttt?tttttttttg?ttttttgttt?tttttttttt?gagatggaat?ctagctttgt????9240
tgcccaggct?ggagtgcagt?ggcacgatct?cagctcactg?caacctccgc?ctcccaggtt????9300
caagcgattc?tcgtgcctca?gcccccttag?tagatgggat?cacaggtgcc?cgccaccatg????9360
cctggctaat?ttttgtattt?ttagtagaga?caaggtttca?ccatattgat?caggctggtc????9420
ccaaactcct?gacctcaagt?gatctgccca?cctcagcctc?ccgaagtgct?aggattgcag????9480
gcatgagcca?ccgtgcccag?ccagtcttct?acttttccat?tcatcccaaa?caccattggc????9540
aggtggaact?tcctaaaatg?cagctgtacc?ttatcttttc?aaaaccaacc?acagtgacag????9600
aatctcacaa?attaagtaaa?ctgccttgac?ttggcacaca?gaggccctcc?tctattgccc????9660
caacttacag?ctccagttct?gtttcctgct?tcctctctct?aagcaggttt?gcttgaattt????9720
atttctgctt?tacctcctct?gggcttttgc?tcctggtctt?cccacctcct?agaatattct????9780
ccctttatgt?caccaattaa?agttctgtcc?atccttcaaa?gtccttatga?agagctcttc????9840
aagactcctc?ccaaggaaac?catttctcct?cctaagaaga?tgcttctctc?ctcacttcag????9900
tatcattttg?ctttggtctt?ttcttccaag?tgaatcaaat?tcagcatctg?tgatagtcag????9960
tcatctactt?gtcttttcct?tcttataagt?ttacaaactc?ctgtaacata?ggacctatct???10020
tttatgcatc?ttttttcttt?ctgcaatata?aatactttgg?tcctggattg?aagcacatct???10080
cttgtctgaa?tgggaagcaa?actttagtct?tttagggtgg?tttccatgaa?agcctgacaa???10140
catagaatct?ctatcaagcc?tcaggctatg?tagatactct?gcaaaagtat?ctgaggttaa???10200
ggttaacttg?gcaggcacat?gtgtagtatg?atggtccttt?agatcatgtg?gcaggaatgt???10260
ttccctcagg?aggcagagtg?tctaatttta?gatgcaactt?tgatatatta?cttgcatgtt???10320
ctgtagcctg?taagtaatga?gggatagatg?ggctctaaac?atgctctaaa?atgttcttaa???10380
atattttcac?tgcatatggt?ctatgtgtga?ttcctattac?aacaaaaaat?tcacgtgctt???10440
atcacatcat?atctttatgg?tggtgcatac?tttatttgtc?tagaatcgaa?cagatgtgca???10500
gtgccagcac?cgatggcaga?aagtactaaa?ccctgagctc?atcaagggtc?cttggaccaa???10560
agaagaagat?cagagagtaa?gttctttctt?cattggtgtg?tgactcataa?ttaagaatat???10620
tcccaaaatg?ctaatatttt?cctatttgag?gaagcaggta?aattgggcaa?acaggaagta???10680
gaggactcta?ttcccatatt?tccagtgaat?gaaagcaaat?tttggaaatt?ttctaaagat???10740
cttgtaacac?tgaagaatga?ttatactgac?tcattacata?actttaaaac?ataggttatt???10800
tttgtgtgtt?tatctgaagg?tgatagagct?tgtacagaaa?tacggtccga?aacgttggtc???10860
tgttattgcc?aagcacttaa?aggggagaat?tggaaaacaa?tgtagggaga?ggtggcataa???10920
ccacttgaat?ccagaagtta?agaaaacctc?ctggacagaa?gaggaagaca?gaattattta???10980
ccaggcacac?aagagactgg?ggaacagatg?ggcagaaatc?gcaaagctac?tgcctggacg???11040
gtaataatat?gtcaaaaaat?atattgatcg?ggaagaatga?gggagtgggt?attgaacatt???11100
ctgctttaaa?tgtatggtga?gtaaattata?tttcatcagt?cgtaagtgtt?ttattagata???11160
aaccagctac?atagctttta?gagattgaag?acatcttaga?gtttatttgg?ttgagaactt???11220
ccaggatcca?cctgacagtg?agccctgggt?gaagacattt?agctttcccc?agcctcaatt???11280
tccccatttc?caaaatgaat?catacagggc?cagtgttagg?atcaagtaaa?atgaatgtca???11340
aaggacttgg?taaactataa?aactctaaat?gcatagaaag?aaactgaaga?cagtttgtct???11400
ctggcagttc?acgtagctgg?ttaagttact?caaaggaact?cagctaccat?tttcctgata???11460
accatcattc?atttctttta?ttttttttaa?ttattatttt?ttagagacag?aggcttgttc???11520
tgtcacacat?gagtgacaga?gtgcagtggc?atgatcatag?ctcaatacat?ccttgaactc???11580
ctgggctcaa?gggatcctcc?cacctcagct?tcttgagtag?ctaggactac?aggtgcaccc???11640
cactacaccc?agctgatttt?aaaatatttt?ttgtagagat?ggaatcctgc?tatgttaccc???11700
aggctggtat?caaactcctg?gcctcaaaca?atcctttcac?ctcccaaagt?gctgggatta???11760
taggcatggg?ccaccatacc?tggccaccag?tattcatttc?attataccac?tttgcttaat???11820
tttttacttc?attgattaaa?gaaataatgt?atttcttgaa?ttctcatact?acttccaggt???11880
ttttaaataa?tgtcagggta?ataatgcttt?ctttttaatg?aacaaggcaa?gaaaatgacc???11940
tcattatgag?ggataactat?ggaagctcat?tttgtcccat?ttttcccact?taagtagtat???12000
ataacagatt?ttgttccttt?ggaaagcaat?acttttaatg?ataaatcttg?cattaaaatg???12060
atgtagatca?aaaatattaa?aaatagtttt?attcttgagc?ttcataatcc?aggagaaagt???12120
ttatatagta?ttctctagtg?atgctccaaa?gcatttgcac?ccttgtggtt?cagtataaac???12180
ttagaaattt?taagaggttg?gtaaaaaaag?gcctttcctc?ccatatggcc?tgagaggata???12240
cactgctgat?tcttctcaaa?attaacaggg?ctattgaact?tcctctttct?ttagcataca???12300
ttcccccacc?cccattgaaa?tagaaaaagg?gagacttaaa?cggttttttt?gaattgagaa???12360
actgttttcc?tgagaatgga?aagactttag?taaaaatgcg?ttgccttttg?aaaatatact???12420
gggggttagg?atctttccct?ttgggatccc?attttccact?ctcgtctctg?tctgattcag???12480
aaaacaattg?gggtggcttt?caaagagtag?gaggtttttt?ggtctcagaa?gttctcacta???12540
gtgctttgag?agataaagag?tgaagtattc?ttcatcagca?gcatcttatc?atccaccaga???12600
cttcaccatg?tccctctcgt?ggtgtgtagg?ccctgctgtg?ccacattcaa?aggagtaatt???12660
tccccaaggc?agctggaact?ttacattgtg?ttggagtctt?gtaccgcgac?cggtcagaat???12720
aaacatgaag?cgttatcgta?ttgaaaatac?tcctttacca?aggaaatgga?atggttaaaa???12780
gatactgata?tttctagaaa?acgacatgca?ttccagattg?tgtaactaag?agaaatctaa???12840
ctaggcaata?gagaaagcct?gatctcagag?ggtggagaag?aaggaaacaa?ttaaaaagag???12900
aaaacaacta?ataggcccaa?gggaaaggtc?tctgttagaa?acagttttct?gtcaattcac???12960
tttaatctga?attgaaattt?ttgtttgtga?aatttgtgtg?atatatttct?gtgcagaact???13020
gataatgcta?tcaagaacca?ctggaattct?acaatgcgtc?ggaaggtcga?acaggaaggt??13080
tatctgcagg?agtcttcaaa?agccagccag?ccagcagtgg?ccacaagctt?ccagaagaac??13140
agtcatttga?tgggttttgc?tcaggctccg?cctacagctc?aactccctgc?cactggccag??13200
cccactgtta?acaacgacta?ttcctattac?cacatttctg?aagcacaaaa?tgtaagccat??13260
tcctgtgaat?ctagtttaat?gagagagacg?gttagtttag?ctccaggtaa?taatcatact??13320
ttgcagttgt?atacttttca?gagaactttc?ctgagcatga?tcatggccag?tcttcaccta??13380
tgccctttga?agtagatagg?gtgtaggtat?ggtgtgttac?ccctggttta?tgtctccaga??13440
aacgaggatg?gtaagcgctt?tgcccagggt?gacacagctc?atagagatag?tgctgactag??13500
aaaacgagct?gtccgattct?aaattgctgc?ttcaaatcta?atttgaaggc?aacctttgaa??13560
tgtaaattta?cttaaggtac?tattgaagaa?taggggaatg?gagtcgcatt?tgtagatgta??13620
atgtttttct?cttccaaaac?gaaaagggaa?ggtagtatta?tgtttaaaga?ggagggagtt??13680
tgacactgaa?attcagtggt?tatggtatgt?ttcattctgg?cagtgatcag?gcagacatgt??13740
cttaatttat?tcactcagta?agtgtttacc?ttcttactct?gagtcaggag?cagaactttg??13800
tgacattcat?tttctgatgt?ctccttttag?tctccgttgg?atcagtgtga?tcccttcctt??13860
atttctagat?atttatattt?agggaaagtg?ataatccata?ataggtggaa?aaagaagaag??13920
aaagaggaaa?aaaatcgaga?aactttaatc?ttaagaataa?caatattaaa?tgataaaact??13980
tacccaagaa?tagagagctc?atatgcaaat?tatatgcaaa?acatgttcat?aaatagtttt??14040
tattcttctc?tgtccatgga?tttgactaca?aactttacag?aaaggtctca?gatgaataaa??14100
ttatcataag?aagtcctgtg?ctttttatta?aaactcaaaa?ttattccaag?aaaaatattt??14160
ttatttacaa?acagctggtg?ccatcaattt?ttcccataga?aacaattttg?aaacattcat??14220
taatgtattt?tataatatat?tttttattgt?ttttttcatt?gtgtttttct?ctaattaagg??14280
aacacttccc?aaaatttaaa?gtgctcatca?acacctccca?aggatttttc?accatgttcc??14340
tttagctgtt?tagccatttt?aaaggcctgg?ctaaatttca?cattaaaagg?ttttaaaaat??14400
ttgtttagtg?tattttatca?gtaaagtgtt?ttttttctct?cagtcccaga?acgaaacctc??14460
aggaagccaa?gtcccctgaa?gcatatgtag?ccctgaatga?cgtggccttt?gttttgtctt??14520
ttgcatctag?gtctccagtc?atgttccata?ccctgtagcg?ttacatgtaa?atatagtcaa??14580
tgtccctcag?ccagctgccg?cagccattca?ggtaagatca?ttgatcattc?actgttcaaa??14640
gcaccacttt?taagaaatgc?ttatttcttt?catctaaaca?catattttta?cctgagcaac??14700
taaagtaaaa?aggaaaggga?acatggagag?gagagcagag?tcttggccag?caatctatta??14760
atcttgtctt?ctagaatgag?tttgctctgg?aattttctga?ttctttgatg?ttgcaaggtg??14820
aaataactca?tcagtcagca?tccactctag?cctgagattt?cacatcactc?ccttaagtct??14880
gaaataacat?tcacaaagat?ttctgcagga?gccaaatttg?gttggtggtg?ttgtcacagt??14940
gaggctcttt?gggctgtgcg?gcacctcatg?accatattgg?ctacacccat?tgtatttgca??15000
gcttccaatc?agacctttgt?ctttatttcc?cttactttgt?aatttcagag?acactataat??15060
gatgaagacc?ctgagaagga?aaagcgaata?aaggaattag?aattgctcct?aatgtcaacc??15120
gagaatgagc?taaaaggaca?gcaggtgcta?ccagtaagac?tgtcatcatg?tgcttgaatg??15180
aggggatagc?agctttgcct?cagtttacct?aagcgctctt?ctcttctaaa?tattacactt??15240
agcaaggctc?catatatcca?ttcagaatgt?ctcaacacaa?gaagttgctt?gtagtaaaat??15300
gtagttggta?tcagattata?tgctgattaa?attggaagca?gtctttttgt?aattgcaata??15360
aaaatgcaat?atccacttat?gattgttatc?atcaattttt?ttcttcagaa?ggactataat??15420
cagtttaaag?actagcaaag?ataaaatgaa?tgaaaatctg?cattttctac?agagctatgt??15480
atctatatgg?taccattttg?gtttttttct?attattttgt?taacctatat?tttcatattt??15540
tataatttca?gacatgattt?gatgcatgtt?cagtatatac?atgtaaaagg?ttatatataa??15600
aacctcttca?taaataacat?tagctgctct?cttttatttg?catgtgtgaa?agttatgtgg??15660
gccattactg?aattctgaca?tctttagcga?ctgggcagcc?aactgggatg?gctccttgtg??15720
ctttgcaaca?tacatagtta?accaacaaag?acaataaaag?agaagtgatg?cccagtaggt??15780
attcctaggg?gtaaatttat?tttttgttaa?ataataagat?gtttatgtgc?acatgctagt??15840
tcgaccactt?tttaacttta?gttaatgttt?tgcaatttct?cctgttgtgt?ggtcatactg??15900
taaggagact?tctgttaatt?tgctcttctg?ctttcctaac?cacgttgatt?tggcagtgct??15960
tcactgcatc?agtgcaatct?gtagcattag?aaagggatag?aagcaacaag?aatatgtgta??16020
gatcaaagca?actctaactc?ctgagtcctc?tagctttttt?gtggttagaa?tgcaaatagg??16080
acagttcttc?agttcttcac?tcacagcaag?catttatgga?cctttggcaa?attatattaa??16140
agtggaattt?taagacaata?tgataattat?acttcgaaat?atagacctat?gatttgacat??16200
ttttacaaaa?tgataacaag?gtaatcatat?aggtacaacc?tctagcacta?ttaaaaacag??16260
ggtcttgcat?tgataaaagg?aaacatcttg?acaactgttt?cataggcgta?agtttgggat??16320
gatgcaacta?ctcttatctt?tcctccaaca?gcatctgata?ccttgtgcaa?cttcattgct??16380
aagttccttc?tccctttctt?ctgtcctctc?tttatttcta?cacccttccc?ccttccttag??16440
acacagaacc?acacatgcag?ctaccccggg?tggcacagca?ccaccattgc?cgaccacacc??16500
agacctcatg?gagacagtgc?acctgtttcc?tgtttgggag?aacaccactc?cactccatct??16560
ctgccagcgg?atcctggctc?cctacctgaa?gaaagcgcct?cgccagcaag?gtgcatgatc??16620
gtccaccagg?gcaccattct?ggataatgtt?aagaacctct?tagaatttgc?agaaacactc??16680
caatttatag?attctgtaag?tagaattgtg?aagggaagtt?aacgattctg?gaagataaat??16740
tagaaaaccc?atttcttaaa?tcattgccat?tttctatagc?caagctgtta?ttagcatata??16800
ttaatgtaaa?ggtagaagta?tgattttcat?cttcatgaat?atgttttttc?aaagatctga??16860
tttcatagcc?taccgagaga?gacaaaagta?tttataaata?cttacaaaat?aggacttttg??16920
aaggaagagt?tttttttcac?attttcacac?ttttccttca?aaagtcatat?ttttaaaaat??16980
agtcagattg?atgacttata?aagcagagct?tggattatgg?gaacggaatg?gtgtttgagt??17040
tacttttaac?tttttttttt?ctttgcttac?ttcatggtgc?ttctctagaa?ttttgtcttg??17100
cttttgaaca?gcacattaat?gatttggctt?actcagagct?gaaagcaatc?ttttagtgta??17160
tttgaagtaa?taatcagctc?cttggtcata?cctggctctt?tccacaaaat?ggtgattatt??17220
gattactggt?cccattgctt?cactgagatg?aatcatcttt?cgtgaaacaa?atttgtattc??17280
agcacctcct?cagcgaacac?ttggttcctt?tcccagttga?ttaaaaatca?tttattgcac??17340
actcattggt?taggagatga?ccattgccgt?aatatgccat?ctagaaaagg?tcttcatttt??17400
gctttccatt?gcatgcagat?gtagagtgtc?gggaggtccc?tgcagcacat?cagagggcca??17460
gccttgaggc?agcccactag?accctgctct?actgcagtat?aatagagcaa?ctgctcatct??17520
tgaagctgtt?gctcaagaag?ccaaaccaac?gggcctagtg?tttgctttgc?gttgagcatc??17580
tctctctcag?tgctgtttca?ggtgatgatg?gcacacacta?tctcaaagtt?ctgtgcctcc??17640
cacattgttt?caggattctt?catcatggtg?tgatctcagc?agttttgaat?tctttgaaga??17700
agcagatttt?tcacctagcc?aacatcacac?aggcaaagcc?ctacagcttc?agcaaagaga??17760
gggcaatggg?actaaacctg?caggagaacc?tagcccaagg?gtgaacaaac?gtatgttgag??17820
tgagagttca?cttgacccac?ccaaggtctt?acctcctgca?aggcacagca?caattccact??17880
ggtcatcctt?cgaaaaaaac?ggggccaggc?cagcccctta?gccactggag?actgtagctc??17940
cttcatattt?gctgacgtca?gcagttcaac?tcccaagcgt?tcccctgtca?aaagcctacc??18000
cttctctccc?tcgcaggtag?aacacaattt?ctgcacagct?gttactcatt?ttcaacagac??18060
acctgagcct?taataatcta?gaaactaata?cttcggaaca?aatgactaat?tactgctgcc??18120
tttgctgatt?tcattctgtc?gttgaatcta?gctgttgcta?agttatactg?cctcccaaag??18180
tttaggctgt?ctgcaggcta?actgttgatt?ttggcatcat?tctctctctt?ttacttttgg??18240
gtggttttga?aagatagttg?catgaaacgt?acttttattt?ccttcaacac?tagagtttaa??18300
catttctgtc?cctttctttt?tctcccatct?gatccctccc?tttaaaattg?ttaacatcat??18360
ttcttcctcg?ttgttcattt?attcttatgc?gattcactga?cccaggggag?gagggaacca??18420
aattgtgttt?atataacatt?tgggttgcaa?ctagaaaatt?taaagctaga?aaatttaaag??18480
gttccaattc?atctttaaat?ggaagagttt?gggaagagga?tgagaaacaa?atgcttaagg??18540
aaaagaacat?atttttaatt?tcagtttctc?aacattggct?cttttgagtt?gtgccatact??18600
attttactag?tgcagttttt?gccaaggaat?atttttattt?catacagctt?ttttaaggca??18660
tgaaaatcac?aattcttatc?agagatttgt?gttcctaaat?tatttaaata?attaaattaa??18720
tagataattt?ggcactttaa?aacatttttc?caattgtatc?tgttgaattt?gttttaagaa??18780
cagatttagg?tatgatatag?gtatatacat?ttgggaaaca?ctgaatcaaa?caaagttaca??18840
agtaggtttc?ttagctgggc?atagtggcat?gggcctgtag?tcctagctac?tggggaagct??18900
aaggcaagag?gattgcttaa?gcacaggagt?tcaagttcag?cctggtgaac?atagcgagat??18960
cccatttctt?taaaaacttt?ctttttttta?actgcaggcc?ttctcagagt?tattaatatg??19020
ccgttgtgcc?ttgtgaattt?ccaggaggag?attatgatat?gttgtgtttc?ccaaatttat??19080
tttaccttgg?aattgtttaa?ggaacaccta?gttttttccc?agaactttag?tgttctgaga??19140
gacacagttt?attattcaag?aattttccat?ataatgtaaa?ccttctgtaa?tactaatttt??19200
ggttcataga?ttattaaaga?gaaaaaacaa?agtatgaaag?gaaactatta?attattaact??19260
attattttcc?ccaaaatcac?taaagccttt?taaatgttaa?taaagaacac?atctcattct??19320
ctttaatgac?aagtgtcatt?tattgagagt?attttctcac?agttttgcaa?gtttttcagc??19380
aatgacccta?aattttattt?cttggaaacc?acacccaaat?cctgggtgct?caattgaaat??19440
aaaacaatac?cctagtcaat?ataacatgct?tgttagcatc?tttattattt?aatatagtgg??19500
gtcaggaaaa?cattcttgtt?atctaggtgt?gatttttaaa?ttggggagaa?taaagaatta??19560
ccatctaatc?taagtatttt?ttctttctct?ccatatttag?ttcttaaaca?cttccagtaa??19620
ccatgaaaac?tcagacttgg?aaatgccttc?tttaacttcc?acccccctca?ttggtcacaa??19680
attgactgtt?acaacaccat?ttcatagaga?ccagactgtg?aaaactcaaa?aggaaaatac??19740
tgtgtaagtc?tttggttcag?gaataaaatg?atttatctta?tttacttata?tttaattaat??19800
ggaatatagt?tcccagatgt?ctgatccact?tgtatgtgca?taattttact?gaccatttat??19860
atataatcat?gtctattccc?acattgaata?tatctgtcct?gggcatatta?caactaccta??19920
caggtagaag?atgggaagat?aaactagatt?ccttttgtaa?cttactccaa?aatttcctat??19980
atatgcacaa?gtagttttca?taaaactttt?ccattcggtt?tgtttgttcc?ctccataagg??20040
ccttcttatg?aaagccagat?tttcaacctc?ctttcctctc?taattgtaag?cttcagaata??20100
agaacgagta?tggtttctta?cctagtttgt?atttttattt?aattttattt?gtttattatt???20160
tatttatttt?tagtttgtat?tttttaatct?tattttttct?ttttgaattt?atgtgtagat???20220
aggacctctt?ctgacatccc?caggaatatt?atatgattag?aagccaaggg?atgagtaagc???20280
agtcattttt?attcaataaa?gccttaatca?attcagagaa?atggtagaga?aagtgtgaat???20340
tatcagaaac?cttgaaaaga?aatgcactgt?tatttcgaag?tacaaaaagt?aaattttaaa???20400
agtttaacat?gttttgtagt?atgggctctg?ttttcatgaa?tagggaaacc?tgatttgtaa???20460
atgacatcag?gtcttcttga?tgctcatagt?cttatacaga?tcataggcac?tacatgtgtc???20520
ttcagagcca?ttaaggaaag?atgttaaatc?atttaatgct?tttagtcttc?gaggtataat???20580
attttgaaat?atttgaaaat?atgtgatatg?catattctct?ttttcttaac?gaataacgtg???20640
attaatcagc?acaccccaat?tccatatccg?gaacagagtt?tattgtattc?agtggaaaaa???20700
tgttaaatgt?aacaggtaaa?gccatgtagg?gacatttatt?cttttgtatt?gagccactgc???20760
ttgttttctt?tttaaagttt?tagaacccca?gctatcaaaa?ggtcaatctt?agaaagctct???20820
ccaagaactc?ctacaccatt?caaacatgca?cttgcagctc?aagaaattaa?atacggtccc???20880
ctgaagatgc?tagtaagttc?tagaaaagtt?tttggatttc?attggtttat?tagtcccatt???20940
aggagttctc?tctgatgcaa?aaatacccac?tcttccgttt?agcctcagac?accctctcat???21000
ctagtagaag?atctgcagga?tgtgatcaaa?caggaatctg?atgaatctgg?aattgttgct???21060
gagtttcaag?aaaatggacc?acccttactg?aagaaaatca?aacaagaggt?aaacacgcaa???21120
ccctttggaa?gcaacaagct?gagaatcctg?ccccctttat?tccttgtgtg?cagcttgatg???21180
tgtctgccat?tggcactgtg?caacaccacg?acttttcgtg?caagattttc?atacaaggtg???21240
cagcgaaaag?gtactgccag?actgtaggca?tcatcaacct?ttcctttggg?aagccaagcc???21300
atctctatct?acttcatgtt?tggacagaaa?acatactaga?gagtatcaag?aaaagggagg???21360
gaaagaggtt?tttaatgtac?ttttaaacat?tactgattat?ataaagttga?tatttcattt???21420
ttagatgtga?agaattggaa?tttaaattaa?aataatagta?tctttaatta?attgtattaa???21480
aaaaagggag?gctgggcgcg?gtggctcata?cctgtaatcc?cagcactttg?ggaggctgag???21540
gtgggcagat?cacaaggtca?ggagttcgag?atcatcctgg?ctaacatggt?gaaaccccgt???21600
atccactaaa?aatacaaaaa?aattagctgg?gtttggtgcg?ggcgcctgta?gtcccagcta???21660
ctcaggagtc?tgaagcagga?ttatggtgtg?aacccgggag?gcagatcttg?cagtgagcca???21720
agatcgtacc?actgcactcc?agcctgggag?acagcaagac?tccgtctcaa?aaaaaataaa???21780
aattaaaatt?aaaaaagaga?atctaacctg?caaggaacaa?agttttagct?tgttggcaac???21840
agctgttttc?caccaggtta?gatacaagtg?cttcttgaat?ttattttcat?gtaaagataa???21900
gtattttttt?ttatagctaa?aaatctgatt?tgattattta?gtaatggaga?attttaccag???21960
aatgtgatta?aaaaccagtc?ttttgtcacc?aaaagatctt?ttatgtcacc?aaaataaaat???22020
ccttgacgga?tacaacacta?ctaggtgtat?cattaaaata?tattcttaat?agaataaaca???22080
cctagaatca?gaacattggt?tgaaaacgta?ggagctttac?aggggcagta?aatttggagc???22140
tagtcacatt?tcaaatttgc?catcagtgtt?tctttgttga?atccagaaga?tagagcaaat???22200
tgagctgata?gtgtaagtta?gcaacatagt?tttataaaag?tatttgaggg?actggccaga???22260
aatatactcc?tgactgtaca?tgtttcatag?gaagttctag?aaacaacaaa?gcactttcta???22320
tatgctttta?ggtggaatct?ccaactgata?aatcaggaaa?cttcttctgc?tcacaccact???22380
gggaagggga?cagtctgaat?acccaactgt?tcacgcagac?ctcgcctgtg?gcagatgcac???22440
cggtaagtac?gtgtgcacca?gcccccaagt?tgttatgtga?cactggtgcc?tcatgaaatc???22500
ctgtgtgtgg?catagggctg?ctgcggtttc?agcactccct?ctttaacaca?tcttatttct???22560
aaatgttctc?ctgcacatga?tttttaaaaa?atgattatta?atgtagagta?taaagttgag???22620
attttttcat?gtattagaaa?cattgcagac?tttttcatta?tttgttttct?cattatttga???22680
gtgccttggc?catttaaaaa?aatctacatg?gatataaaaa?ctatgttcat?ttatatattt???22740
tcctttattt?gtcttctaaa?taatattgtc?ttcttgttaa?gttgtctctt?gtttggtttg???22800
tgtagcatct?catatggtat?caaccacgtt?gtctatagta?gatgcccagt?attgtaccca???22860
attgctttgg?gttactacca?ccatttttaa?agtttataat?ttgtgaaggg?ttgtccttat???22920
gtcttaagga?aaatcagagc?taataacagc?tgctaagatt?tatttagtag?gtactaagtg???22980
gtaagtactg?ctctaggggc?tttcgatgaa?ttgattcagt?taattctcac?aattatcctg???23040
taaggttggt?attattttta?ttaattttta?ttttttttga?gacggagttt?cgctcttgtt???23100
gcccagactg?gagtgcaatg?gcacgatttc?ggctcactgc?aacctctgcc?tcccgggttc???23160
aagcgattct?cctgcctcag?tctcccgaga?agctgggatt?acaggcaccc?gccaccacgc???23220
ccagctaatt?ttgaattttt?agtagagacg?gggtttctcc?atgttggtca?ggctggtctt???23280
gaactcctga?cctcaggtga?tctacccgcc?tcggcctccc?aaagtgctag?gattacaggc???23340
gtgagccacc?gcgcccggcc?aggtgggtat?tattatcatt?tctattatgt?caactggaaa???23400
acagaggcac?aaagaggctt?agtcatttgc?tcaagattgt?atacctaata?agggatggtg???23460
ctgggttgga?actcagaagg?tcttgttcta?aattccctac?ttttcaccat?tttttgccaa???23520
taatttatag?ctctaaaata?ttgatagttc?tgtaccaata?tatgaaataa?agtgttctct???23580
ccccagaact?tagtaaatct?tctattttga?aaggcatgta?tttattaatt?catttagcag???23640
atgttttttg?agtgcctaac?acatgcctag?aactctggga?tcccaaagga?gctttgtgtt??23700
gtggtgatgt?ttgccacaat?gggattaagg?ttcagacata?gttgatttct?ccttttagct??23760
catagtgggg?agaatttatg?gcagatgact?gcattttttt?atgctaatgt?gtatcatctc??23820
atagtaatct?actctccaca?gtgttagaaa?aatgtggctc?tcatattatc?caacctcatt??23880
taaatttcaa?attattctct?tccctttaga?atattcttac?aagctccgtt?ttaatggcac??23940
cagcatcaga?agatgaagac?aatgttctca?aagcatttac?agtacctaaa?aacaggtccc??24000
tggcgagccc?cttgcaggta?attactattc?caacattggt?attttaaaat?tcattcactg??24060
aaaaactgtc?atctttggtg?gtggtggtgg?tggtggtgat?ggtagtgctg?gtggtctgtt??24120
tttcgttttc?aacattttag?acatagggga?gtaatgcttt?tatacaattc?ttgatgtcta??24180
gaaaaatcca?ataattcctt?tttgctactc?atatgagaca?tgctagaaat?ttttatcagt??24240
gcagattccc?caaagcatga?tgaaatgaaa?catttcttac?tgtgactgag?actaagatgt??24300
attacacgtg?tgtcactatc?ttcttctgcc?ctcaaatgtt?ttataaaaat?atatcaatat??24360
agtttacttt?atgttccaaa?ttttttattt?tttatgctgt?atccctaggc?aaccaaagct??24420
cagagactgt?tccaatttta?aatgaagtct?gaaattagtc?agacagaaaa?taattgcaat??24480
gtatttgact?gtcagaataa?gaaagtcaac?tgtcaatgtt?gtgaggccga?tctgaagttg??24540
accatctgct?gtcaggtttt?aatatcaaaa?gagatttccc?ttgtaaccct?aaagtgggtg??24600
tgtgttgatg?aaatataacc?aattccaaat?taatcctgag?ccaaatgtaa?ataagaatgc??24660
aaattttgct?tccctaaatt?atttaaagca?gtttctgaca?ttaaatagcc?caatacccag??24720
ccacctgcag?agccctctcc?tccatccccc?tgtgccatgc?agagggaaac?ttcataaccc??24780
tagaggaagt?gaagagcctg?gattcagaag?ctgaaggtca?tatttcttgc?tgtaagttaa??24840
atggcacgat?catcggacag?cagcctttga?gtctgcccat?taccttaaag?ggatttccca??24900
ttcttgtgat?agtttctcct?tccaaattgt?gagaatctga?aaacttgccc?ggtaagtatt??24960
tgtgatgctt?agtttttcag?ggttttttgt?tgatgttgct?ttgttttttg?aaacagggtc??25020
ttacccggtg?gcccaggctg?gagtgtagtg?gggtgatctc?ggctcactgc?aacctccgcc??25080
tcccagattc?aagtgattct?tatgcctcag?cctcccaatt?agctggaatt?acaggtgtga??25140
gccaccggca?acccagccgt?ttttttgtat?ttttggatag?agacaaggtt?ttgccatgtt??25200
ggccaggcag?gtctcgaatt?cctggtttca?agtgatctgc?ccacctcggc?ctcctacagt??25260
gctgggatta?caggtgtgag?ctaccgcacc?tagccagtgc?gtagtattat?tgtaaaataa??25320
ggattgcaag?cacattttgt?ttaattaaat?cagattgctt?ccttcctggc?ctctctctga??25380
cagcaattgt?tttggtaaaa?tggctgtgga?ctggaatgtg?agacagggag?tcagtattta??25440
tggggaatct?ggacaagttc?ctgcgtcagt?gaggctggag?ttggccccgt?gtggagaaga??25500
ctcagaatac?cacaaagagg?ccagacgcag?tggctcatgc?ctgtaatccc?agcactttgg??25560
gaggccaagg?cgggtggata?acttgaggtc?aggagtttga?gaccagcctg?gccaacacgg??25620
tgaaacccca?tctctactaa?tcccagctac?tcgggaggct?gagacaggag?gatcgcttga??25680
acccaggagg?cagaggctgc?agtgagccga?gatcatgaca?ctgcactcca?gcctgggtga??25740
cagaatgaga?ctccatctca?aaaaaaagaa?tatcacaaat?aattgggaga?tgaggaagag??25800
aatgaactcc?acccagacat?aagaggaacc?cagaaaggga?gttacaaaga?tttcaaggca??25860
aagggcagcc?atttcaaaat?tttcctgaaa?gctgttctta?ggtccagaag?aaaagctttt??25920
gctttttttt?ttttttttaa?ttattcccac?ctaagttact?tggctcttgg?tttctagaga??25980
acagaattca?actactgcta?ttcgtgtttt?aattaagtta?gattaagttt?tgcatatatt??26040
ttctaaccta?gtctggggcc?aacttgtatt?tgtagtagta?cagaaaaagt?ctgttctaaa??26100
tttcaaaaag?ccaacacatc?aatatacttg?gggacaatta?ccattacata?agttgaaacc??26160
catagttcag?tattgagctt?tatcagtata?tttgtatttt?atttaatgga?aatattggtt??26220
atctttgaaa?ctcaaaatta?agttatagct?ctattttatt?aatctgtatt?ctcctgtcta??26280
tcagtgagtc?atattggcac?tttatgaatt?ctaacatctg?tacactgaga?ttaagttgtg??26340
tatggtgaag?gtaatatttg?tcaattctat?caaactgaat?aattgagact?tgaaagttaa??26400
ttttagagtc?aatactcctg?tctaaaccat?atagtttact?ggtttacact?gaccagatat??26460
attttaataa?tctgctataa?agccaggctc?agtggctcat?gcctgtaatc?acaatacttt??26520
gggaagccaa?gactggagga?tagcttgaga?ccaggaattt?gagaccagcc?tgggcaacat??26580
agtgagactc?agtctctatt?aaaaaataat?tagctgggct?gggcgtggtg?gctcacgctg??26640
taatcccagc?acttttggag?gccgaggtgg?gcggatcacg?aggtcaggag?atcgagacca??26700
tcctggctaa?cacggtgaaa?ccccgtctct?actaaaaata?caaaaaaatt?agccgggcgt??26760
agtggcgggc?acctgtagtc?ccagctactt?gggaggctga?ggcaggagaa?tggcatgaac??26820
ccaggaggcg?gagcttgcag?tgagctgaga?tagcgccact?acactccggc?ctgggtgact??26880
gagcgagact?ccatctcaaa?aaaaaaaaaa?aaaaaaaaaa?aataataata?ataataataa??26940
ttagctgggt?gtagtggtac?atacctgtaa?tcctagctac?ttgggaggct?agggcaggag??27000
gatcccttaa?gcccaggagt?tcaaggttac?agtgagctat?gattataccc?ctgcactcta??27060
gcctgggaga?caagaccaag?accttgtctc?taaaaaaaat?tttttaaggc?cagccgtggt??27120
ggctcacacc?tgtaatccca?gcactttggg?aggcagaggc?gggtggatca?caaggtcagg??27180
agtttgagac?caacctggcc?aacatggtga?aactctgtct?ttactaaaaa?tacaaaaaat????27240
agccaggcgt?ggtggtgcgc?acatgtaatc?ccagctactt?gggaggctgc?ggcaggagaa????27300
ttgcttgaac?ccaggaggtg?aaagttgcag?tgagcccaga?tcgtgccatt?gaactccagc????27360
ctaggcaata?gagtgagact?ctatctcaaa?caaacaaaca?aaaatttaat?ctgctataat????27420
agatactaat?ttattttaag?aaatacttag?tgaatgttgt?tctacatact?tgccaattgg????27480
gaggaaaaaa?gaaatattca?ctaaatgaat?gaatgttata?caccaagtat?ctccttcatt????27540
ttatgtcatg?gagttctagc?ttctgatatc?acagaaatat?gtgatttcta?aaatgtttca????27600
gactattgtg?ataagtattg?gaaaatgcat?caagtgactc?taaatattta?aacatacatc????27660
acctatattt?catataatcc?tgattcaata?atatttctat?agatttttta?tgcaatttgg????27720
taattttgtt?tgtttgaaac?acagtgctat?ttcatatatt?tctattgata?atccaatttg????27780
acctaattag?gcatatttct?cacacatggt?attattgaac?atattgcaca?tttattaaat????27840
gatatgtatt?ttacttaaga?gtactttata?taatctataa?tttttgtaat?ttgtacttat????27900
aataaatcaa?atattaagtg?ggattttagc?caaaccatct?agacattttt?gtcataatgt????27960
ttttattaaa?acagcaaaat?aggagtttca?aaatatttta?aaatttgaat?acttctaaaa????28020
tgtaaaatgt?ttgcaactgg?tgtaatgcag?ttgattgtct?atccctctaa?actgtctaaa????28080
taacttaggt?tcaagaacag?tttattgaag?aacataggaa?ctgcgcaatt?ctgtgtttta????28140
tcatttcagt?tttaatcacc?ccggcttgat?tatggcaaca?acattttaga?caagcaatta????28200
ttactatttt?tgaggcccat?atactgggcc?attatggttt?tgcttctaac?gtacatataa????28260
atgcacatgc?aatatatata?aattatgttt?gctgttttaa?aattagaaat?gccgagaagc????28320
tatgtgtttg?acattttatt?ataaattttg?tacacacata?gatgtatgga?aaatatacta????28380
tgttctagag?tcaacaattg?ttatcatttt?gccatattta?cctcatctct?tttttttttt????28440
ttttgagatg?aaatctcatt?ctgttgccca?ggctggggtg?cagtgacgca?atctcagctc????28500
actgtaacct?ctgcctccca?ggttcaagca?attctcctgc?ctcagtcccc?tgagtagctg????28560
ggactacaga?tatctgcctg?gctaattttt?gtatttttag?tagagatagg?gtttctccat????28620
gttggctagg?ctggtctcga?actgctgacc?tcaagtgatc?cacctgcctc?ggcctcccaa????28680
agtgctggga?ttatagatgt?gagccaccag?cctgggctac?ctcatctctt?ttttatgaag????28740
tttttatttt?tattttttaa?tttttttttt?aatttttttt?tttttttgag?acagcgtctt????28800
gttctgtcac?ccaggctgga?gtgcagtggc?atgatctcgg?ctcactgcaa?cctccacctc????28860
ttgggttcaa?gtgattcttc?tgccttagcc?tctcgagtag?ctgggactac?aggcgcttgt????28920
caccatgcta?gctaattttt?ggtattttta?gtagggacgg?ggtttcaccg?tgttagccag????28980
gatggtctca?atctcctgac?ctcatgatcg?gcccgccttg?gcctaccaaa?gtgctgggat????29040
tacagacatg?agccactgca?cccggccagt?tttttatttt?ttattttttt?gagacaaggc????29100
cttgctctgt?cacccaggct?ggagtgcagt?ggcaagatca?tggctcactg?cagcctacct????29160
cctggactca?agcaatcctc?ctccctcagc?ctcctgagta?gctgggacta?taggcacatg????29220
ccaccatgcc?cagctagttt?tttttttttt?ttttagagat?ggggtctccc?tgtgttgccc????29280
agcctggcct?caaactcctg?ggctcaagca?atccttctgc?cttgacctcc?caaagtgctg????29340
ggattacagg?caggagccac?cctgcccagc?ctgaagtatt?ttaaagtaaa?ttccaactaa????29400
tataacattt?tattctcatg?tacttccagc?atgtgtctct?aaaaatagac?atttttatat????29460
ataacctaat?accatcatta?catatttaac?aataattctt?taatcttctc?attctcaggt????29520
atatttaaat?tttcccatta?atccaaaaaa?gaatcaaaat?tgaaacaatt?tcacacattg????29580
catttggtta?agcctcttaa?atctctttta?ctctctagtc?cagtgctgtc?taatagaact????29640
tctacagtga?tgtgaaggaa?atatctaata?tgctgctatc?cactatgata?gccagtggct????29700
tttgagcact?tgaaatatgg?caagtgctgc?tgaggagcag?aattttcaat?ttgaattaat????29760
ttaagttgct?ttaatttaat?agccacattt?ccttactctc?ttactggaat?cagccatttc????29820
cccatggtgc?tctgaatcct?cttagtgaga?aatggtacat?agacaccaaa?atcagggtgc????29880
taagtctgtc?cattgttatg?tggtttgtca?ttgagataca?aataattttt?tttgttttgt????29940
tttgagacag?agtctcactc?tgtcacccag?gctgcagtgc?agtggtacaa?tcttggctta????30000
ctgcaacctc?cacctccttg?gtccaagcta?ttctcctgcc?tcagtttccc?aagtagttgg????30060
gattacaggc?acccaccacc?acactcagct?aattttcgta?tttttagtag?agacatggtt????30120
tcaccatgtt?ggccaggcca?ggctcaaact?cccaacctca?ggtgatctgc?ctgtctcagc????30180
ctcccaaagt?gctaggatta?caggcgtgag?ctgccacacc?tagccaagac?tcagtgaatt????30240
ttaataatcc?agagctctcc?atgtatcccc?taatctataa?tctacagtgc?ttaaatagag????30300
gcttttataa?gccattatgt?acactcatgt?tatgtttgtt?tttttaatta?cttagtcttt????30360
ttccaaaagg?aatttgcaac?agctacataa?tcttataatt?gttatgtttc?ttttatattt????30420
tcacagttgt?tttaatttta?aattgtaaaa?gcaaacagga?tattcttata?ataaaaatgt????30480
tctgagatgt?ttttcatatt?tgtgtctttc?atgtggctca?gaatatatga?gattttcatg????30540
tcatatcatt?gtcagtattc?agccttttct?ccacattgtg?tggggtagag?gatagagtct????30600
agaagcttat?aaaatggtca?caaatgagga?agtagagaaa?acattcagcc?tagaaagcgt????30660
gcatttgtgg?tatgaaaggc?actgtggaat?gctgctggat?tctgcccttc?agtagttacc????30720
cgagagagac?cgaggcagac?gcatagctcc?ctcctcagca?aggagaaaag?agttgcctgc????30780
tttcctcagc?gtgctttcca?tttcctttgt?tttgaaaaaa?gcacattatg?ctaatcaact????30840
ttccttaagg?tagacatttt?tattggtaga?tcaaaaaccg?tgctttaagt?tataagcctt????30900
ctgaacttct?gtatttttct?cttaaattat?aatttccttt?tattcctatt?taaaacccaa????30960
gtgtaaaaga?tgtgttcata?ggctttctac?tcactcactt?gcctgttttt?cgtaaccaca????31020
gaatgacagc?cttaaatttt?aatttcctga?cttggcttgc?ccatggcctg?acttttttaa????31080
aacaaggagt?ttaagttaaa?gaaatttctc?cagccattat?tatttaaaca?catttacaca????31140
gtatcaaaga?atttaaaaat?gttgctagca?gttgatactc?ccctcagctt?tctgttcagc????31200
gtgttttaga?ttgctcctat?tttgttattt?gtctatgaac?ttttatattt?ttcaaaaaaa????31260
taaggcaagc?cctttttgcc?tgataggcac?tgctgagcag?tataataaac?aagcatgggg????31320
gcaacagaac?ctgggttcca?ggctagactc?caccgttttc?aaacggcatg?actttaattc????31380
atcaagtgaa?cacaattaag?caaagtataa?gtttgggacc?atggaatatc?cttcactcat????31440
cacacaggtt?actgtgaaga?tcaaatggat?ataatattgc?tttcaagatg?gcggcacact????31500
tgccactata?cacaggtgat?attattaggc?agagtcgcca?catagtccct?gtccttgaaa????31560
tgcaggcaag?gagccctcac?caaaggccag?ggtggttttt?tgtttttcgg?gtttttgttt????31620
gtttttttca?ttgtggtata?ttatttcctc?tggcatgcta?ttccctgtcc?cactcttaca????31680
gaactgtgtc?agtgggtcac?caaaagagag?agctgattca?actgattcct?tgaaataaag????31740
gtagtcagac?ctcgcggtat?gatggagatg?aagcaactga?tttactggtt?ttctctattt????31800
ttctgctgtt?cgtgttgtca?tttaaacaga?catggaaacc?gtctgcacac?gacagagatc????31860
tttccgagcc?tttttcatct?agtgtgctgt?gctgacccgc?tcccctggat?gcctgcacac????31920
tcatttggtt?ctcccttttg?gtcacttttt?tccacttacc?ctggcagaaa?ttcacgtggc????31980
tgtcagtagt?aattcagtga?cctgctttca?aaaaatgtta?taaattaagc?agcttttctg????32040
tgtatagctt?ttgggttgaa?ctgagaaggg?ctaagtgaag?ggagaagcta?tgttgagctc????32100
agagtcagaa?tgccctgccc?cagacatttt?gagatgtgaa?tttttcattt?gttttgtttg????32160
gtgacactta?gagagaataa?acattctgag?ataaaaatct?tccttgggca?ggccagcaaa????32220
gatagaattg?ctttagagca?atttggcact?tggtcttaat?cttaatagtt?tggaactcat????32280
atgtgactaa?ggaaaaggat?attgcaaaaa?gttgtgccac?cctaggctat?atgatataaa????32340
cttagaaagt?ctatcaacgt?gaaaggtgaa?gagacctcta?gagacatcgg?ggacaaactc????32400
caaatttggg?aaaggtccat?ccacatttgc?aattgttagt?taggcttggg?aagtggagtc????32460
attggtggat?gaggcaactt?ccaatcacca?tctcgatttt?tatttcccta?acttttaaga????32520
ttattttcta?tagaaaatca?gtagtgctga?agttggagtc?atttaattga?tttgtgccaa????32580
cctagggaag?catttactct?cgagaaaagt?ttggtaacca?ttaatcttac?cttagcaaca????32640
tttcagttta?agcttatctc?tgactttcca?aagacaacta?aagatttcta?cagtctctct????32700
tataactgat?ccattttttc?aaaagatgtc?cctttgggaa?ggaattctca?tttttagaat????32760
tttagtccag?aggaagtatt?tttttttcag?ataggtgaaa?taggataagc?agaaaattct????32820
tattttttac?tgttttagat?accattgatg?ccatgcttat?tttaaaacaa?aacacaataa????32880
aaacatgata?ccatctgatg?agttgctttg?gttttacttt?tttttttttt?tttttttttt????32940
ttttttttag?tttatacaag?tttcttatta?ggtcagcaaa?ttgtaaaaca?cataatttgc????33000
caaggctttt?gaagccagca?gatactgggt?gaagttgctg?tatgaatata?ataataagaa????33060
ccgtttttgt?tcagcttgat?gaaaaaacta?ttcatgttct?aaatgaaaat?gtgaagatca????33120
tctaattctg?acagaaaacc?acctgtggac?atggtcttgt?ttctttttat?ttcttctgtt????33180
ttctacaaaa?gatggcaata?tttatttaaa?gtgctttaac?aggatatccc?agttaaagct????33240
ttgacagact?gtttatagtt?cacttctgcc?caggggcaat?taaaggcaca?ttacaggata????33300
agcgagatga?ctccccggcc?acaggaggag?gatgtgtggg?taggattatt?aatgttgaaa????33360
tgaattatga?tagagtgaca?tgtctgcaag?agcaccatgg?gttagtaact?taacaagaat????33420
tcatatgcag?aaacccataa?ttctcagcag?ttctttaggg?taaatgaaac?taaagtttag????33480
ggagagagaa?caaggagaat?ttgcaaagat?atgtatgaca?gtatttaaaa?atgttcttcc????33540
agcacaaaga?cattaatctt?cctttgtcaa?aataattctt?tctgcatgaa?aagttggcat????33600
ggggctccca?ggcttcaggt?aagatgggag?aaagggagcg?tttctttttt?tttgtaccaa????33660
agtactcaat?cttctgatct?tccttgactt?tcacgacata?ggcatatgtc?tactctgacc????33720
agtttgtttc?atttattgtg?gtaagatgtg?cttgtcttgt?gctttcctgg?tgcagaaaag????33780
gagctgcctc?gctcatgctt?ggtggccttt?ttgttcctac?acacagtggt?tctcgtacat????33840
ggtggctacc?acatcctggt?gtttgtctat?actgggagaa?tcaggcgttc?caagtcatct????33900
tggctgccct?agctctgagc?ttcaggggat?atatgactca?gaaatgctat?gctttctgga????33960
atttggatat?ttcattttta?tttgtttctt?ggtaaattct?cttttgactt?aggagaagct????34020
aactatttgg?aaaggtctct?cagaactcta?attacaaata?tatggtacct?tctttatatt????34080
gatctcattt?ttaaatacct?agtcctggag?gaagagctaa?atatgaatac?agtgatgagc????34140
tgtgtagggg?atctcacagg?aacacagtca?gatttcaagt?tgaaaataga?ttcagttgat????34200
aatacttagt?actagattct?cctgaatcga?ctttgggaat?tctgagtcaa?cctaaggact????34260
atattacaaa?ggatcagacc?ttgactttta?ggccaggtgt?agtggctcat?gcctgtaatc???34320
ccagcacttt?aggaagccaa?ggcaagtgga?tcacttcagt?ccaggaggtt?gagaccagcc???34380
tgtgcaacac?agcgagaccc?tgtctctacc?aaaaatacaa?aaattagcca?ggtgtagtgt???34440
agcacctgcc?tgtagtctga?gctactgagg?aggctgaggt?tggagaatca?actttaaccc???34500
cggatatgga?ggctgcagtg?agctgtgatc?atgccagcct?gggcaaggag?caagaccctc???34560
tctcaaaaac?aaaaacaaaa?aagacacaga?acttgacttt?taacaggagg?catagtaata???34620
gcaagcataa?gattgtttta?acatagcatt?taagataatt?tacgcattta?gaaaattaat???34680
tctggctggt?tgtagtagct?tatccttaat?cctaacccta?tgagaggcca?aggcaggaag???34740
atcacctgag?gccaggagtt?tgagaccagc?ctaggcaaca?tagcgagacc?cctgctctct???34800
acaaaaaatt?ttttttaaaa?aacatagctg?ggcatggtga?cacactcctg?tagtcccagc???34860
tactcaggag?gctgaggcgg?gaggcttgct?tgaggccagg?agtttgaggg?agctgtgatc???34920
acgcccctgc?actccagcct?gggcaacaga?atgaggccct?atatctaaaa?aaaagaaaaa???34980
tggacaagaa?aattctatac?tgattgaagc?ccagctaaat?agaaaaaaaa?aaattctaat???35040
aggccatttt?aaagactttg?gaaaaattct?tagctagagt?aaaacagaag?caatcctttc???35100
tttcctggtt?ctcacatctc?cattctcacc?ttcattcccc?ttcccctcca?tttgtttcaa???35160
gtatttttct?tttctatgtg?tgtctttatg?taaatttaaatttacctaca?agatattatg????35220
atattaagat?atttcattct?gcttcctgtt?ttgtcacttg?ttctttaaga?tccaagtcct???35280
tggctccatg?tgcatctaat?ccttgccata?agccctgcat?cgtacttcat?attcaccata???35340
ttgaactatt?caccctcaag?tagtcactat?taaatgtcac?actgttgtag?cgactacaaa???35400
tgcaagctca?gtaactgaaa?agataacatt?aaattttgtt?ttttaattgg?tttcctgtgt???35460
caatttcatg?tttcgggaat?ttgtatggac?agatagacac?cagagagata?tgcgtatttc???35520
acctgcatgc?ttgaggctag?agaaacatga?tttattcaga?actatttcaa?atactgtaac???35580
taacctggta?attttgttta?ggaaaacatt?aaattcggaa?tagaaggcaa?ttcagaatac???35640
ctgtctgtta?tctttgtctt?gtcatttttg?agatactcag?ttcctggaaa?atattatgat???35700
aatccatctt?gctatgtagt?atttcaagtt?atatgttgag?cctctgagct?tgcagagtga???35760
acatttctat?tcatagacag?tgctgttgac?atcagtgggg?agatggcaca?agctttgtgc???35820
cctaggaaag?tttaaaagct?agaacattac?actgcaaacc?ctatgtcagg?aaactttgtt???35880
gcagcaacta?ggcaaggaaa?ccctaccact?gtacccatac?cttgttttag?tagaattatg???35940
tttctttgct?aagaagtctg?gtgactctca?caccccatct?cagcactttc?ctggattcca???36000
aggccctggt?gccctgagca?cacaacttcc?caggttcctt?ttattgttcc?tgataagaac???36060
tcgaattaga?ggcagcgtct?ggtactcacc?aggggtgtgg?tcccatgggt?gacattcttg???36120
ctccatcatt?tcttacccgg?gtaaccttga?gtatggcgcc?taaatcctct?cttcctttca???36180
aattgcctgg?tctgtaaaat?ggatgcagtg?aatggtagct?atgccacagg?tgttttgtgt???36240
gagaattaaa?tgagaggagt?tatatcaata?acacttagag?gcatgcggtg?cctatagtga???36300
gggcttgttt?tatgttgtcg?tttaaccctt?tctttctcag?ccctccccca?ctccacatcc???36360
cctcccactg?gcaatgtaca?aattgcagct?tcctgcctct?ttcctgctat?ctcgctccct???36420
ggatgacatc?cctgcgtttc?cacctctctc?tcccctaatc?ttagctgttt?tcaccctttc???36480
ccctaatctt?agctgttttc?accctttccc?cacagcattg?ccttgacagt?tcctcagtgt???36540
cccatcccct?cttcccatgg?acaacaccac?aaacctgcgc?acacggcctg?ttattattcc???36600
tcctgtttca?cctgtgtcca?ttctgtctca?taaaggacac?tgtaggctct?gaacagctaa???36660
ggcctttctt?attcttttgt?cccatggaaa?ataatccctc?cagatagcaa?taccaggaat???36720
agtgcctttt?ctaacccact?ggtttccttt?tgagcttaca?gccaccaaat?cttatatctg???36780
tttgtttctg?ttcccctctc?tgcattgact?gctagagaga?tcagagtcaa?gcaggaggtt???36840
acatctagct?ggggcactgt?gctttatgct?ggtgtttcgt?gtgcaaatct?cataccatct???36900
tcatgttaca?gttgttcctt?agtgtccatg?ggggatttgt?ttcagaaccc?gcaaggacac???36960
caaagtccag?ggatgctcaa?gtccctgata?taaaatagca?ttgtgtttgc?atataatcta???37020
cacacatctt?accatataat?tttttttatt?atttgtatac?attcgtgggg?tacaagtgta???37080
attttgttac?atggatgtat?tgcatagtgg?tcaaagtcga?ggctgtgagt?gtatttatcg???37140
ttggaataat?gtacattgta?cccgttcatt?cctgtatact?ttaagtcatc?tctagattac???37200
ttataatagc?taatacaatg?taaatgctat?ggaaatagtt?gttatattgt?taggggataa???37260
tgacaaggaa?aacacctata?catgttcaat?acacagcaat?tatttttttc?tgaatatttt???37320
tgatctgcca?ttgattgaat?ctatggatgt?ggaacccaca?gatatgggac?ccacagatgc???37380
agagggccaa?ctgtgtttat?tttttccaaa?tttgtttccc?tgttatgact?cttctttata???37440
cacattttta?aactctgttg?tcttgtattt?gttttctgat?catcttacgt?ccttttggaa???37500
accaagtggg?gcataaatag?tacatgtttg?catttgaggc?aacacagagg?catcgcagga???37560
gctggagaca?gcaaagttca?aagccagtcc?accacttacc?tacagagtgg?ccttgggcaa???37620
gttacctcca?ttttctgtct?ccattctcga?gcctgtctag?agagagaatg?aacctgcctc???37680
ccgaggtgat?ggtgctgttt?taatgaaata?atgcatgtga?aatgtgaagt?gccactgtcc???37740
ctggcatgga?gatgctgtcc?ttagatatta?ggtccatctt?gtatcttcta?gtgtatcttt???37800
aaaaattaaa?ctatactggc?ttcatgcctg?taatctcagc?actttgggag?gccaagaccg????37860
gcagatcact?tgagtccagg?agttcaagac?caacctgggc?aacatggtga?aaccctgtct????37920
ctacagaaaa?tacaaaaatt?agtggggctt?ggtggcacgt?gcctgtagtc?ccagctgctt????37980
gggtagctga?ggtaggagga?tcacttgagc?ccaggaggct?gaggctacag?tgagctgtga????38040
ttgcaccaat?accgatcagc?ctggtatgac?agagcatgac?tctgtctcaa?aaaaaaaagg????38100
aaaaaaatta?aactatactg?ataaatattt?ctaaagtaat?tgatgaaaac?atatatatga????38160
cacttagcag?gtgttgatca?attccagtgc?gtgcatgagc?tgctttgcac?tgtggctaca????38220
gcactcagca?gtttgcttag?tgagtagatg?gccatagctc?actttaggta?tctggagaat????38280
aatgctcatt?cctgtctcca?gctttgtaaa?attgcctaat?agacggaagg?cattaaaata????38340
tgaagatggg?attatatctt?atgaagctgc?ttggaatggg?ttttccctct?taacaaataa????38400
tttctgatca?tccatatccc?ttaagaacac?cggatttctg?ggggccaggg?aggtaagatt????38460
ctatctgaca?aagccttcct?ggtgtcaacc?acttgccatc?tgttggtcag?tgctggccct????38520
gctgggtcta?tagaatgagc?ttctttgtct?gacgctcctg?ttgccatccc?tttctccatc????38580
agccttgtag?cagtacctgg?gaacctgcat?cctgtggaaa?gatggaggag?cagatgacat????38640
cttccagtca?agctcgtaaa?tacgtgaatg?cattctcagc?ccggacgctg?gtcatgtgag????38700
acatttccag?aaaagcatta?tggttttcag?aacacttcaa?gttgacttgg?gatatatcat????38760
tcctcaacat?gaaacttttc?atgaatggga?gaagaaccta?tttttgttgt?ggtacaacag????38820
ttgagagcag?caccaagtgc?atttagttga?atgaagtctt?cttggatttc?acccaactaa????38880
aaggattttt?aaaaataaat?aacagtctta?cctaaattat?taggtaatga?attgtagcca????38940
gttgttaata?tcttaatgca?gattttttta?aaaaaaaaca?taaaatgatt?tatctgtatt????39000
ttaaaggatc?caacagatca?gtattttttc?ctgtgatggg?ttttttgaaa?tttgacacat????39060
taaaaggtac?tccagtattt?cacttttctc?gatcactaaa?catatgcata?tatttttaaa????39120
aatcagtaaa?agcattactc?taagtgtaga?cttaatacca?tgtgacattt?aatccagatt????39180
gtaaatgctc?atttatggtt?aatgacattg?aaggtacatt?tattgtacca?aaccatttta????39240
tgagttttct?gttagcttgc?tttaaaaatt?attactgtaa?gaaatagttt?tataaaaaat????39300
tatattttta?ttcagtaatt?taattttgta?aatgccaaat?gaaaaacgtt?ttttgctgct????39360
atggtcttag?cctgtagaca?tgctgctagt?atcagagggg?cagtagagct?tggacagaaa????39420
gaaaagaaac?ttggtgttag?gtaattgact?atgcactagt?atttcagact?ttttaatttt????39480
atatatatat?acattttttt?tccttctgca?atacatttga?aaacttgttt?gggagactct????39540
gcatttttta?ttgtggtttt?tttgttattg?ttggtttata?caagcatgcg?ttgcacttct????39600
tttttgggag?atgtgtgttg?ttgatgttct?atgttttgtt?ttgagtgtag?cctgactgtt????39660
ttataatttg?ggagttctgc?atttgatccg?catcccctgt?ggtttctaag?tgtatggtct????39720
cagaactgtt?gcatggatcc?tgtgtttgca?actggggaga?cagaaactgt?ggttgatagc????39780
cagtcactgc?cttaagaaca?tttgatgcaa?gatggccagc?actgaacttt?tgagatatga????39840
cggtgtactt?actgccttgt?agcaaaataa?agatgtgccc?ttattttacc?tacttttgtt????39900
ttctctctta?tttgtcacta?tggacaacag?ttacacttta?aagaaaatgg?agcagactca????39960
aagcactttc?ctctaataat?tgcacaccat?atgcacttcg?tgtgaagaag?ccaggaatca????40020
ttacaatgga?gaagaaagaa?caagcaaact?gagttaaata?gttcacaaac?aatcacatgg????40080
acaatgggga?ggaatcttcc?agaatgcatc?agtcagggtc?tagcggcatt?tgagtgcgga????40140
tgctgtaagg?ggaattcgtt?cacaaatgta?ctgataccac?agatgtgggc?aggggctagg????40200
agagcaacag?ggtgcagcct?gaaggcacct?ggaaggccgc?ctggaggggg?acagtcagga????40260
aagcaggagc?ccttgagagg?agcactagcc?tttgatgaga?ccactgccat?cgaaggagac????40320
ctcacaggga?gggagccaag?ggtaaaccct?aactttgttt?ccccatttct?cctatctctg????40380
cctaggggtc?ctcactggca?gaaccaacca?gaagctggag?gacaagggag?ctc???????????40433
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>2
ttttaatggc?accagcatca???????????????????????????????????????????????????20
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>3
catcatgctt?tggggaatct???????????????????????????????????????????????????20

Claims (10)

1. whether a vitro detection sample exists the method for the single nucleotide polymorphism of myeloblastosis proto-oncogene MYB, it is characterized in that, comprises step:
(a) with the myb gene of myb gene primer amplified sample, obtain amplified production; With
(b) detect whether there is following single nucleotide polymorphism in the amplified production:
24071 A → G;
Wherein, the nucleotide position numbering is based on SEQ ID NO:1.
2. the method for claim 1 is characterized in that, described gene-specific primer has the sequence of SEQID NO:2 and 3.
3. the method for claim 1 is characterized in that, the length of described amplified production is 100-3000bp, and contains among the SEQ ID NO:1 the 24071st.
4. one kind is detected hypertensive test kit, it is characterized in that it comprises the primer of specific amplification myb gene or transcript, and it is 100-3000bp that described primer amplification goes out length, and contains among the SEQ ID NO:1 the 24071st amplified production.
5. test kit as claimed in claim 4 is characterized in that, it also contains the reagent that is selected from down group:
(a) with SEQ ID NO:1 in the 24071st sudden change bonded probe;
(b) the 24071st sudden change restriction enzyme among the identification SEQ ID NO:1.
6. test kit as claimed in claim 5 is characterized in that, described sudden change is following single nucleotide polymorphism:
24071 A → G;
Wherein, the nucleotide position numbering is based on SEQ ID NO:1.
7. test kit as claimed in claim 4 is characterized in that described primer has the sequence of SEQ ID NO:2 and 3.
8. method that the hypertension susceptibility of individuality is diagnosed is characterized in that it comprises step:
Detect this individual myb gene, transcript and/or albumen, and compare with normal myb gene, transcript and/or albumen,
There are differences and just show that this individuality suffers from hypertensive possibility and be higher than normal population.
9. method as claimed in claim 8 is characterized in that, detection be gene or the transcript of MYB, and with normal MYB nucleotide sequence comparing difference.
10. method as claimed in claim 9 is characterized in that, described difference is following single nucleotide polymorphism:
24071 A → G;
Wherein, the nucleotide position numbering is based on SEQ ID NO:1.
CN 200410016467 2004-02-23 2004-02-23 Relativity between proto-oncogene of leukaemia in myeloblast class essential hypertension Pending CN1661048A (en)

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CN 200410016467 CN1661048A (en) 2004-02-23 2004-02-23 Relativity between proto-oncogene of leukaemia in myeloblast class essential hypertension

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 200410016467 CN1661048A (en) 2004-02-23 2004-02-23 Relativity between proto-oncogene of leukaemia in myeloblast class essential hypertension

Publications (1)

Publication Number Publication Date
CN1661048A true CN1661048A (en) 2005-08-31

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