CN1548556A - Reagent kit and primer for predicting susceptibility of type-II diabetes - Google Patents
Reagent kit and primer for predicting susceptibility of type-II diabetes Download PDFInfo
- Publication number
- CN1548556A CN1548556A CNA031369804A CN03136980A CN1548556A CN 1548556 A CN1548556 A CN 1548556A CN A031369804 A CNA031369804 A CN A031369804A CN 03136980 A CN03136980 A CN 03136980A CN 1548556 A CN1548556 A CN 1548556A
- Authority
- CN
- China
- Prior art keywords
- primer
- diabetes
- gene
- snp
- intron
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to reagent kit and primer for predicting susceptibility of type-II diabetes, and is especially reagent kit and primer designed based on one mononucleotide polymorphism site of newly distinguished type-II diabetes susceptibility gene PANK4 and used for predicting susceptibility of type-II diabetes. The present invention also relates to the application of PANK4 gene in preparing diagnosis preparation for predicting susceptibility of type-II diabetes.
Description
Technical field
The present invention relates to be used to predict the test kit and the primer of diabetes B susceptibility, more specifically, the present invention relates to utilize the test kit and the primer of prediction diabetes B susceptibility of a single nucleotide polymorphism (SNP) site design of the diabetes B tumor susceptibility gene PANK4 of new discriminating.The invention still further relates to the application of PANK4 gene in the diagnostic reagent of preparation prediction diabetes B susceptibility.
Background technology
The massive epidemiology data shows that diabetes are a kind of disease of multifactorial inheritance with complexity of obvious genetic predisposition.Inherited genetic factors can obviously influence the susceptibility of body to Regular Insulin.Recent years, most researchers is devoted to seek the diabetes genes involved by the positional cloning method, promptly seeks interregional chain of the chromosomal DNA shared between ill family member.Found some the relative more rare single-gene forms in the diabetes B, (maturity-onset diabetes of the young MODY), is because sudden change has caused the β cell function low to be called young late hair style diabetes.These patients fall ill to the adult is early stage in pubescence usually, show as the glucose reactivity insulin hyposecretion.6 glucokinases of encoding respectively, transcription factor HNF1 α, 1 β, 4 α and insulin promoter factor (IPF1) are arranged, neurocyte differential protein (NeuroD1) in these Disease-causing genes.These transgenations finally cause the hyperglycemia mass formed by blood stasis.The sickness rate of these single-gene diabetes is also imprecise, estimates to account for 5% of all diabetes Bs.But, utilize the strategy of positional cloning to determine that the successful report of disease of multifactorial inheritance is also few merely.
Except the positional cloning method, the genes involved research to disease of multifactorial inheritance also has the most frequently used method of a kind of method at present, i.e. candidate gene method, the i.e. variation of direct study of disease candidate gene and the relation between the disease phenotype.This method is mainly based on the case-contrast association analysis to family or crowd.Association analysis does not need big family research but relatively certain or a certain cover are marked at the distributed degrees of patient and normal individual.If it is fairly obvious that certain mark distributes, so just can think that this mark is associated with disease phenotype in diseased individuals.But, because the Disease-causing gene position is not enough understood,, be primarily aimed at some metabolic pathways relevant and study in the candidate gene method with disease development selecting there is bigger blindness on the candidate gene.The positional candidate cloning method that above-mentioned two kinds of methods are combined has remedied the limitation of any single a kind of method to a certain extent, is the general strategy of employing of institute in the disease of multifactorial inheritance research at present.
Corresponding with the inherited disease research strategy, its research tool-polymorphism mark is also from restriction fragment length polymorphism (the Restrictive Fragment Length Polymorphism of the first-generation, RFLP), the microsatellite marker of the s-generation (Microsatellite Marker) develop into the third generation single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP).Especially disease of multifactorial inheritance is being carried out need utilize a large amount of highdensity markers in case-contrast association analysis, preceding two generation polymorphism mark can not satisfy this requirement.Therefore, in recent years, SNP more and more causes people's extensive concern as third generation construction of genetic atlas person.
Single nucleotide polymorphism (SNP) mainly is meant on the genomic level by the caused dna sequence polymorphism of the variation of mononucleotide.It is modal a kind of in human heritable variation, accounts for more than 90% of all known polymorphisms.SNP extensively distributes in human genome, just has 1 in average every 500-1000 base, and its Estimate of Total Number can reach 3,000,000 even more.In recent years, along with the progress of human genome and genetics research, SNP also more and more demonstrates its significance in depth understanding between individuality and colony genomic variation or aspects such as polymorphism and disease research comprehensively.It is exactly its widely distributed property that SNP is used for the localized biggest advantage of diseases predisposing gene.Next is that it has the stability higher than little satellite, can reduce to greatest extent in the genetic process because the information loss that sequence variations caused.In addition, SNP is easy to realize high-throughout somatotype, can produce a large amount of results in the short period of time, and this point is that little satellite is incomparable.The These characteristics of SNP has determined it to be very suitable for multigenic disease, the especially Position Research of the disease that a plurality of minor gene caused resemble diabetes.Selecting appropriate cases and normal control, take the research method of association analysis, for SNP, is fit closely.
The inventor was engaged in Han People of North China diabetes B (non insulin dependent diabetes, NIDDM) assignment of genes gene mapping research in recent years always.In the full genome scanning work of formerly carrying out, the inventor has identified a plurality of tumor susceptibility genes site respectively, and they lay respectively at No. 1, and No. 12, on No. 18 and No. 20 karyomit(e).Wherein on No. 1 karyomit(e), have 5 zones that have tumor susceptibility gene.On this basis, the inventor attempts selecting suitable candidate gene from locating area, utilize SNP to carry out case-contrast association analysis, finally identified a diabetes tumor susceptibility gene (being the PANK4 gene) finally, and a SNP site relevant especially with the Han nationality in China diabetes B on this gene.Finished the present invention through further investigation thus.
Summary of the invention
According to an aspect of the present invention, a kind of test kit of predicting the diabetes B susceptibility is provided, and described test kit comprises based on the special primer that is used for pcr amplification of the sequences Design of Figure 1B or Figure 1A and the contained conventional assembly of test kit that detects by nucleic acid amplification.Described primer designs at the SNP site shown in the figure, and length is 18bp-21bp.Preferably, described primer is selected from a group that SEQ ID NO:1-21 forms, and more preferably, described primer is for having SEQ ID NO:22, single-basic extension (SBE) primer of sequence shown in the SEQ ID NO:23.
According to a further aspect in the invention, the primer that is used to predict diabetes susceptibility is provided, described primer is that described primer designs at the SNP site shown in the figure based on the special primer that is used for pcr amplification of the sequences Design of Figure 1B or Figure 1A, and length is 18bp-21bp.Preferably, described primer is selected from one group that SEQ ID NO:1-21 forms.According to a preferred embodiment of the present invention, primer of the present invention is SEQ ID NO:22, single-basic extension (SBE) primer shown in the SEQ ID NO:23.
In accordance with a further aspect of the present invention, provide the PANK4 gene to be used for the application of the diagnostic reagent of diabetes diagnosis as the diabetes tumor susceptibility gene in preparation, the nucleotide sequence of PANK4 gene is shown in SEQID NO:24.
Description of drawings
Figure 1A illustrates the nucleotide sequence of the PANK4 gene region at place, SNP of the present invention site (R).
Figure 1B is the complementary sequence of Figure 1A sequence
Embodiment
Definition:
Single nucleotide polymorphism (SNP): mainly be meant on the genomic level by the caused dna sequence polymorphism of the variation of single Nucleotide.It is modal a kind of in human heritable variation, accounts for more than 90% of all known polymorphisms.
Microsatellite locus: is length that the tandem repetitive sequence (VNTR) of 2-6 Nucleotide calls little satellite (Microsatellite), microsatellite locus has two advantages the most outstanding: the one, and its frequency of occurrences is very high, be dispersed throughout whole genome, and, because short sequence is not selected on evolving, thereby variable repeating unit number of variations is very big on same site, and a site has nearly tens kinds allelotrope, information content height; The 2nd, the specificity single-copy sequence stability of STR both sides is high, can be used as the PCR primer that detects its polymorphism.With the round pcr operation, can realize the automatization large-scale operation
Gene type and gene frequency distribute: in the same population biology, the sequence analysis of phase isoallele nucleotide sequence is represented determining of the moiety of identical SNP site in Different Individual at this, is gene type.Gene frequency is meant in the same kind allelotrope of being studied, and the allelic proportion that changes just appears in the ratio that the allelotrope of desired appearance accounts for.
Disease susceptibility (diabetes susceptibility): the crowd experiences degree easily to disease.
Case-contrast association analysis: be a kind of a priori retrospective study.It is earlier to determine the respondent by morbid state, is divided into case and control group, and the difference by the genetic marker frequency of occurrences studied between case group relatively and control group obtains information related between this sign and disease.
Single-basic extension (SBE) reaction: it is meant and is lacking four kinds of dNTP, existing under the situation of four kinds of ddNTP, Sequenase can add a ddNTP at 3 ' end of primer, but,, promptly come to an end after base is extended one and end so can not continue to extend because can not form phosphodiester bond.Only needing to detect this base gets final product.Claim little sequencing (Minisequencing) again.
Parameter linkage analysis and Non-Parametric Linkage Analysis Methods: calculate two chromosome maps between the gene apart from being linkage analysis according to the recombination fraction of gene, analyze with classified variable (discrete variable), measurement index is sorted out by a certain standard, counting, analyze then and be the parameter analysis, the analysis of carrying out with the result after the actual measurement is a Non-Parametric Linkage Analysis Methods
The mechanism that glucose level is regulated is very complicated, relates to numerous genes.Up to now, quite a few blood sugar metabolism related gene is cloned, but the definite function of a lot of gene is also not fully aware of at present.In order to separate, clone and evaluation blood glucose regulation genes involved, we have made a SD rat blood sugar by jugular vein right atrium intubation and have regulated model automatically, use the fragment that DDRT-PCR (difference shows reverse transcription-polymerase chain reaction) and Northern hybridization have obtained a differential expression, called after EST135, and from SD rat skeletal muscle cDNA library, be sieved to 3 positive colonies as probe with EST135, but be not full-length cDNA, sequential analysis shows that they and human AC133 antigen and mouse Prominin have homology.Use RACE (rapid amplifying cDNA end) method and obtain full-length cDNA, called after Fudenine, the Genbank accession number is AF263368.The cell transfecting experiment shows can be with a key enzyme GADPH of blood sugar metabolism (glyceraldehyde 3-phosphate dehydrogenase) up-regulated.The homologous sequence of Fudenine in the mankind is the PANK4 gene.
The PANK4 gene is positioned at chromosomal 1p36.32, belong to pantothenate kinases family member, be the regulation and control enzyme of a key in the biosynthesizing of coenzyme A in bacterium and Mammals, the final product that its catalysis extensively exists is the biosynthetic pathway of coenzyme A, and is subjected to the feedback regulation of coenzyme A.PANK4 exists very abundant in muscle, also extensively exists in its hetero-organization.The sudden change of this gene may directly cause glycometabolic disorder, causes diabetes.
Working in advance of this seminar shows, 1, all has the tumor susceptibility gene site of Han nationality in China diabetes B on 12,18 and No. 20 karyomit(e), wherein there are 4 zones (1p36,1p31,1q22 and 1q42-43) to show and diabetes B linked (this group forefathers make, and do not need document) on No. 1 karyomit(e).Shown in following embodiment, further the fine scanning result that this zone is carried out shows wherein have 3 zones (1p36.33-36.23,1q24.3-25.1 and 1q42.12-42.13) still to exist chain.
Have a plurality of public databases to store millions of SNP information at present, people can be in several ways, as with gene name, registration number, karyomit(e) number, functional class etc., searches.But, the SNP in these databases most just " candidates ", but not real SNP through verifying.They utilize computer to excavate out when a plurality of cloned sequences are compared, and wherein certainly exist a part of false positive SNP.In addition, these SNP great majority are the SNP that utilize Caucasian and Black people to check order and seek, and some is not necessarily identical for the distribution in Chinese population, and we utilize the diabetic subject and the normal control of Han People of North China, and SNP is sought in order-checking again.And utilize the SNP of the high frequency that we find in the northern Han nationality of China diabetic population to carry out gene type.
For seeking the diabetes B tumor susceptibility gene, the inventor chooses 34 candidate genes of diabetes B that are positioned at 1p36.23-36.33,1q24.3-25.1 and 1q42.12-42.13 zone according to the information that announce at American National information biology (National Center for Biotechnology Information) center, exon to these genes, Promotor district 2kb, and check order SNP in the examination Han People of North China of the zone of the intron that closes on.And the SNP of part high frequency wherein carried out gene type, (comprise direct SBE reflection, Snapshot by the SBE reaction
TMTest kit, Snupe
TMTest kit), enzyme is cut, and methods such as sequencing are carried out somatotype and case-contrast association analysis to it in case group and control group.
Go through as following examples, in the SNP site of selected 124 high frequencies, choose 6 sites in the PANK4 gene and carried out gene type, find that this site is relevant with diabetes, lay respectively at a SNP site (Y at 65 places of the ten No. three exon, be C/T), this site is not in the news at first to be found at Chinese population.The difference that its gene frequency is distributed in case group and the control group has statistical significance (P＜0.05), point out this SNP site relevant with diabetes B, further reach a conclusion, its place gene PANK4 gene is the tumor susceptibility gene of Chinese northerner's diabetes B.
Therefore, one aspect of the present invention relates to the application of diabetes B tumor susceptibility gene PANK4 gene in the prediction diabetes susceptibility.Based on the PANK4 gene, can obtain various diagnostic reagents and test kit to be used to predict diabetes susceptibility.
PANK4 place genome sequence is shown in SEQ ID NO:24, total length is 18,040bp, disclosed SNP site (Y) is positioned at 12 of this sequence, 801-14, between 400, this regional nucleotide sequence is shown in Figure 1A, and Y is the SNP site among the figure, and it represents base C/T polymorphism, promptly this site can be C, also can be T.This regional complementary sequence is shown in Figure 1B, and R represents the complementary base in SNP of the present invention site among the figure, and it represents bases G/A.
The invention still further relates to the primer that is used to predict the diabetes B susceptibility.Preferably, described primer designs based on the sequence of Figure 1A, and thus, the product order-checking behind the pcr amplification can directly record the SNP site.Certainly, primer of the present invention also can be based on the sequences Design of Figure 1B, and the order-checking of the product behind the pcr amplification obtains the complementary base of SNP like this.Preferably, primer of the present invention has the sequence shown in the SEQ ID NO:1-21, particularly preferably is SEQ IDNO:22, the SBE primer shown in the SEQ ID NO:23.Following table 1 shows some combinations of primer of the present invention, the product size that the melting temperature(Tm) of primer (Tm) and these combinations obtain for example.It will be appreciated by those skilled in the art that primer of the present invention is not limited to these primer of listing and combinations thereof.
Table 1
| ????SEQ?ID ????NO： | Primer sequence and Tm | The product size |
| ????1 ????2 | Justice is arranged: gtggtatggaacaggctgc (61.03 ℃) antisense: gcagacacctacGCAGACAC (61.45 ℃) | ????253bp |
| ????3 ????2 | Justice is arranged: agtcttcgatcctgagggg (62.24 ℃) antisense: gcagacacctacGCAGACAC (61.45 ℃) | ????273bp |
| ????4 ????2 | Justice is arranged: ggcctgagtcttcgatcct (61.24 ℃) antisense: gcagacacctacGCAGACAC (61.45 ℃) | ????279bp |
| ????5 ????2 | Justice is arranged: gcctgagtcttcgatcctga (62.40 ℃) antisense: gcagacacctacGCAGACAC (61.45 ℃) | ????278bp |
| ????6 | Justice is arranged: actcttgtcctgcccttcac (61.17 ℃) | ????418bp |
| ????2 | Antisense: gcagacacctacGCAGACAC (61.45 ℃) | |
| ????1 ????7 | Justice is arranged: gtggtatggaacaggctgc (61.03 ℃) antisense: aacctgaggggacagacaag (61.02 ℃) | ??450bp |
| ????8 ????7 | Justice is arranged: ctgaggccacgtcttcact (60.96 ℃) antisense: aacctgaggggacagacaag (61.02 ℃) | ??408bp |
| ????9 ????2 | Justice is arranged: cacccactgctcagtcacat (61.75 ℃) antisense: gcagacacctacGCAGACAC (61.45 ℃) | ??401bp |
| ????10 ????2 | Justice is arranged: cttcacccactgctcagtcac (62.82 ℃) antisense: gcagacacctacGCAGACAC (61.45 ℃) | ??404bp |
| ????1 ????7 | Justice is arranged: gtggtatggaacaggctgc (61.03 ℃) antisense: aacctgaggggacagacaag (61.02 ℃) | ??450bp |
| ????3 ????11 | Justice is arranged: agtcttcgatcctgagggg (61.09 ℃) antisense: AGCGACCTGTAAGACATGACC (61.02 ℃) | ??470bp |
| ????12 ????7 | Justice is arranged: gataggcttgtgcctggtg (61.16 ℃) antisense: aacctgaggggacagacaag (61.02 ℃) | ??507bp |
| ????13 ????7 | Justice is arranged: tgagtcttcgatcctgaggg (62.24 ℃) antisense: aacctgaggggacagacaag (61.02 ℃) | ??472bp |
| ????4 ????7 | Justice is arranged: ggcctgagtcttcgatcct (61.24 ℃) antisense: aacctgaggggacagacaag (61.02 ℃) | ??476bp |
| ????14 ????7 | Justice is arranged: cttcacccactgctcagtca (62.01 ℃) antisense: aacctgaggggacagacaag (61.02 ℃) | ??601bp |
| ????15 ????7 | Justice is arranged: ttcacccactgctcagtca (60.98 ℃) antisense: aacctgaggggacagacaag (61.02 ℃) | ??600bp |
| ????16 ????7 | Justice is arranged: ccactgctcagtcacatgg (60.84 ℃) antisense: aacctgaggggacagacaag (61.02 ℃) | ??595bp |
| ????9 ????17 | Justice is arranged: cacccactgctcagtcacat (61.75 ℃) antisense: cagaacctgaggggacagac (61.60 ℃) | ??601bp |
| ????10 ????17 | Justice is arranged: cttcacccactgctcagtcac (60.02 ℃) antisense: cagaacctgaggggacagac (61.60 ℃) | ??604bp |
| ????6 ????18 | Justice is arranged: actcttgtcctgcccttcac (61.17 ℃) antisense: tgacaatagaaaatgccagcc (61.81 ℃) | ??701bp |
| ????1 ????19 | Justice is arranged: gtggtatggaacaggctgc (61.03 ℃) antisense: gtgaaggtaaatgatggtggg (60.92 ℃) | ??720bp |
| ????1 ????20 | Justice is arranged: gtggtatggaacaggctgc (61.03 ℃) antisense: agcttctttgggagtgaaggt (61.09 ℃) | ??733bp |
| ????8 ????19 | Justice is arranged: ctgaggccacgtcttcact (60.96 ℃) antisense: gtgaaggtaaatgatggtggg (60.92 ℃) | ??678bp |
| ????21 ????19 | Justice is arranged: aggccacgtcttcactcag (60.96 ℃) antisense: gtgaaggtaaatgatggtggg (60.92 ℃) | ??675bp |
The invention further relates to the test kit that comprises one or more primer of the present invention, this test kit is used to detect the susceptibility of diabetes.Except primer of the present invention, described test kit also comprises the utilization pcr amplification and the conventional assembly of the test kit that detects, reagent, damping fluid etc., and those skilled in the art are familiar with these conventional assembly and detection methods.
Further describe the present invention hereinafter with reference to embodiment.
The Fine Mapping of embodiment 1 Han People of North China diabetes B tumor susceptibility gene
1, the collection of diabetes B family
Collect and satisfy the following diabetes B family that requires in the Han People of North China:
1) in continuous 2 generations or occur the diabetes B patient more than 2 generations in the family, in per generation, have a patient, a normal people at least.
2) diabetic subject should make a definite diagnosis in regular hospital, to insulin insensitivity person (except type 1 diabetes).Selected contrast Individual Age should be in (to get rid of as yet the not family member of morbidity as far as possible) more than 35 years old.
3) except that diabetes, other significant organic disease of nonjoinder is as hypertension, coronary heart disease (except complication that diabetes caused) etc.
Should guarantee that before gathering family all family members all understand the blood sampling purpose, promptly have right to know, blood sample to provide the people after having understood all situations,, go up signature, the investigation with other personal considerations of could implementing to draw blood at " Informed Consent Form " if agree.If disagree with, then respect its individual opinion.The data of collecting is holded in close confidence, externally do not announce all data such as name, age, disease of blood supply member.
To each member of selected family, except that the diabetes B patient who has clarified a diagnosis, (whether Oral Glucose Tolerance Test OGTT), can be used as normal control to confirm each member all should to carry out the oral glucose carbohydrate tolerance test.OGTT process of the test: extract the venous blood of venous blood and oral 75g glucose (finishing off in 5 minutes) on an empty stomach back 30 minutes, 60 minutes, 120 minutes and 180 minutes, observe the dynamic change situation of blood sugar concentration.In addition, write down each individual generalized case, comprise sex, date of birth, native place, age of onset, height, body weight, heart rate, breathing, blood pressure, medicining condition, have or not complication and blood fat, project such as blood electrolyte, go special examined in case of necessity, be associated with the family of diseases such as hypertension, heart trouble, atherosclerosis with eliminating.
The diagnosis of diabetes B is carried out in strict accordance with the standard that WHO issued in 1985.That is: fasting blood glucose concn 〉=7.8mmol/L or 2 hours after the meal blood sugar 〉=11.1mmol/L are diabetes.The judging criterion of OGTT is: back 2 hours blood glucose concentration＜7.8mmol/L of clothes sugar are for normal, and 〉=11.1mmol/L is diabetes, between 7.8-11.1mmol/L be impaired glucose tolerance (impaired glucose tolerance, IGT).
Collect 60 diabetes B familys that meet above condition altogether, the member amounts to 367 people.Diabetes B patient 180 examples wherein, normal people's 171 examples, IGT patient's 16 examples.These familys are respectively from the crowd of Han nationality on ground such as Beijing, Shandong, Hebei, the Inner Mongol, Henan.Draw the pedigree chart of all familys, and each family and family member are numbered, this numbers with DNA warehouse-in unanimity.In collected family, 15% (9/60) is three generations's family, 85% (51/60) be two generation family.The male sex is 83 people among the patient, and the women is 97 people; IGT person 16 people.Family member's mean age is 50.03 years old, and its distribution situation (dividing ill and normal) sees Table 2:
Table 2 diabetes B family member age composition
| Age bracket | The case group | Normal group | ????IGT | Amount to |
| ????＜30 | ????10 | ????17 | ????2 | ????29 |
| ????31-40 | ????26 | ????45 | ????2 | ????73 |
| ????41-50 | ????51 | ????56 | ????7 | ????115 |
| ????51-65 | ????42 | ????26 | ????3 | ????70 |
| ????＞65 | ????51 | ????27 | ????2 | ????80 |
| Amount to | ????180 | ????171 | ????16 | ????367 |
The peripheral blood sample of getting all each members of family is standby.
2, the little satellite fragment of pcr amplification, gene type
Ordinary method is extracted the genomic dna in the above-mentioned blood sample, and the DNA after the extraction all is dissolved in the TE damping fluid, indicates data such as the affiliated family of sample, numbering, and-20 ℃ of preservations are stand-by.
, distribution comparatively uniform microsatellite locus higher at full genome scanning hot spot (1p36,1p31,1p12,1q22,1q42-43 district, 20p12,20q13 district, 12p12 district and 18q23 district) selection heterozygosity in the past carries out second wheel scan.60 sites have been selected altogether, wherein: interior 34 of 5 positive regions of No. 1 karyomit(e), mean density 3.2cM; 4 of No. 12 karyomit(e)s, mean density 2.2cM; 4 of No. 18 karyomit(e)s, mean density 1.9cM; On No. 20 karyomit(e)s in 3 zones 18, mean density 2.6cM.The minimum heterozygosity of these microsatellite locus is 0.64, is up to 0.88, mean value 0.748.Gained (under " Human PhysicalMapping Project " hurdle) is searched from http://www-genome.wi.mit.edu in the genetics position of part microsatellite locus, and part obtains on the data at the human polymorphism center of French Genethon.In 60 used microsatellite locus, 16 existing primers in the Version2.0 of ABI company test kit, 46 need newly design synthetic primer.In the new a pair of primer of synthetic one with fluorescent substance FAM or HEX mark.The size and the possible reasonable grouping situation of all primers comprehensive consideration pcr amplified fragment length when synthetic are determined fluorescently-labeled color.React used primer sequence Microsatellite Markers Data Set referring to French Genethon genetic map.Each microsatellite locus expanding fragment length and primer information see Table 3.
The magnitude range and the mark fluorescent of 60 microsatellite locus of table 3
Site title clip size (bp) mark fluorescent site title clip size (bp) mark fluorescent
D1S243??????142-170?????????HEX?????????D1S2709?????191-197?????????FAM
D1S468??????173-191?????????HEX?????????D1S2800?????195-225?????????FAM
D1S2845?????193-233?????????FAM?????????D1S235??????175-195?????????FAM
D1S214??????122-152?????????NED?????????D1S2850?????145-153?????????FAM
D1S2663?????183-205?????????FAM?????????D12S1625????264-280?????????FAM
D1S472??????228-244?????????FAM?????????D12S77??????163-193?????????FAM
D1S2892?????93-129??????????FAM?????????D12S336?????117-135?????????NED
D1S2722?????195-223?????????FAM?????????D12S89??????254-288?????????HEX
D1S463??????223-233?????????HEX?????????D18S465?????233-251?????????HEX
D1S211??????170-198?????????HEX?????????D18S61??????213-243?????????NED
D1S2627?????265-279?????????FAM?????????D18S1125????268-286?????????FAM
D1S2868?????210-224?????????FAM?????????D18S488?????239-264?????????HEX
D1S497??????250-278?????????EAM?????????D20S906?????229-247?????????FAM
D1S206??????208-226?????????NED?????????D20S842?????156-180?????????HEX
D1S495??????138-164?????????FAM?????????D20S889?????93-129??????????FAM
D1S2707?????137-159?????????FAM?????????D20S849?????214-236?????????FAM
D1S2878?????152-178?????????NED?????????D20S95??????82-100??????????FAM
D1S196??????322-338?????????HEX?????????D20S892?????205-223?????????FAM
D1S2815?????210-237?????????FAM?????????D20S115?????238-250?????????NED
D1S218??????266-286?????????NED?????????D20S851?????128-150?????????FAM
D1S466??????155-175?????????FAM?????????D20S901?????260-268?????????FAM
D1S2692?????187-217?????????HEX?????????D20S186?????121-143?????????HEX
D1S245??????235-253?????????FAM?????????D20S852?????152-176?????????HEX
D1S205??????94-112??????????FAM?????????D20S112?????217-241?????????FAM
D1S425??????336-360?????????NED?????????D20S912?????267-283?????????FAM
D1S419??????162-194?????????FAM?????????D20S857?????204-220?????????FAM
D1S2860?????168-184?????????FAM?????????D20S840?????141-165?????????FAM
D1S2880?????113-135?????????FAM?????????D20S120?????213-241?????????FAM
D1S213??????107-133?????????NED?????????D20S100?????213-239?????????HEX
D1S2833?????92-108??????????FAM?????????D20S102?????169-177?????????FAM
Behind the pcr amplification, on 377 electrophoresis apparatuses (ABI company), carry out electrophoresis, with 377 electrophoresis image collection software (ABI Prism
TM377-96 Collection or ABI Prism
TM377XL Collection) collects the result, go up computing from http://cmag.cit.nih.gov, calculate relevant site and chain P value, the Z value of phenotype at GENEHUNTER2.0 software (Massachusetts Institute Technology's hereditary system is write, and can freely download).The result is presented in 10 zones being studied has 3 zones to continue to demonstrate and the diabetes Evidence for linkage, as shown in table 4:
Hereditary position, P value and the Z value in meaningful site in 3 positive regions of table 4
The site title is added up P primary system meter Z value apart from the p end-to-end distance from (cM) cytogenetic location
D1S243??????0.0????????????????1p36.33???????????0.00834????1.653
D1S468??????6.2????????????????1p36.33???????????0.0015?????2.135
D1S2845?????11.1???????????????1p36.32???????????0.033??????1.292
D1S214??????16.4???????????????1p36.31???????????0.0025?????2.005
D1S2663?????16.9???????????????1p36.23???????????0.00205????1.996
D1S2815?????193.8??????????????1q24.3????????????0.00126????2.17
D1S218??????196.5??????????????1q25.1????????????0.043??????1.2
D1S2800?????256.2??????????????1q42.12???????????0.0128?????1.579
D1S235??????258.7??????????????1q42.13???????????0.0547?????1.363
As can be seen from the above table, the positive site of 1p36.33-36.23 district meaningful (P＜0.05) is maximum, has 5, and minimum P value is 0.0015, and 2.135,5 sites of Z value are distributed in the zone that reaches 16.9cM.1q24.3-25.1 there are 2 sites in the district, 1q42.12-42.13 the district has 1 site to satisfy the standard of P＜0.05, therefore though the P value of D1S235 is a little more than 0.05 in addition, its Z value has reached statistics dividing value (Z＞1.36), can think that this site and diabetes still have linkage relationship.Fine Mapping has been distinguished to the narrow range of 2.7cM (D1S2815-D1S218) and 2.5cM (D1S2800-D1S235) in these two zones.Noticeable is in the 1p36.33-36.23 district, and continuous 5 sites the P value all occurred less than 0.05 situation, so next step selects this regional cloning tumor susceptibility gene.
The correlative study of SNP in the evaluation of embodiment 2:2 type diabetes tumor susceptibility gene PANK4 and this gene
1, the collection of Han People of North China diabetes B patient and normal control sample
Normal group and diabetes B group crowd peripheral blood sample all pick up from the outpatient service of Endocrinology Department of BJ Union Hospital.The sample source is the northern area of China, comprises ground such as Beijing, Shandong, Hebei, Inner Mongolia Autonomous Region, based on Beijing.Diabetes can be distributes, and also can be member's (but have only in each family this moment a patient selected usually, the member without any genetic connection in the same family can select more than) of diabetic pedigree; Normal control requires the three generations of its family with the interior diabetic subject of not having (comprising type i diabetes) from normal population, and control group and ill group of age and sex are complementary.
The diabetes B diagnosis is with embodiment 1.Each selected member record generalized case, comprise sex, date of birth, native place, age of onset, height, body weight, heart rate, breathing, blood pressure, medicining condition, have or not complication etc., each member is checked projects such as blood fat, blood electrolyte, capable in case of necessity special examined is associated with diseases such as hypertension, heart trouble, atherosclerosis with eliminating.Each selected member should go up signature at " Informed Consent Form ".
Collect 325 parts of samples altogether, wherein the case group is 173 parts, 152 parts of control groups.Two groups in sex, be complementary on the age, and diabetic subject's mean age is 51.99 years old, and the mean age during the normal control sampling is 48.8 years old.In all members, men and women's ratio is 169: 156, the case group male sex 92 people, women 81 people; The control group male sex 77 people, women 75 people.Two group memberships' age structure situation is as follows:
Ill group of table 5 and control group member's age composition
| Age bracket | The case group | Control group |
| ????＜30 | ????2 | ????4 |
| ????30-39 | ????12 | ????18 |
| ????40-49 | ????30 | ????54 |
| ????50-65 | ????91 | ????62 |
| ????＞65 | ????38 | ????14 |
| Amount to | ????173 | ????152 |
2, be used for the selection in the SNP site of gene type in the definite and candidate gene of candidate gene
Result based on embodiment 1, be that karyomit(e) 1p36.33-36.23 zone is linked with diabetes B, utilize bioinformatics method from NCBI snp database (www.ncbi.nlm.nih.gov/SNP), to select to be positioned near the promotor district 2kb of this zone or it 37 and glucose metabolism genes involved mensuration gene, exon and contiguous subarea and the 3 ' non-translational region of including thereof.And the snp of wherein 80 high frequencies carried out gene type, described 34 candidate genes are as follows:
(1) CASP9 is positioned at chromosomal 1p36.3-p36.1, is caspase family member's halfcystine arginine proteolytic enzyme, and the proteic sequential activation of caspase plays important effect in the execute phase of apoptosis.Caspase albumen is with the preceding leach protein form village of non-activity, and by producing two subunits in conservative arginine residues place fracture, these two subunits are combined into activated proteolytic enzyme.
(2) CDC2L2 genes encoding p34Cdc2 protein kinase family member is positioned at 1p36.3.This kinases family member plays a part crucial to eukaryotic cell cycle regulating.
(3) glucose phosphate dehydrogenase (PGD) is second desaturase in the pentose phosphate alternative pathway, is positioned at the 1p36.3-p36.13 of human chromosome.
(4) PRKCZ is protein kinase C ζ member, is positioned at chromosomal 1p36.33-p36.2 position.Be Serine, the ζ member in the Serineprotein kinase family, it participates in several cellular process,
(5) one of SAC genes encoding and the typical different adenylate cyclase of Mammals are positioned at chromosomal 1q24.
(6) Urotensin II gene is positioned at karyomit(e) 1p36, and the sophisticated polypeptide of encoding has active ring 12 peptides, has very high conservative property from seven cheek eels to the mankind.
(7) the GNB1 gene is positioned at karyomit(e) 1p36.33, accepts to transmit signal between molecule and the effector molecule at signal, is made of this genes encoding beta subunit three different subunits.
(8) the GPR52 gene is positioned at karyomit(e) 1q24, the receptor family member of G protein coupling.
(9) amiloride susceptibility sodium-ion channel δ subunit (dNaCh, SCNN1D) gene: be arranged in 1p36.32 district NT_002209, coded product belongs to non-voltage-sensitive type channel protein, can be suppressed by the diuretic(s) amiloride, ionic electrodiffusion in the mediation chamber is present on the film with the tetrameric form of allos.
(10) acetyl-CoA thioester hydrolase (HBACH) gene: be arranged in 1p36.23 district NT_002530.Coded product belongs to one of acetylcoenzyme family member, the CoA thioesters of hydrolyzable palmitinic acid CoA and other longer chain fatty acid.
(11) Urotensin II: be arranged in 1p36.23 district NT_002530.At present its protein function is known little, but known its conservative property is very high.A g protein coupled receptor GPR14 can be used as its acceptor.
(12) DJ-1 (rna binding protein adjusting subunit): be arranged in 1p36.23 district NT_002163.
(13) 5,10-Methylene tetrahydrofolate reductase (MTHFR) genes: be arranged in 1p36.21 district AL354994 clone.5, the 10-Methylene tetrahydrofolate reductase participates in folic acid metabolism, is the key enzyme of the process that methylates again of homocysteine folic acid dependence, with homodimer form performance catalytic activity, regulated by the other structure of S-ademetionine.
(14) chloride channel B (CLCNKB) gene: be positioned at 1p36.13 district bA254I4.00143 (long 47068bp, belong to unfinished sequence) in, the valtage-gated type chloride channel protein Type B of encoding is mainly expressed in kidney, and it and A type have 94% homology.Chloride channel can participate in regulating cell volume, membrane potential stability, signal conduction and transepithelial transhipment etc.
(15) TNFSF4 is positioned at chromosomal 1q25, the individuality of a tumor necrosis factor receptor super family of coding.
(16) TNFSF6 is positioned at chromosomal 1q23, the individuality of a tumor necrosis factor receptor super family of coding.
(17) SLC19A2 is positioned at chromosomal 1q23.3, and the translocator of coding VITAMIN b1 belongs to the member that folic acid is transported superfamily.
(18) CDC42BPA is called PK428 again, is positioned at karyomit(e) 1q42.11, and is similar with the protein kinase that myotonia atrophica is relevant, Serine, and Serineprotein kinase, similar with the protein kinase that cAMP relies on.
(20) KCNK1 is positioned at chromosomal 1q42-q43, the member of a potassium-channel proteins superfamily of this genes encoding, include 2 p structural domains that form porin, the product of this gene seems not to be a functional passage, and he needs other proteic participation just to have activity.
(21) ATP2B4 is positioned at chromosomal 1q25-q32, a plasma membrane calcium ion of this genes encoding pump.
(22) CACYBP is positioned at chromosomal 1q24-q25, a calcium binding protein of this genes encoding conjugated protein, and this proteic bonded effect is not clear.
(23) chloride channel A (CLCNKA) gene is positioned among the 1p36.13 district bA254I4.00143 (long 47068bp belongs to unfinished sequence), and the valtage-gated type chloride channel protein A type of encoding is mainly expressed in kidney.
(24) ENO1 is positioned at chromosomal 1p36.3-p36.2, in 3 Hydratase, phosphoenolpyruvate isomerases in this genes encoding Mammals one, its alpha Hydratase, phosphoenolpyruvate of encoding, a homodimer lyoenzyme.
(25) DFFA is positioned at chromosomal 1p36.3-p36.2, a dna fragmentation factor D of this genes encoding FF, and this is that an allodiploid albumen comprises DFFB and two subunits of DFFA.
(26) G albumen gamma 4 albumen (GNG4) are components in the heterotrimeric G protein complexes, trimer protein from the acceptor birth canal signal of G protein coupling to intracellular effector.
(27) ICMT isopentyl halfcystine carbonyl methyltransgerase is positioned at chromosomal 1p36.21, proteic transferring enzyme of posttranslational modification of this genes encoding.
(28) RE2 is the acceptor of G protein coupling, is positioned at chromosomal 1q23.2, and this gene is the G protein coupling receptor of rhodopsin family, assists regulation and control to attack and feed.
(29) PKLR is the carbohydrate-splitting enzyme of the L type of liver pyruvate kinase, is positioned at chromosomal 1q23.2, and phosphoenolpyruvic acid is transformed into pyruvic acid, and generates ATP.
(30) ALPL is a kind of non-tissue-specific alkaline phosphatase, is positioned at chromosomal 1p36.1-p34, and at liver, bone, kidney all have distribution.Physiological function is unknown now accurately.
(31) PLA2G2D is phosphoesterase A2, and the IID group is positioned at chromosomal 1p36.12, and perhaps the sn-2 ester bond of preferential hydration's phosphatide has important function in the process that infects.
(32) CA14 is in the carbonyl dehydratase family one, is positioned at chromosomal 1q21, and the reversibility aquation of catalysis carbonic acid gas participates in several different physiological processs.
(33) CA6 is in the carbonyl dehydratase family one, is positioned at chromosomal 1p36.2, and the reversibility aquation of catalysis carbonic acid gas is mainly found in sialisterium the inside.
(34) PANK4 is the member of pantothenate kinases family, is positioned at chromosomal 1p36.23, the biosynthesizing of catalysis coenzyme A in bacterium and Mammals.And be subjected to the feedback regulation of coenzyme A.
Selected 80 SNP sites that are used for gene type are as shown in table 6.
| The gene title | The segment name | The site | The position | The Snp type | The site function | ????Pvalue |
| Gene frequency | ||||||
| ????CDC2L2 | ??L11N1 | ????SNP1 | ??Intron11 | ????A/G | ????Intron | ????0.181 |
| ????SNP2 | ??Intron11 | ????T/C | ????Intron | ????0.942 | ||
| ????SNP3 | ??Intron12 | ????T/C | ????Intron | ????0.158 | ||
| ????SNP4 | ??Intron12 | ????T/C | ????Intron | ????0.746 | ||
| ??L13N1 | ????SNP1 | ??Intron17 | ????A/G | ????Intron | ????0.331 | |
| ????SNP2 | ??Exon18 | ????C/T | ????Intron | ????0.234 | ||
| ????SNP3 | ??Exon18 | ????C/T | ????Intron | ????0.217 | ||
| ????SNP4 | ??Exon18 | ????C/T | ????Intron | ????0.280 | ||
| ????SNP5 | ??Exon18 | ????A/G | ????Intron | ????0.670 | ||
| ??L5N1 | ????SNP1 | ??Exon4 | ????T/C | ????Tyr→Arg | ????0.493 | |
| ????SNP2 | ??Splicing ??site | ????A/G | ????Splicing ????site | ????0.421 | ||
| ??L6N1 | ?DELETION | ??Intron5 | ????A/ | ????Deletion | ????0.269 | |
| ??L9N1 | ????SNP1 | ??Intron9 | ????A/G | ????Intron | ????0.024 | |
| ????SNP2 | ??Intron9 | ????C/G | ????Intron | ????0.035 | ||
| ????SNP3 | ??Intron9 | ????A/T | ????Intron | ????0.112 | ||
| ????SNP4 | ??Splicing ??site | ????A/G | ????Splicing ????site | ????0.016 | ||
| ????GNG4 | ??L3N1 | ????SNP1 | ??Intron1 | ????G/A | ????Intron | ????0.477 |
| ????SNP2 | ??Intron1 | ????C/A | ????Intron | ????0.865 | ||
| ????SNP3 | ??Intron1 | ????G/A | ????Intron | ????0.606 | ||
| ????SNP4 | ??Intron1 | ????G/C | ????Intron | ????1.000 | ||
| ????SNP5 | ??Exon2 | ????G/A | ????Ala→Ala | ????0.863 | ||
| ????PGD | ??L13N2 | ????SNP1 | ??Exon12 | ????G/A | ????Ser→Ser | ????0.073 |
| ????SNP2 | ??Intron10 | ????G/A | ????Splicing ????site | ????0.021 | ||
| ????SNP3 | ??Intron | ????G/C | ????Intron | ????0.539 | ||
| ??L1N2 | ????SNP1 | ??Promotor | ????A/T | ????Intron | ????0.496 |
| ????SNP2 | ??Promotor | ????C/T | ????Intron | ????0.226 | ||
| ????SNP3 | ??Promotor | ????A/G | ????Intron | ????0.247 | ||
| ????SAC | ??L1N2 | ????SNP1 | ??Intron12 | ????A/C | ????Intron | ????0.017 |
| ????SNP2 | ??Intron12 | ????C/T | ????Intron | ????0.137 | ||
| ????SNP3 | ??Intron12 | ????G/C | ????Intron | ????0.011 | ||
| ????SNP4 | ??Intron12 | ????A/T | ????Intron | ????0.034 | ||
| ??L6N1 | ????SNP1 | ??Intron16 | ????A/G | ????Intron | ????0.126 | |
| ????SNP2 | ??Exon17 | ????C/T | ????Phe→Ser | ????0.832 | ||
| ????SNP3 | ??Exon17 | ????G/A | ????Val→Ile | ????0.739 | ||
| ??L10N2 | ????SNP1 | ??Exon11 | ????T/C | ????Gly→Gly | ????0.003 | |
| ??L26N1 | ????SNP1 | ??Intron28 | ????A/C | ????Intron | ????0.202 | |
| ??L17N1 | ????SNP1 | ??Intron18 | ????G/A | ????Intron | ????0.243 | |
| ????SNP2 | ??Intron18 | ????C/T | ????Intron | ????0.595 | ||
| ????ALPL | ??L8N1 | ????SNP1 | ??Exon7 | ????T/C | ????Tyr→His | ????0.685 |
| ??L9N1 | ????SNP1 | ??Intron8 | ????C/T | ????Intron | ????0.474 | |
| ????SNP2 | ??Intron8 | ????G/T | ????Intron | ????0.360 | ||
| ????SNP3 | ??Intron8 | ????G/A | ????Intron | ????0.254 | ||
| ????SNP4 | ??Intron8 | ????C/T | ????Intron | ????0.528 | ||
| ????CASP9 | ??L3N1 | ????SNP1 | ??Intron2 | ????A/T | ????Intron | ????0.878 |
| ????SNP2 | ??Intron2 | ????C/T | ????Intron | ????0.829 | ||
| ??L2N4 | ????SNP3 | ??Intron1 | ????G/T | ????Intron | ????0.358 | |
| ??L8N1 | ????SNP4 | ??Intron7 | ????G/A | ????Intron | ????0.386 | |
| ??L9N1 | ????SNP5 | ??Exon8 | ????T/C | ????Phe→Phe | ????0.256 | |
| ??L4N1 | ????SNP6 | ??Intron8 | ????A/G | ????Intron | ????0.795 | |
| ??L2N4 | ????SNP7 | ??Intron1 | ????A/C | ????Intron | ????0.014 | |
| ????SCNN1D | ??L1N1 | ????SNP1 | ??Exon13 | ????A/G | ????Arg→Gly | ????0.741 |
| ??L1N3 | ????SNP2 | ??Intron10 | ????C/T | ????Intron | ????0.157 | |
| ????INSRR | ??L7N4 | ????SNP1 | ??Intron13 | ????G/A | ????Intron | ????0.106 |
| ??L1N2 | ????SNP2 | ??Promotor | ????G/T | ????Intron | ????0.437 | |
| ????KCNJ9 | ??L4N4 | ????SNP1 | ??Exon3 | ????C/T | ????Intron | ????0.280 |
| ????ATP2B4 | ??L13N1 | ????SNP1 | ??Exon12 | ????C/T | ????Asn→Asn | ????0.513 |
| ????SNP2 | ??Intron11 | ????C/T | ????Intron | ????0.749 | ||
| ??L17N1 | ????SNP1 | ??Intron16 | ????C/T | ????Intron | ????0.387 | |
| ??L6N1 | ????SNP1 | ??Intron5 | ????C/T | ????Intron | ????0.979 | |
| ????PK428 | ??L4N1 | ????SNP1 | ??Intron4 | ????C/T | ????Intron | ????0.171 |
| ????SNP2 | ??Intron5 | ????C/T | ????Intron | ????0.065 | ||
| ????TNFSF6 | ??L3N1 | ????SNP1 | ??Promotor | ????C/T | ????Intron | ????0.104 |
| ????CACYBP | ??L6N2 | ????SNP1 | ??3’UTR | ????C/T | ????Intron | ????0.600 |
| ????SLC19A2 | ??L1N1 | ????SNP1 | ??Promotor | ????A/G | ????Intron | ????0.943 |
| ????MTHFR | ??L6N3 | ????SNP1 | ??Intron4 | ????A/C | ????Intron | ????0.399 |
| ????SNP1 | ??Exon6 | ????A/C | ????Glu→Ala | ????0.595 | ||
| ??L4N1 | ????SNP1 | ??Intron3 | ????A/G | ????Intron | ????0.979 | |
| ????SNP2 | ??Intron2 | ????C/T | ????Intron | ????0.570 |
| ??PANK4 | ??L4N2 | ????SNP1 | ??Intron3 | ????C/T | ?Intron | ????0.304 |
| ??L1N1 | ????SNP1 | ??Promotor | ????C/T | ?Intron | ????0.487 | |
| ??L9N1 | ????SNP1 | ??Exon13 | ????C/T | ?Val→Ala | ????0.063 | |
| ????SNP2 | ??Intron13 | ????A/G | ?Intron | ????0.537 | ||
| ??L8N2 | ????SNP1 | ??Intron12 | ????C/T | ?Intron | ????0.962 | |
| ??L11N3 | ????SNP1 | ??Exon18 | ????A/G | ?Gln→Arg | ????0.609 | |
| ??CA6 | ??L3N1 | ????SNP1 | ??Exon1 | ????C/T | ?Intron | ????0.656 |
| ??L3N1 | ????SNP2 | ??Exon2 | ????C/T | ?Thr→Met | ????0.971 | |
| ??L3N1 | ????SNP3 | ??Exon2 | ????A/C | ?Met→Leu | ????0.780 | |
| ??L3N1 | ????SNP4 | ??Exon2 | ????G/C | ?Gly→Ala | ????0.957 | |
| ??L3N1 | ????SNP5 | ??Exon2 | ????G/C | ?Gly→Gly | ????0.641 | |
| ??L5N1 | ????SNP1 | ??Exon4 | ????A/G | ?Gly→Ser | ????0.501 | |
| ??L9N1 | ????SNP1 | ??Exon7 | ????C/T | ?Asn→Asn | ????0.641 |
3, SNP locus gene phenotypic analysis
The main somatotype that adopts 3 kinds of methods to carry out SNP: length multiplicity single-basic extension (LengthMultiplexed Single Base Extension, LM-SBE) reaction, the SnaPshot method, restriction fragment length polymorphism (Restrictive Fragment Length Polymorphism, RFLP-PCR) and sequencing and typing.
1) .LM-SBE reaction carrying out SNP somatotype
1.1) design of primers and synthetic: at 3 primers of each SNP site design in above-mentioned 23 SNP sites, wherein 2 is the PCR primer, is used to increase comprise one section sequence in SNP site, and other one is the SBE primer, is used for single base extension.The PCR primer is by the online design of Primer 3.0 programs, and expanding fragment length is 150～300bp.
SBE design of primers principle: primer will be positioned at 5 of SNP site ' end, and last base of its 3 ' end is the previous base that is close to SNP just.Primer length is between 18～46base, and the Tm value is between 55～80 ℃.Preparation several sites on the same group are dissimilar alternately appearance, and its length differs 4 (or its multiple) bases, and can not form hairpin structure between them, and long SBE primer can not form secondary structures such as inflection.Reference literature (Lindhlad-Toh K, Winchester E, Daly MJ, et al.Lange-scale discoveryand genotyping of single-nucleotide polymorphisms in the mouse.Nat.Genet..2000,24:381-386).
In addition, design is as the dna fragmentation of the FAM mark of molecular weight size criteria, and its size is respectively: 19bp, 23bp, 27bp, 31bp, 35bp, 39bp, 43bp, 47bp, all sequences are AT and repeat to form.
1.2) PCR reaction and product evaluation: a plurality of SNP site (being generally 3-5) of dissimilar and different SBE primer lengths is combined as one group, carries out the respective number dna fragmentation that the multiplex PCR amplification comprises the SNP site earlier.Reaction system 5 μ l, each component is as follows:
Title original liquid concentration application of sample amount (μ l) system final concentration
Mg
2+????????25mM??????????0.6??????????????3mM
Damping fluid 10 * 0.5 1 *
dNTP?????????2mM???????????0.075????????????30nM
Each 0.1 μ M of primer 10 μ M 0.05 * n
AmpliGold????5U/μl????????0.05?????????????0.05U/μl
Template 50ng/ μ l 1 5ng/ μ l
DdH
2O polishing to 5 μ l
Adopt Touch-down PCR, its program is as follows: after 94 ℃, 12 minutes, carried out 94 ℃ 30 seconds, 63 ℃ of 15 round-robin 30 seconds (each circulating temperature descends 0.5 ℃), 72 ℃ 50 seconds, 94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ of 24 round-robin are 50 seconds subsequently, last 72 ℃ 10 minutes twice.Reaction finishes the back to be identified with 1% agarose electrophoresis, observes to have or not product under the UV lamp, and takes pictures writing time, site title, sample number etc. at ultraviolet gel imaging instrument.
1.3) remaining dNTP and the strand primer that participates in reaction can influence next step SBE reaction in the system of PCR product purification: PCR reaction back, need remove the two so be used as the PCR product of SBE reaction template.(Shrimp AlkalinePhosphatase SAP) can remove the phosphate group of dNTP 5 ' end shrimp alkaline phosphotase, makes it to form phosphodiester bond, thereby can not participate in the extension of DNA chain; (Exonuclease I ExoI) can influence next step SBE reaction thereby avoid PCR to react remaining primer from 3 of single stranded DNA ' hold 5 ' extreme direction Nucleotide of degrading one by one to exonuclease I.
In per 5 μ l PCR products, add 2 μ l enzyme mixed solutions, wherein contain SAP 0.5U/ μ l, Exo I 1U/ μ l, mixing is inserted 37 ℃ of water baths digestion 1 hour, and it is centrifugal to take out the back, and 85 ℃ of 15min deactivation SAP and Exo I are as the SBE reaction template.
1.4) the LM-SBE reaction: owing to have only fluorescently-labeled ddNTP (to be respectively JOE-ddATP in this experiment in the system, FAM-ddGTP, TAMRA-ddCTP ROX-ddUTP), use is reacted than higher hot Sequenase (ThermoSequenase) fluorescent mark ddNTP incorporation efficiency, can make each SBE primer only extend a base in reaction and only promptly come to an end.Although amplification is a linear growth, after 40 circulations, under the situation of ddNTP capacity, its total amount has also reached 40 times of template, is enough to detect.
Reaction system is 5 μ l, and each component is as follows:
Title original liquid concentration application of sample amount (μ l) system final concentration
Mg
2+????????????????25mM??????????0.5??????????????2.5mM
Damping fluid 10 * 0.5 1 *
JOE-ddATP????????????0.12μM???????0.1??????????????2.4nM
FAM-ddGTP????????????0.12μM???????0.1??????????????2.4nM
TAMRA-ddCTP??????????0.12μM???????0.1??????????????2.4nM
ROX-ddUTP????????????0.60μM???????0.1??????????????12nM
SBE primer 2 μ M 0.05 * n 40nM
Theromo?Sequenase????10U/μl???????0.025????????????0.05U/μl
Template 1
DdH
2O polishing to 5 μ l
Response procedures is: after 95 ℃, 3 minutes, carried out 95 ℃ 30 seconds, 50 ℃ 30 seconds, 60 ℃ of 40 round-robin 40 seconds, last 60 ℃ 2 minutes.
1.5) electrophoresis: SBE product 1 μ l mixes with the sample-loading buffer equal proportion, carries out electrophoresis on 377 sequenators.30 minutes 2 times application of samples in the time of can be respectively at electrophoresis during application of sample and behind the application of sample respectively add the size criteria in a hole respectively at the two ends of well.Electrophoresis finishes the back by observing SBE product electrophoresis position and comparing with size criteria, can judge it is which site.
1.6) genotype judgement and data preparation: judge the SNP genotype according to the band color, blueness is G/G, and green is A/A, and yellow is C/C, red T/T, indigo plant/green is A/G, Huang/redness is C/T.With each site somatotype result arrangement is that the Excel form is to carry out statistical treatment.
2) SnaPshot
TMCarry out gene type:
SnaPshot
TMBe the test kit of PE company based on the gene type of the principle exploitation of single-basic extension (SBE), the extension of multi-primers specificity is used for a plurality of SNP site and does gene type simultaneously, must be at ABI
Move on 3700 sequenators.
2.1) need the SNP site of somatotype, generally same system is put in 7～8 sites, primer 3 ' terminal must design in the upstream in SNP site or the contiguous site in downstream, regularly react with common PCR.The Tm value of each primer is best approaching, can not be lower than 50 ℃;
2.2) before primer, add and make the polyT of suitable length to differ 4bp between each site at least, and make two contiguous sites not have the base that overlaps as far as possible, (for example 3 sites are C/T successively, A/G, C/T);
2.3) contain the dna fragmentation in each SNP site respectively with ordinary method PCR, demarcate balanced mix after the concentration, purifying;
2.4) with the extension primer balanced mix in each SNP site and be diluted to and be respectively 0.5 μ M.With the 1 μ l mixing of the PCR product mixtures behind SNaPshot reaction mixture 2 μ l, primer mixture 2 μ l and the purifying, 96 ℃ of 10s, 50 ℃ of 5s, 60 ℃ of 30s, 30 circulations are carried out primer specificity and are extended.Add 70% ethanol of 9 times of volumes in the reaction product, left standstill under the normal temperature 20 minutes, 4 ℃ 4000 left the heart 45 minutes, abandoned supernatant, added 70% ethanol of 9 times of volumes again, left standstill 20 minutes, and similarity condition is centrifugal, abandons supernatant, and 1300 commentaries on classics are inverted centrifugal.Dried in the air 30 minutes, and added 6 μ l distilled waters.
2.5) last sample of SNaPshot and interpretation: every hole adds methane amide (Hi-Di Formamide) 9 μ l, length mark 0.2 μ l, SNaPshot reaction product 3 μ l, 95 ℃ of sex change 5 minutes, capillary electrophoresis is being carried out in the cooling back on ABI PRISM 3700 dna sequencing instrument, operation Genescan3.7 carries out data analysis, and Genotyper3.7 exports the genotype of everybody point of each sample.
3) RFLP-PCR carries out the SNP somatotype: for the SNP that changes restriction enzyme site, can advanced performing PCR amplification, amplified production digests with corresponding restriction endonuclease, behind the electrophoresis according to band location determination genotype.Complete degestion or can not be digested be two kinds different homozygous, not exclusively enzyme is cut is heterozygous.
4) sequencing and typing: carry out sequencing and typing with above two kinds of method somatotypes or the unfavorable site of somatotype result for being not suitable for.
More than the somatotype in each SNP site all in the case of picked at random and 96 individualities of normal control, carry out earlier.
In case group and control group, above-mentioned 80 sites all meet the Hardy-Weinberg balance.Analyze through SPSS statistical software 10.0 editions (free download is in http://linkage.rockefeller.edu/soft/), there is 1 SNP site to be positioned at the 66th of No. 13 exon of PANK4, the Ala that is in the 547th in amino acid becomes Val (GCG → GTG), this site is a brand-new site, and nobody reported.Gene frequency is distributed in that difference has statistical significance (P＜0.05) in two groups, and is as shown in table 7.
Table 7 case group and control group SNP somatotype SPSS statistic analysis result
Allelotrope
SNP C T A G amounts to the P value
Case group 283 161 444 0.028
PANK4L9N1SNP1 control group 195 77 272
Amount to
There is significant difference in the gene frequency of 1 SNP site GCG → GTG that is arranged in the L9N1 segment of PANK4 gene in case group and control group, therefore points out this site relevant with diabetes B.And then prompting PANK4 gene is a kind of new diabetes tumor susceptibility gene.
The genome sequence of PANK4 is shown in SEQ ID NO:24, total length is 18,040bp, the SNP site (Y) of being disclosed is positioned at 12 of this sequence, 801-14, between 400, this regional nucleotide sequence is shown in Figure 1A, and Y is the SNP site among the figure, and it represents base C/T polymorphism, promptly this site can be C, also can be T.This regional complementary sequence is shown in Figure 1B, and R represents the complementary base in described SNP site among the figure, and it represents bases G/A.
Based on this SNP site, can design the primer of suitable length, be used for predicting diabetes susceptibility through pcr amplification.
The prediction of embodiment 3 prediction diabetes susceptibility primer design and diabetes susceptibility
1.SBE primer
Primer will be positioned at the 5 ' end in SNP site, and last base of its 3 ' end is the previous base that is close to SNP just.This primer has one.Length is between 18-46 the base.Avoid containing too much G+C, the Tm value is between 50～65 ℃.Because conversion hysteria is more in the SNP site, therefore should avoid the identical sequence type of continuous several appearance (as continuous several C/T types), as when running into several sites of preparing to put into same group and all being the C/T type, wherein several can be designed from anti-chain, promptly become the A/G type, preferably C/T and A/G occur at interval.Formation such as hair fastener can not be arranged between preparation several sites on the same group in addition, and long SBE primer can not form secondary structures such as inflection.A primer according to above principle design is: 5 ' CCG TTC CTC CCA GCC CAG 3 ' (SEQ ID NO:22), perhaps 5 ' TGCGCT CCC TGG ACG C 3 ' (SEQ ID NO:23) its be the same preface primer of this chain, promptly combine with its complementary strand.Genes of individuals group DNA to be measured is carried out the sequence in SBE reaction detection SNP site with this SBE primer.
2, regular-PCR amplimer
According to the sequence (Figure 1A and 1B) at place, SNP site, the primer of design shown in SEQ ID NO:1-21.As shown in table 1ly carry out various combinations.With the described primer that is combined as, be that template is carried out pcr amplification with the genomic dna that derives from individual blood sample to be measured, amplified production is checked order detect the single nucleotide polymorphism in described SNP site.
SEQUENCE?LISTING
＜110〉Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences
Sinogenomax Co., Ltd.
Nanfang Research Centre, State Human Gene Group
The Chinese Academy of Medical Sciences Beijing Union Medical College Hospital
＜120〉be used to predict the test kit and the primer of diabetes B susceptibility
<130>I20030512CB
<160>24
<170>PatentIn?version?3.1
<210>1
<211>19
<212>DNA
＜213〉artificial sequence
<400>1
gtggtatgga?acaggctgc
19
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<400>2
gcagacacct?acgcagacac
20
<210>3
<211>19
<212>DNA
＜213〉artificial sequence
<400>3
agtcttcgat?cctgagggg
19
<210>4
<211>19
<212>DNA
＜213〉artificial sequence
<400>4
ggcctgagtc?ttcgatcct
19
<210>5
<211>20
<212>DNA
＜213〉artificial sequence
<400>5
gcctgagtct?tcgatcctga
20
<210>6
<211>20
<212>DNA
＜213〉artificial sequence
<400>6
actcttgtcc?tgcccttcac
20
<210>7
<211>20
<212>DNA
＜213〉artificial sequence
<400>7
aacctgaggg?gacagacaag
20
<210>8
<211>19
<212>DNA
＜213〉artificial sequence
<400>8
ctgaggccac?gtcttcact
19
<210>9
<211>20
<212>DNA
＜213〉artificial sequence
<400>9
cacccactgc?tcagtcacat
20
<210>10
<211>21
<212>DNA
＜213〉artificial sequence
<400>10
cttcacccac?tgctcagtca?c
21
<210>11
<211>21
<212>DNA
＜213〉artificial sequence
<400>11
agcgacctgt?aagacatgac?c
21
<210>12
<211>19
<212>DNA
＜213〉artificial sequence
<400>12
gataggcttg?tgcctggtg
19
<210>13
<211>20
<212>DNA
＜213〉artificial sequence
<400>13
tgagtcttcg?atcctgaggg
20
<210>14
<211>20
<212>DNA
＜213〉artificial sequence
<400>14
cttcacccac?tgctcagtca
20
<210>15
<211>19
<212>DNA
＜213〉artificial sequence
<400>15
ttcacccact?gctcagtca
19
<210>16
<211>19
<212>DNA
＜213〉artificial sequence
<400>16
ccactgctca?gtcacatgg
19
<210>17
<211>20
<212>DNA
＜213〉artificial sequence
<400>17
cagaacctga?ggggacagac
20
<210>18
<211>21
<212>DNA
＜213〉artificial sequence
<400>18
tgacaataga?aaatgccagc?c
21
<210>19
<211>21
<212>DNA
＜213〉artificial sequence
<400>19
gtgaaggtaa?atgatggtgg?g
21
<210>20
<211>21
<212>DNA
＜213〉artificial sequence
<400>20
agcttctttg?ggagtgaagg?t
21
<210>21
<211>19
<212>DNA
＜213〉artificial sequence
<400>21
aggccacgtc?ttcactcag
19
<210>22
<211>18
<212>DNA
＜213〉artificial sequence
<400>22
ccgttcctcc?cagcccag
18
<210>23
<211>16
<212>DNA
＜213〉artificial sequence
<400>23
tgcgctccct?ggacgc
16
<210>24
<211>18040
<212>DNA
<213>Homo?sapiens
<400>24
gcgagcggca?gcgggagcag?cggggacagt?ctggacaaga?gcatcacgct?gccccccgac
60
gagatcttcc?gcaacctgga?gaacgccaag?cgcttcgcca?tcgacatagg?tagccgcagg
120
ccgggcgccg?ggcgcgggct?gcgggggcgg?gcggggcgcc?ggcggtgggc?cggcgagcgg
180
gggcggcggg?aaggacgaag?ggcgggggcg?cgggcctcgc?gcacagcctc?acgggccttg
240
tagtccgcgg?gcccgcgcac?agcctcacgg?gccttgtagt?ccgcagggtc?gctgatgcgc
300
gggctcgggg?cgacggccgg?actacagcta?ccggcaggcg?ccgcagggac?cagagcgggg
360
cctgccgggc?ctggtagttc?gcgcggccgt?cctgggtggc?tgggggccgg?ggtccagcgt
420
tacccgccgc?gaggtgggag?ggtcggcgga?attttatgtc?ttcggccaca?tcggcatctg
480
ggcgccccgg?ctccgccgct?tggctggtgg?ctggcctgag?atttggggtc?cttagttctg
540
gtcgagacga?ggacgagctc?ggcgggaagc?ccttggcaga?gtggccccga?aaaggccgcg
600
cgttcccgcg?gcgtggccag?ctgtggcccg?cgggccggac?agtccgtgca?caggccccgc
660
tctctggaga?gtggacgccg?ggcgtgagtt?caggcggccg?tggacgtggg?acctgggaac
720
tcgtgcctct?ggtttaaaag?ccacccgtgc?cttccgtggc?ctttaaggcg?gcctgcactt
780
ctgccctggc?ctctccaggg?tccgctgcag?accctcctgc?ctgatggctg?tgctctcagt
840
aatgacggcg?ggccaggctc?tgttctagac?ctttttccca?cgctcctgac?tcccggacac
900
ctcccagcca?cctctagggg?aggaacgcga?attactccct?tttcgcagat?gaggatacca
960
agccccagaa?aggtgaacta?atttgcccag?ggtcatcggg?ctagaaagtg?gcagaaccag
1020
gatttgaacc?agccactccc?tgctctccta?ctctgcccat?gtttcaggca?tgagggcttc
1080
ctgggggcag?cttcacctgg?agaaaccccc?cttgaatagc?cgctgcggct?tcacatacct
1140
ctccctctta?gcactcatct?cggtcgtagg?ctgagtctgc?ttgtgtgtga?atcccacaag
1200
agcagcggtg?tgtcttgtag?ccccagcccc?cgaaaaggat?gcccaacctg?tctttgccgc
1260
ccagtaaata?ctgggatgaa?tgggcctgcc?gctgactgct?tcccacctcc?ctggaaaagc
1320
tgggactcag?agagacccag?gccattccct?ctgagctgtg?tgctcctggt?gcatctgaca
1380
tgtttgctgc?tgtggtgtcc?tcggggttgt?tggtgtcatc?atctctctga?gatgtccagc
1440
cagggaaggg?tgtcctctag?ggaagtgggg?ggcgatgaga?atggaagtag?ctataggtcc
1500
tggctcccac?ctttcagtga?gcttatggga?ggcaatgctt?cttagggggt?gtggcctggt
1560
gccaggtatg?agaacgtgtg?agtagcatct?ttagggaccg?ctgataggac?ttgggactga
1620
ctggttattg?ggctgcaggt?gctgagcttg?cagccctagc?ctaggggatt?gtctaccgca
1680
ggcctcacca?gaggctcccc?agccatgctt?cccagccttc?tgagaccttg?aggcccggta
1740
gagcgtgtgc?tctgcaaggt?tctgctgccc?ttacactggg?ggattctgtt?actggaagca
1800
agtcgggatt?aaatgtttct?gaaatgcaat?tgcagttgac?ctgcataagt?cacttgggga
1860
atggccagga?gaacagggct?caccgtggat?gttctgtgtg?gccccgagag?gccagtgaag
1920
ccgcagagct?ggggctgtcc?aggaagggct?gtcgcagccc?tagagggccc?tggagtacca
1980
gcctgttctc?caggctgagc?ccagggggtt?tcttggtaga?aggatactct?aagaggtctg
2040
tttagtgctg?gtatttattt?cccctcttca?aagtttctcc?ctgcgtctgg?gtcctgtgtc
2100
tctggttctt?aagaggacct?gttgactgat?actgagagac?cttttgtttg?ttactcctga
2160
ccgagctgct?gggaggccac?gctcagggtg?gggtgtcttc?aacccaggaa?ccctcaaagc
2220
caaagtgctt?gtcaacagtg?ttgtttaaaa?gcaggcttct?cccatttcct?cttcccttgc
2280
ctgtgtctgg?atctaggaaa?gctcacctgc?cctgtgttct?ggcaagctgt?tttcctctcg
2340
atgggacgct?gatgtctgct?gagcagattc?acctctttga?aagcagcagg?gacgttgtat
2400
ttctcaaaac?atactgcagc?ttcgatctga?ttttaggttt?ctgcgggctg?tggggagggt
2460
tcctcagagg?caggtgagac?aggaaggggc?cacggggcgg?tggtgtctgc?ttggctgtcc
2520
tgccctccat?tggtgcccaa?agaggaaaca?caggaccaga?gagaatggca?gaggggatgg
2580
ggcctgtggg?acagggagaa?ctagaaggcc?atggctcaga?tgtgaagagc?acaggtgacc
2640
aaacctctgg?gaagccccct?tcccccacca?gctgttggcc?tcagggccga?gcgcgggcag
2700
cccggagagc?cgccaggagg?cactcctgtc?actgccttgc?caggcgcggt?ttgctgtgat
2760
tccgaggggt?ccctgagctc?agcccggaag?ggtccccgcc?cgcaggcggt?tccctctgct
2820
ctaacagatc?tccctgctgt?gcgtgcccct?cgcgcctcag?gtcccgtttt?gtccccccgg
2880
gtgtggggag?taggcagacg?gaacactggc?cttgctgggc?ctctccgtgc?aggggtctca
2940
gggagggagg?atgcgtatct?tgcagaaacc?aacccacccc?cacctctgtc?ccaaccccag
3000
tacatcctgt?ttgggcatgt?atttaacaca?aagaacccag?ggcctgaaac?tggatttctg
3060
tgacctgaga?gtttgtttat?actgactttt?gatgagtctc?ttaaaataga?tcttcatcct
3120
cttgcatcat?ctgtcgtttt?agaaaacgat?ttatttactg?aggtgagtcg?gcggcatggt
3180
gcatgttagt?gagactctcc?gggcccggcc?agcactgggg?gcaggggagg?ggataaggag
3240
gcttaaagcc?acaggctagg?ggatcagatt?gagcctgtga?cgcgtccacc?agactgtacg
3300
ccagccgtcg?gggagcctgc?cctcaccacc?aggcaggagg?tgccctggga?tggctgttgt
3360
ggggcctgga?cacggtgggt?tttgctacat?gaacagcatt?ggctgtttgc?ttggtgtttc
3420
caaatctttt?tactgacctg?agtgcttgtt?ctcagacatc?cttatagtat?tttgtcaaaa
3480
aacaataatt?tgaaaataac?tttagcctca?ttactctcat?gatgtgggat?ttttgcctct
3540
ttttgtcagg?ccctgtaaga?aatgcactgt?gttaagtcac?tatagcacca?tccagtgccc
3600
tccgttaagt?cactataaca?ccacacggtc?ccctctgtta?agtcactata?gcaccatacg
3660
gtgccctctg?ttaagtcact?atagcacaca?cagtgctctc?cgttaagtca?ctatagcacc
3720
acacggtccc?ctccgttaag?tcactatagc?accatccagt?gccctccgtt?aagtcactat
3780
agcaccatac?ggtcccctcc?gttaagttac?tataacacac?acggtgccct?ccgttaagtc
3840
actataacac?acacagtgcc?ctctgttaag?tcactgtagc?accgcgcaat?gcactcaaag
3900
tcacattttg?tttctttttc?tatttgtcat?tcatgttgtt?tcaaatctat?gaatacaact
3960
ctagcctcaa?caatgtctgt?tttttttttt?aacttagaag?aatttgtcct?tgaaaaggcc
4020
ttcttcaaga?atcttttcac?ttttccctga?aatctggttc?aggtacatag?agccccttcc
4080
atttcctgat?atttattcaa?cctttgatca?caggcggcat?cctggaagcc?acagggcttc
4140
tcgagagcat?cagagagact?ttggcctgtg?gggaaacgga?ctttggtggc?ctctgacttc
4200
tggctacttt?tggggctggg?gctgcatttt?ctgcctggag?cctctcaaga?gtagccagcg
4260
gcaccgtttg?cctctccagg?gtaggggagc?tgctcaggcc?ccgttaccgc?cctgggctgt
4320
gaggagagcc?tcctttgtaa?gcagggacgg?tcgagggtgt?acttcttaaa?acgaaaacag
4380
cctggatttg?tcagcttatc?gctttgggtc?attagcaaga?tcacttgaaa?ctgaattttc
4440
ttaaagcagt?gaggataacc?aaggttaccc?aaagcaagac?agctctccct?ttcctctgca
4500
ggacttggtg?ccaaggggaa?gcgggacctc?ttgagctcag?tttggccagg?gagctgagga
4560
ggagcagaat?ttgagaaaga?aaagtgtaga?tttgattgtt?cgttttcctg?tcctctcttt
4620
tgtgcccaca?gggtttttaa?gttccagtag?ccccaaaccc?ctcatctaca?gagactttag
4680
tctccaggac?acccagagag?tgtggggcat?ggacccccag?gccccgctgt?gtcctggtcc
4740
tgaatcctgc?ccggtttgtg?tctcccctgc?aggcgggtcg?ttaaccaagc?tggcctacta
4800
ttcaacggta?cagcacaaag?tcgccaaggt?gcggtctttc?gaccactccg?gaaaggtgag
4860
cctgcactgt?ggccacccgg?ggcagggcag?ccgcttctct?gtcgtcttgg?acctggcact
4920
tgtctcgcag?tcatccctct?gctgctgcag?acctcgactt?tagtgtcctg?ggagtttcta
4980
gcagggacag?cctcccctgc?cttccgccgt?gtcccttcaa?gaccctttgt?aggaacctgc
5040
gcccgcctgt?gctcccccgg?gagaggcttc?cgctcctgct?gggtggccct?gaggtcgcca
5100
cggtcctagg?ctcctccctc?ttgcccagga?ttctgcctca?gagcgccgcc?tggttgaagg
5160
ctggcgcgaa?cagccagctc?cactagggca?gggtgcgcac?agcccagccc?ggcgcggtgt
5220
cccacacaca?tgcggcctcc?cctactctgt?tttccaggac?acagaacgtg?aacatgagcc
5280
gccctatgag?atttcagttc?aagaagagat?cactgctcga?ctgcacttca?ttaagtttga
5340
gaatacctac?atcgaagcct?gcctggactt?catcaaagac?catctcgtca?acacagagac
5400
caaggtcatc?caggcgaccg?ggggcggggc?ctacaagttc?aaagacctca?tcgaagagaa
5460
gctgcggctg?aagtgagtgg?ggatctcaag?ggcgagaaag?gaacatgtgt?ctgcccccga
5520
gtccctgggt?gtcccagagc?cgcgtccctg?gcgctcgtgt?gtcagattgc?gcatggggca
5580
tggctgcccc?ttcggaccag?gcaggcttgc?atggttgcac?ctgtctgtgg?cccagactct
5640
ttaaggggtt?ggcgcttcct?tttcagagtc?gacaaggagg?acgtgatgac?gtgcctgatt
5700
aaggggtgca?acttcgtgct?caagaacatc?ccccatgagg?ccttcgtgta?ccagaaggat
5760
tccgaccctg?agttccggtt?ccagaccaac?cacccccaca?ttttccccta?tcttcttgtc
5820
aatatcggct?ctggagtctc?catcgtgaag?gtaagacccg?gcttcatgaa?tgaatgagtg
5880
gatggtttag?ccatagtttg?ttaagccctc?gctgtgtgca?ggagccaggg?ctgggcacat
5940
ggatggggcc?cagtgggcgg?gaggggggtg?gcgaggctgt?gagctcagag?cctctgtggg
6000
aggggcatca?gcgcccccag?gcttggccat?gagagtcctc?ccactggcgg?ttggggtggg
6060
ggcggggttc?accccagcgc?agcacatggg?gcggggggcg?ggggagcctg?tgtggctgag
6120
ggcccactga?gggcacacct?gccctggctc?tgttgcaggt?ggagacggag?gacaggttcg
6180
agtgggtcgg?cggcagctcc?attggaggcg?gcaccttctg?ggggcttggc?gctctgctca
6240
ccaaaacgaa?ggtatgcggc?agctgccaga?gaccttccag?gggtctgcgg?agatgtctgc
6300
ttccttcccc?cgaaggcctg?cagctgggcg?gtgcaaaagc?tgcttccggg?cctccctcct
6360
gactcgcgtc?agtgggtctc?tggcctctgc?ggcttcactc?tttgcgccct?gagggttggg
6420
tgtcccagca?acccagagct?tctatcctgg?ctgggtggcc?cgagggtccc?gcttgccgcc
6480
tcctgccttt?ggtcccacgc?gatgagggcc?catttacccc?ctgcccgcgc?gtgcctcctg
6540
ccatgggctt?ggtttctggg?gtcgtgggga?ttccagcagc?tcctggcgcc?tcacccgccc
6600
cctcgccgtg?tcctgcagaa?gtttgacgag?ctcctgcacc?tggcctcgag?gggccagcac
6660
agcaatgtgg?acatgctggt?gcgggacgtc?tacggcggcg?cccaccagac?tctcgggctg
6720
agcgggaacc?tcatcgccag?cagcttcggg?aagtcggcca?ccgccgacca?aggtgctcac
6780
cccggcctct?gccgccagag?agcaggatgg?tggggacact?tggggtctca?cggacaggag
6840
cttcccccac?cattgctttc?ccacaactgc?tccctggaga?gtcggggtct?tgggtgtcag
6900
ccctgtaacc?tcttcctgcc?gagtcgctgc?agctcaggcc?cactgctcag?aacgtcggca
6960
gataaacgcc?acggtcttgg?ttttggaaga?aaaaatagtt?tcctgattgg?gttttttcct
7020
ccttcaaaac?aaagcttaat?ccgtccagga?atgattcaca?catcacacgc?agcctcccgc
7080
acttgggctc?cagttccccc?actcagctct?ctctccccct?cccctcccac?tcagctctct
7140
ctccccctcc?cctcccactc?agctctctct?ccccctcccc?tcctgctcgc?tctcatgtcg
7200
tgcacttgct?gtacttggag?atgagtgcct?tttccttccc?ttcctcagag?ttctccaaag
7260
aagacatggc?gaagagcctg?ctgcacatga?tcagcaacga?cattgggcag?ctggcctgcc
7320
tccacgcacg?gctgcacagc?ctggaccgcg?tgtactttgg?aggcttcttt?atccggggcc
7380
accccgtgac?catgcgcacc?atcacctata?gcatcaactt?cttctccaag?gtaacggatg
7440
cgccgccctc?cccagcctct?aacgcagacc?ccccaggatc?ttcccaatca?gtcatgctgg
7500
ttagacaccg?cagcaccccc?agggaggcct?ggagggtggg?ggccaagaag?cccagcaccc
7560
ctcccttctg?actgattctg?ggcagcctac?attttgtggc?accgtggcca?cctgagtttg
7620
agtacatcac?cagtgatgag?cattctagag?agctccaggc?aggcctgctg?atctgcgctg
7680
gggtcagggt?gcccctgccc?ccgcagaggc?acgcccaccc?acatgcctga?cgcagagact
7740
tgtgggccag?ggactcgctg?catgggagaa?gctatctcac?ccttagcttt?cttcaaacag
7800
cagcccgccc?tctggtgcca?ggcgcgattc?cactggcaag?tatgccgtgg?gtgccatggc
7860
gcctcttcga?gggtttgggc?cgcgcggtgc?gaggcccgcg?cacgttgagc?tggtttgcgg
7920
aggcaccggg?tgttgtgtgg?ggccaacagc?agcacgtgtg?ctttggccac?ggcaggggga
7980
agtgcaggcg?ctgtttctga?ggcacgaagg?ctacctggga?gccatcggag?cgttcctgaa
8040
aggagctgag?caggacagtg?agtcgcggtg?ctggcgcccc?tgagcagcgt?ggggcctcag
8100
ctgtggcctg?ggcctgtgct?ccaggccagg?gcggcccagg?gtggaggtgg?ggtggggagc
8160
gtgagtcctc?agaaagtgac?agcagcaggc?atgtctaaca?ttttaacaat?ttagttaata
8220
agcgatttta?taccctttcc?tcctccactc?tcctgctttt?gaatcaaggg?caaggttaac
8280
acaagtgacc?tggcttcttg?ctttccagat?cctaaccagt?acagctgggg?agagaactat
8340
gcaggcagct?ccgggctgat?gagtgcatca?cccgagctcg?gcccggcgca?gcgggcgcgg
8400
agtggcactg?tgagtagtgg?gctctggccg?ccccgggccc?caggggagca?gcctatgccg
8460
cactcacctc?gcctgtccct?gagcgaagcc?tcttgtggct?gtcccggctg?aaaggcgcct
8520
ctgcccatgg?cgaatcctcc?catggacctg?gggtataact?aggggcaaga?gaaggacact
8580
gtggcttgct?ttcccatctc?cctcattggg?tgctttcgta?ttttggaaac?attcagcagg
8640
aattacggag?tctgtgtgat?ccggccttct?tgtttgcaga?gagattgcta?aagttcaggg
8700
gtgacgggga?cctctcccca?cgccagaagg?cccgtccact?ttggagttga?aacccgaaac
8760
cgcaggagcc?acccagggcc?ctgcctagga?ctgcgcgctc?tgccgcacac?tgccgcgcac
8820
tgtggctgat?ggtctgtcgg?ttacagtctg?ctctcctcgg?ggtgtcccgg?ctcattcctt
8880
ggatccccag?tgggtttgcc?ctggcctgag?ctgggtggca?ctggcgggct?aggagacgca
8940
ccccatgtct?gccgagttgg?agctgctccg?gaagggtccc?ccgccaggac?ctgcgtcagc
9000
cacgggatgc?ccccagcaca?gccccacatg?gcaggaaccg?ccccgtgtca?caggctgtca
9060
gggtcctcga?ccgtgccccc?ccgcggggct?cattccttct?gtagctgtga?gcagttaacc
9120
tgaggtcacg?cacaaagcta?gtcggacccc?tcaggacagg?ccgctggcat?tgttggcagg
9180
aagttgcttg?tcatttgtag?acatggggtt?cagtgtcagt?attttcaaga?ccgcgacttg
9240
ttgagttttg?gcttctcagg?agggcgcctc?ccttttgtgt?gggtcctgca?gttcacacca
9300
ggccacggcc?tcgcacagac?gcaccacact?ggccgcagcc?ctggtctgcc?tttcccaccg
9360
ccagcactag?gcaggggagt?gggggcgagg?ctctgggggc?ctggcagagc?cccaggctct
9420
cagcttttgc?cctgctgttg?ggcagagggg?cttggtgtct?tccagcctca?gtttctgatt
9480
tgccaagtgt?gcataatttg?ccaaatgtcc?tggactgtaa?cctggagtga?ctgcgtggac
9540
ggccacagtg?cttcgggggc?cccgttcagg?aggcgcttgt?tacctatttg?gtaccccacc
9600
agtggcacag?ccctgccagg?caggaggggc?ccccacctta?ctgaggatca?aactgacatg
9660
cagagagatg?gagtcattta?tttcagttta?tattgtccaa?aaaagtcaaa?gcgaagattt
9720
gaacccattt?gtcaggatag?aaggagcctg?cgacctggca?aggcagcacc?aggcgatccc
9780
ggctgtggtc?cgtggtagtt?aaggtggggc?tcctgccgcc?ttctgcactg?gctttgggaa
9840
gtcactggct?ttttccacgt?ggagcttctg?cccggcatgg?gcttcccacc?tggaaggcgt
9900
cccctttgct?ctggaggtcc?ctgcccaccc?agaggctgct?tctccagccc?caggggttgc
9960
cagcaggggt?gccccagtgg?agaagggcag?agggccagca?tgtgacctgt?ggggtagcca
10020
ccggagggtg?ggaagaggcg?gtcagctaca?cagttcccct?catagcccct?ctggtcccag
10080
agcagcggct?gctgcggtga?gcgtgcagag?agccgagtac?tgatgtgtgc?gtcggctcag
10140
ttcagttcac?ttcctcccgg?cataaggtag?aaaaagccag?cagggcccgt?gagcgcgcct
10200
gtgctgggtg?caggtggccc?cggggtgccc?tgggtgcata?atggcctggc?cctgccatag
10260
tccttcatgg?acagaacgtg?ctgggaggcc?cggcagccat?ggaggaagaa?gggtgcatgt
10320
ggggcgtggg?gactccctgc?ccggcacgga?gtggaacact?gcctggtgct?tagtgaggac
10380
agaaccccaa?acctctgtgt?gcagacacgg?ccacctggag?gaccagaggt?gggaacagtg
10440
tgactgcagg?ggtcacggga?ggaggtgaaa?ctgtaggggt?gacagggagg?ggtgactgca
10500
gggaggtggt?gatgggggag?ggagttacta?tagggatggt?ggaggagggg?tgatgggagg
10560
gtgactgcag?cggtgattgg?ggaggtgtgg?ctgcagcggg?gaggtgctgg?gggcaggagg
10620
cgtgtggtgg?gagcagacgc?aaccccagtg?tcaaaccagg?gggtaagtca?aggtatccgg
10680
ctcaggccgc?cgggcagctg?agggggccca?gtgggggtct?cgtctgtggc?ccagagacgt
10740
ggcggaagaa?ggcagtacat?ctcccttctt?agagagagag?tggaagcttc?tgagtgtggc
10800
ttgggtcgtt?ctgaaccatg?gtgacgtttc?caccctgcca?ctgcctgtct?tccagtttga
10860
cttgctggaa?atggaccggc?tggagaggcc?actggttgac?ctgccgctcc?tcctggaccc
10920
gccctcctac?gtgcccgaca?cggtggacct?caccgatgac?gctctggccc?gaaaatactg
10980
gctcacctgc?tttgaggagg?ccctggacgg?ggtgagggct?ccgggtgccg?tctagacgat
11040
tccaaggccg?cagccgggtt?aaccttccaa?ggctccgggg?gcaaagccaa?ggtggcgaag
11100
ggggacaggc?cagtgttgtc?cagaaacccc?ttgatctgcg?tctgagtgag?ggggcgattt
11160
tggtttctca?ggcaaaaata?ccgctcctgg?ccacctggtg?ggtcagtgag?gacgtggcgt
11220
ctgcgaggca?cgttgcttct?aagggcagct?gatagttctg?ctagctagca?cattccttca
11280
aaaagcaatt?taggagacgt?ctctgctctt?ttgaggtgtg?actgaggtgc?gggaggcatt
11340
gtgggaccct?gactctgggg?gtggggtgag?actcagcccc?agcaagctcc?cggcgaggcc
11400
accttgggac?tcctgcgtcc?caccttctcc?aaggctgggc?tggtcccgtg?gagcaaatgc
11460
caggccctgg?gccacgctgc?ccctcaggag?ctcagctcgg?ggccagggag?ccccggtagc
11520
aacacgcact?cttcacgaag?ccctcggaga?ggccgggtcg?tgccagggtt?tgtcctgagg
11580
gaggagtccc?agccctgccc?aggacccgga?gtgagctctg?agtgcccaga?gcccctcgtc
11640
cccggcacag?gatgggggct?cgagtctgag?gacgtgggtc?ttggcctcgg?gtttcctggg
11700
gggtgaggga?ccaaggtcac?tcctgctcct?gcctcccagg?gcaactggga?ggggcgaatg
11760
gcaaagggaa?gcctcgggtg?accgtgaact?gtcaccgtca?gcctttcctg?ttccagtaga
11820
aactgccctg?accacagccc?aggcagcccc?caccctgccc?gagtccccca?cagcaggccc
11880
tgaccctgcc?ccgggaagcc?ccccatagct?agcccccacc?ctgccccaaa?cagcccccga
11940
cagccggctc?ccaccctgca?ccaggcctcc?tacagccgcc?cccaccctgc?cccagacagt
12000
cccccacggc?cgacccccac?ccttccccga?gccccctgct?gtcacgtggg?gattcgcaag
12060
cagcagggcc?agcgcttgac?taaccccctc?cttctgtcgc?tggcaggtag?tgaagcgcgc
12120
agtggcgagc?cagccagact?ctgtggatgc?agccgagagg?gcggagaagt?tccggcagaa
12180
gtactggaac?aagcttcaga?ccctgaggca?gcagcccttg?taagtgccca?gcaccccggt
12240
gtgtggccac?ctcttccatc?cagagggccc?ctgcacctct?gagaaagtgc?cgtgatgttg
12300
gcattcggac?cccagctcgc?tgggggtggg?tgtgggccct?ggcggcacag?cccccttgga
12360
ccccgcctca?agcagcgttt?gcactgccgg?gctcctgagg?acgtccgtgg?cacagccgag
12420
gatggcagac?gggcgggtgt?tcggcctgcc?ctgctcagcg?ccctctgccg?tgtctgtccc
12480
cagcgcctat?gggaccctga?ccgtgcgcag?cctgctggac?accagggagc?actgtctgaa
12540
cgagttcaac?ttcccggatc?cctactccaa?agtaagtgca?gtgtgccctg?gccttccgct
12600
cgctcttgtc?cccgtcccgc?tggcaccgcg?tgtcctgtct?ctgtcccgct?ggctcggggc
12660
atcctgtcct?gtctgaggcc?atgcaggttt?ggtagcggcc?ccaactccag?ccctggcacc
12720
acaatagcca?gggcctctgt?tggcagcatc?tgctgaggag?ctgtgttacc?cccacagatg
12780
ctggggccac?ttaggggtgg?ggactgctgt?gactagagaa?gaagggtgtg?aaccccagtt
12840
tggatgtgtt?cctgacagac?acaggctgcc?tgcctgcagg?agccaagggc?ctcccagctt
12900
ctgtgagggt?gggcgattgt?ggttggggac?tgctttcccc?tcatggggag?gggctggaac
12960
cctggaccac?cctggctccc?tcctaggagg?ctggacagtg?accagaggcc?tcctatcccg
13020
gcaacgtgca?gggccgtgca?aggtgggcct?ttccgggcgt?ccgttttgct?tccccaagtc
13080
tggcggccag?agccctcctt?ggccctttct?gccaggaggc?agcgggcagc?agcggaggca
13140
tggactcctc?ccacctcttc?tgggaagggc?cagcgccatg?cctccctccc?ctcacacgcc
13200
gagccttgtg?acagcggaag?tctgggtgtc?gagggttttg?aggccagggc?tgtgggtacc
13260
tgtgggggcc?gtgtcctcca?ctcttgtcct?gcccttcacc?cactgctcag?tcacatgggg
13320
gctgaagttg?tcctgggcct?cgtcctcaga?gccccgtgca?gctgtgcgct?ggccactgtc
13380
ccggaccgat?aggcttgtgc?ctggtggggt?cccagttggg?cctgagtctt?cgatcctgag
13440
gggcgtggta?tggaacaggc?tgcgcctggc?aggtgtccag?ggtgggctga?ggccacgtct
13500
tcactcaggc?ctggcatctg?gcactctcca?caggtgaagc?agcgggagaa?tggcgtggcg
13560
ctgaggtgct?tccccggggt?cgtgcgctcc?ctggacgcyc?tgggctggga?ggaacggcag
13620
ctggcgctgg?tgaaaggcct?cctggcgggg?aatgtcttcg?actggggggc?caaagccgtg
13680
tctgcgtagg?tgtctgcggg?ctcggagcag?gctctgcagc?ctgtgggtgc?ctgtcctgcc
13740
ccaggcttgc?gggcaaggag?gtggagcggg?tcccttgggg?gtcacgtggc?gcttggtggc
13800
ttggtggctc?agggctttgg?ggcccaggga?gtgcacatgg?cagcagcgcc?aggggctggg
13860
cagggcgcag?cgcccttgtc?tgtcccctca?ggttctgagg?aggccccttc?cttaagcatg
13920
ggtgccgcag?aggcctaagg?gtgtggattt?gaaaatcggg?gctggcattt?tctattgtca
13980
gaatccaaat?tcatgtttcc?tatgtgtggt?aggaattaaa?catttctgat?gtgatgatca
14040
aagggtgctt?tgcaaagcag?tcccctagac?tggcctgtag?gacacgcagg?tgtctagcac
14100
ctggcgccgt?ctcctggggc?tctgaatctt?gaaaatgaac?ccccccacca?tcatttacct
14160
tcactcccaa?agaagctgct?tccagtctct?gggcctcccc?aggcggccgc?accacctctc
14220
gtggtccctg?ctttttttca?tgtggggctg?gagggaaatg?cctgggttct?cagccctccc
14280
tgggtcccac?tcgtgtctcg?agacatagat?gagaacgccg?cctccaacca?tggctgcccc
14340
tctgggacac?aggcccctcc?cgcagcctgt?caggagcatt?ggaggagggt?cctgacccat
14400
gggattggcc?ttgtggctgg?gggctcccca?caccgcctgc?atgggtgcca?agcacaggga
14460
ccatactgcg?gctgccagtc?accgagaggc?ttccctggcc?ccgtggctcc?gagtccacct
14520
tgagaaacag?cctgtgctct?ctcagacctt?tgcttggtgg?ccccctgagc?cttctgcgga
14580
tcgcagcatg?ttaagaacgc?tgtatctgtt?ctgcctggga?gtgatttggg?gtcttcacgt
14640
tgagcagctg?cctctgtccc?acctgggagg?gatttggggt?cttagcagct?gcctctgtcc
14700
tgcctgggag?tgatttgggg?tcttcacgtt?gagcagctgc?ccctgtccca?cctgggaggg
14760
atttgggatc?tttacattga?gcagctgcct?ctgtcccgcc?tgggagggat?ttggggtctt
14820
cacattgagc?actgtggctg?aaggtttttg?ttctgttgtc?ttaattttct?tttctcctcc
14880
tccatgcagt?gtccttgaat?ccgaccccta?ctttgggttt?gaagaagcaa?agaggaagtt
14940
acaaggtacg?gatggccaag?taacttgggg?ccttgccttg?tgctggggcc?atgcctggac
15000
gttctctgca?acagcgaatg?cagttgcggt?cggggtccag?gcacctcccc?tgctcaaggc
15060
tctgtcagct?gggcttcggg?ggcggggaga?acttgcttcc?tggagagacg?aacacctgaa
15120
gacgcgtctc?cctgacccct?gacctggagc?accgggctaa?tcatgtgtcc?ctctctctgt
15180
ctcagaaaga?ccctggctcg?tggattccta?cagcgagtgg?cttcagagat?taaaggtatg
15240
tggaggggcc?tcccacctct?gctgctgggg?acacagcggg?gcccgggggg?gtggtcactg
15300
gagggaggtg?ggcatgggct?gcctcgggtg?gccctcccct?cctgttagag?tctcaccatc
15360
ctgcgagcca?agggtctcag?catcccctgc?cccacacttg?ccctcctgtc?gtgactgctt
15420
cttaggtcac?ctgctctagg?cacaaagcct?tgccagaaga?gacaggcact?ggcccctgca
15480
ggccaatccg?tccctggcgc?ctctggggtc?ctgaagcccc?agtctcagcc?cctgcaggct
15540
ggcccgtccc?ggtgcctctg?gggtcctgaa?gtcccggtct?cagcccctgc?agcccagccc
15600
agtcccggca?cctctggggc?cctgaagccc?ctggtctcgg?cccctgcagg?acagcccatc
15660
cctggcatct?ctggggccct?gaagccccca?gtctcggccc?ctgcagggca?gcgtgacggg
15720
cagcaggggt?gcggactgac?tcgcttggat?gcgcctgttc?ccaactgaac?gtcctggttt
15780
tgtcttacag?gggccccctc?ataaatgtgc?cttaattttc?gcagataaca?gtggaataga
15840
catcattttg?ggagtcttcc?cctttgtcag?ggagctactc?cttagaggga?cagaggtgag
15900
tgttgaggtg?gtgggtgggg?cccttcccct?gggcttctct?gctctcggtg?gggttggcgc
15960
cgctgtgggc?ctctggcggt?ggtcggggct?cctccggacg?cagcaggtag?gtgcaggcag
16020
cagcacgggg?gaggcggccc?agagccgtca?tctgctgggg?tggaggctcc?gagccccctg
16080
ggctcaggct?gagggtccat?gtgctgaagc?ccagggttga?cgccaggctc?ctggaagggt
16140
gctcacagca?aggaggcttt?ggggctgtga?cttcacagac?aggcaggtcc?caggggcagg
16200
ctctaaactc?agctctgccc?ctcggctcag?ctccctccca?ggggccacag?agtggcaagg
16260
tcaggcgagg?tccctgctcc?tcagcagcag?gtggctggcc?aggcccgctg?ggcacagcac
16320
cctcacccag?cccagccact?cgaaggccgg?ggtgcacgca?ggctccatgc?tgggccagca
16380
caggttccta?aagagcctgg?gccctcctcc?tgcaggtcat?cctggcgtgc?aactcaggcc
16440
ccgccctgaa?cgacgtgacc?cacagcgagt?ccctcatcgt?ggcagagcgt?attgcgggca
16500
tggaccctgt?cgtgcagtga?gtgttggcgg?gcagtgctgg?gggggccttg?atggggaggg
16560
gcaccagctg?ggcagagtgg?gccatgggga?ttgtcactga?actgggagct?cctgggggca
16620
ctgaccacct?tgtatctggc?agctctgcgc?tccaggaaga?gaggctgctg?ctggtgcaga
16680
cgggctccag?ctccccgtgc?ctcgacctca?ggtaaccccg?caccctgcac?ctgttctctc
16740
cctcctgggc?tctgtgccct?ctgtgccgtg?cacctgcacc?caggtgcatg?cgggacaccg
16800
acctcaggac?tgcccctgcg?gcctctcctg?accccaggcc?cggattgatc?tcaggagggg
16860
acagatatgc?catccccgaa?ggtggcctgg?ggagggtgaa?tggattcagg?gtctgaggtg
16920
gttacacatg?ggctgggttt?ggggacttgg?ggcaagaggg?gccaatgtcc?ctgtggccac
16980
tcagctgaga?ccgagggcga?cctgggcagc?tgcccggtgt?ctgtcacctc?cgtgtcccac
17040
atagatgcca?ggctctgctt?ctgtggttct?ggaggtcatt?agtcaattgt?atgtggtgct
17100
gtctgtcctc?ctgattgcag?aggaggaagg?aaccccttaa?atgagcgggt?tctgagtgct
17160
ggggccgctg?gtctgctctg?cctggtggga?ttctccagtg?ctggcttcat?ctgtgcccca
17220
gccccactct?caccaacaag?gagggcgtga?aaatgacaag?gaatccatcc?ctagagttca
17280
caggagatct?agggcagagt?ttccaagctg?cagctgctct?ggccctgtgt?gagctgctgc
17340
tctgaggaag?ccccaggctg?aggcagctac?caggcggagg?ctgggtttgg?aggcctccac
17400
atcagggaat?tgagcggtag?gggtttcagc?cttcacgttg?gtcgccgcac?tgtatgggaa
17460
gtggggtctg?gggtctgctt?gcccagtctc?accgtcctct?tcctccccaa?agccgcctgg
17520
ataaggggct?ggccgcactg?gtgcgggagc?gtggcgcgga?tctggtggtc?atcgagggca
17580
tgggccgtgc?tgtccacaca?aactaccacg?cagccctgcg?ctgcgagagc?ctcaagctgg
17640
ccgtcatcaa?gaacgcgtgg?ctggccgagc?ggctgggcgg?ccggctcttc?agcgtcatct
17700
tcaagtacga?ggtcccagcc?gagtgaggcg?ctgcagctgc?cggactcttc?tgcttgtcac
17760
ttgtcaggaa?tgtgttttta?ccaccacagg?gaaactgcgt?tcaaatcaac?gtatttatat
17820
ggtactgctg?tgacgcggca?catacacccc?agccgcacag?atgcgtgtga?cccagaggcg
17880
agacgcagct?ttgtcctggg?agacgttcat?attggaatct?atttaactgc?taaagaacct
17940
tttatatata?tatatatata?aatagagaga?tctatacagg?tatgtctgac?gggacgcagc
18000
accgtgggca?cgcaccaaat?agagttttta?aaagaggccg
18040
Claims (5)
1, be used to predict the primer of diabetes B susceptibility, the special primer that is used for pcr amplification that described primer designs for the sequence based on Figure 1B or Figure 1A, described primer designs at the SNP site R shown in the figure, and length is 18-46 Nucleotide.
2, primer as claimed in claim 1, it comprises one group a kind of sequence that is selected from SEQ ID NO:1-21 composition.
3, primer as claimed in claim 2, it is the single-basic extension primer with the sequence shown in the SEQ ID NO:22 SEQ IDNO:23.
4, be used to predict the test kit of diabetes B susceptibility, it comprises each described primer as claim 1-3.
5, the PANK4 gene is used for predicting the application of the diagnostic reagent of diabetes B susceptibility in preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031369804A CN1548556A (en) | 2003-05-21 | 2003-05-21 | Reagent kit and primer for predicting susceptibility of type-II diabetes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031369804A CN1548556A (en) | 2003-05-21 | 2003-05-21 | Reagent kit and primer for predicting susceptibility of type-II diabetes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1548556A true CN1548556A (en) | 2004-11-24 |
Family
ID=34323466
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA031369804A Pending CN1548556A (en) | 2003-05-21 | 2003-05-21 | Reagent kit and primer for predicting susceptibility of type-II diabetes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1548556A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105238861A (en) * | 2015-10-16 | 2016-01-13 | 柳州市妇幼保健院 | Kit for Chinese pediatric asthma susceptibility gene SNP (single nucleotide polymorphism) genotyping and application method of kit for Chinese pediatric asthma susceptibility gene SNP genotyping |
| CN116926092A (en) * | 2022-10-28 | 2023-10-24 | 昆明理工大学 | Pantothenate kinase gene RkPank and application thereof |
-
2003
- 2003-05-21 CN CNA031369804A patent/CN1548556A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105238861A (en) * | 2015-10-16 | 2016-01-13 | 柳州市妇幼保健院 | Kit for Chinese pediatric asthma susceptibility gene SNP (single nucleotide polymorphism) genotyping and application method of kit for Chinese pediatric asthma susceptibility gene SNP genotyping |
| CN116926092A (en) * | 2022-10-28 | 2023-10-24 | 昆明理工大学 | Pantothenate kinase gene RkPank and application thereof |
| CN116926092B (en) * | 2022-10-28 | 2024-04-26 | 昆明理工大学 | Pantothenate kinase gene RkPank and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101056990A (en) | Polymorphisms in the epidermal growth factor receptor gene promoter | |
| CN1942480A (en) | Identification of SNPs associated with hyperlipidemia, dyslipidemia and defective carbohydrate metabolism | |
| CN1635142A (en) | Reagent kit for forecasting susceptibility of intolerance type dementia preaecox and primer used thereby | |
| CN1548556A (en) | Reagent kit and primer for predicting susceptibility of type-II diabetes | |
| CN1761760A (en) | Use of a novel polymorphism in the hsgk1 gene in the diagnosis of hypertonia and use of the sgk gene family in the diagnosis and therapy of the long Q/T syndrome | |
| CN1211486C (en) | A DNA molecule encoding mutant prepro-neuropeptide Y, a mutant signal peptide, and uses thereof | |
| CN1958605A (en) | SPG3A gene mutation, encoded production, and application | |
| CN1552875A (en) | Scar and hyperplastic scar differential expression gene library and its constructing method | |
| CN101033487A (en) | Method of detecting KLK1 gene rs5517 polymorphism correlated to primary hypertension | |
| CN1634963A (en) | Coronary disease related gene, its detection method and use thereof | |
| CN1177048C (en) | Human protein with function of suppressing cancer cell growth and its coding sequence | |
| CN1515686A (en) | Kit for predicting susceptibility of diabetes and its primer | |
| CN1177864C (en) | Novel human protein with expression difference in liver cancer tissue and its code sequence | |
| CN1199998C (en) | Human protein with suppression to cancer cell growth and its coding sequence | |
| CN1548555A (en) | Reagent kit and primer for predicting susceptibility of type-II diabetes | |
| CN1169954C (en) | Human protein able to suppress growth of cancer cells and its coding sequence | |
| CN1515685A (en) | Kit for predicting susceptibility of diabetes and its primer | |
| CN1209373C (en) | Human protein with suppression to cancer cell growth and its coding sequence | |
| CN1222616C (en) | Novel human protein with cancer-inhibiting function and coding sequence thereof | |
| CN1721549A (en) | The test kit and the primer of forecast China people's diabetes B susceptibility | |
| CN1199997C (en) | New human protein having mouse NIH/3T3 cell conversion promoting function and its code sequence | |
| CN1169958C (en) | Human protein able to suppress growth of cancer cells and its coding sequence | |
| CN1169955C (en) | Human protein able to suppress growth of cancer cells and its coding sequence | |
| CN1712545A (en) | Label mononucleotide polymorphic site of II receptor gene of I-type angiotonin and monosomy therefrom | |
| CN101056994A (en) | Methods for identifying a patient as a candidate for treatment with a long acting beta agonist and for predicting a patient's response to long acting beta 2 agonist therapy by analysing polymorphisms |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |