CN1515685A - Kit for predicting susceptibility of diabetes and its primer - Google Patents

Abstract

The present invention relates to a kit for predicting susceptibility of the diabetes and its used primer, in more concrete, it relates to a kit for predicting suscepatibility of diabetes which is designed by utilizing a mononucleotide polymorphism (SNP) site of newly-identified diabetes sueceptible gene UTS2 and its primer. It also relates to the application of UTS2 in preparation of diagnostic reagent for predicting susceptability of diabetes.

Description

Be used to predict the test kit and the primer of diabetes susceptibility

Technical field

The present invention relates to be used to predict the test kit and the primer of diabetes susceptibility, more specifically, the present invention relates to utilize the test kit and the primer of prediction diabetes susceptibility of a single nucleotide polymorphism (SNP) site design of the diabetes tumor susceptibility gene UTS2 of new discriminating.The invention still further relates to the application of UTS2 gene in the diagnostic reagent of preparation prediction diabetes 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.Having found some the relative more rare single-gene forms in the diabetes B, be called young late hair style diabetes (MODY), is because sudden change has caused the β cell function low.These patients fall ill to the adult is early stage in pubescence usually, show as the glucose reactivity insulin hyposecretion.5 glucose metabolism enzyme-glucokinases of encoding respectively, transcription factor HNF1 α, β, 4 α and insulin promoter factor (IPF1) are arranged in these Disease-causing genes.These transgenations make the insulin secretion moderate or significantly reduce, and finally develop into 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 seldom except the positional cloning method merely, genes involved research to disease of multifactorial inheritance also has the most frequently used method of a kind of method at present, be the candidate gene method, i.e. directly the variation of 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 one 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 obviously 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 single Nucleotide.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.Select appropriate cases and normal control, take the Position Research of the disease that caused.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 type ii diabetes (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 UTS2 gene) finally, and a SNP site relevant especially with the Han nationality in China type ii diabetes 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 diabetes 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 18-46 Nucleotide.Preferably, described primer is selected from a group that SEQ ID NO:1-10 forms, and more preferably, described primer is single-basic extension (SBE) primer with sequence shown in the SEQ ID NO:10.

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 R 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 18-46 Nucleotide.Preferably, described primer is selected from one group that SEQ ID NO:1-10 forms.According to a preferred embodiment of the present invention, primer of the present invention is single-basic extension (SBE) primer shown in the SEQ ID NO:10.

In accordance with a further aspect of the present invention, UTS2 gene (Urotesin II is provided, a kind of peptide with strong vasoconstriction function of encoding) be used for the application of the diagnostic reagent of diabetes diagnosis as the diabetes tumor susceptibility gene in preparation, the nucleotide sequence of UTS2 gene is shown in SEQ IDNO:11.

Description of drawings

Figure 1A illustrates the nucleotide sequence of the UTS2 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: lacking dNTP, existing under the situation of ddNTP, Sequenase can add a ddNTP at 3 ' end of primer, but because can not form phosphodiester bond, so can not continue to extend, promptly comes to an end after base is extended one and end.Only needing to detect this base gets final product.Claim little sequencing 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 UTS2 gene is the abbreviation of Urotesin II gene, the mature peptide that 124 amino acid of its coding are formed, and relative molecular weight is 14000, the function of this peptide is the intensive vasoconstrictor.UTS2 only expresses in cerebral tissue, and its sequence all is high conservative from the Lampetra japonica (Martens). to the mankind.This gene has been located in karyomit(e) 1p36 district.But prior art does not disclose the information or the prompting of any relation about UTS2 gene and diabetes.

Working in advance of the inventor shows, 1, all have the tumor susceptibility gene site of Han nationality in China diabetes B on 12,18 and No. 20 karyomit(e), there wherein have 4 zones (1p36,1p31,1q22 and 1q42-43) to show with diabetes B on No. 1 karyomit(e) to be linked.Shown in following embodiment, further the fine scanning result that this zone is carried out shows, wherein there are 3 zones (1p36.33-36.23,1q24.3-25.1 and 1q42.12-42.13) still to exist chain, particularly in the 1p36.33-36.23 zone, parameter linkage analysis and Non-Parametric Linkage Analysis Methods show that continuous 5 sites and disease are chain, illustrate that probably there is the tumor susceptibility gene of diabetes B in this zone.

For seeking the diabetes B tumor susceptibility gene, 23 SNP sites that the inventor adopts bioinformatics method to select to be arranged in the diabetes B candidate gene in 1p36.33-36.23 zone (derive from the NCBI snp database, www.ncbi.nlm.nih.gov/SNP), by length multiplicity single-basic extension (SBE) reaction method it is carried out somatotype and case-contrast association analysis in case group and control group.The information of SNP can be obtained from public database by bioinformatics method.There are a plurality of public databases to store millions of SNP information at present, and there are many biomedical websites to open up special SNP webpage, people can be in several ways very easily, 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.Go through as following examples, find to have 1 SNP site (R who is positioned at the rs228648 place of UTS2 gene intron in selected 23 SNP sites, being A/G) difference that is distributed in case group and the control group of its gene frequency has statistical significance (P＜0.05), point out this SNP site relevant with diabetes B, further reach a conclusion, its place gene UTS2 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 tumor susceptibility gene UTS2 gene in the prediction diabetes susceptibility.Based on the UTS2 gene, can obtain various diagnostic reagents and test kit to be used to predict diabetes susceptibility.

UTS2 place genome sequence is shown in SEQ ID NO:11, total length is 10488bp, disclosed SNP site (R) is between the 10087-10488 position of this sequence, this regional nucleotide sequence is shown in Figure 1A, R is the SNP site among the figure, it represents base A/G polymorphism, and promptly this site can be A, also can be G.This regional complementary sequence is shown in Figure 1B, and Y represents the complementary base in SNP of the present invention site among the figure, and it represents base T/C.

The invention still further relates to the primer that is used to predict diabetes susceptibility.Preferably, described primer designs based on the sequence of Figure 1B, 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 1A, and the order-checking of the product behind the pcr amplification obtains the complementary base of SNP like this.The length of primer of the present invention can be 18-46 Nucleotide.Preferably, primer of the present invention has the sequence shown in the SEQID NO:1-10, particularly preferably is the SBE primer shown in the SEQ ID NO:10.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 this primer of listing and combination thereof.

Table 1

?SEQ?ID ?NO：	Primer sequence and Tm	The product size
			?1 ?8	Justice is arranged: TGTCATTCTTCAGGGATGGC (61.0 ℃) antisense: TCTGGCAAAAGAGGCAACTT (60.0 ℃)	218bp
?1 ?9	Justice is arranged: TGTCATTCTTCAGGGATGGC (61.0 ℃) antisense: AAATCTGGCAAAAGAGGCAA (59.8 ℃)	221bp
			?2 ?8	Justice is arranged: TCATTCTTCAGGGATGGCAG (61.1 ℃) antisense: TCTGGCAAAAGAGGCAACTT (60.0 ℃)	216bp
?2 ?9	Justice is arranged: TCATTCTTCAGGGATGGCAG (61.1 ℃) antisense: AAATCTGGCAAAAGAGGCAA (59.8 ℃)	219bp
			?3 ?8	Justice is arranged: GATGGCAGCCCTAAACACAG (60.7 ℃) antisense: TCTGGCAAAAGAGGCAACTT (60.0 ℃)	204bp
?3 ?9	Justice is arranged: GATGGCAGCCCTAAACACAG (60.7 ℃) antisense: AAATCTGGCAAAAGAGGCAA (59.8 ℃)	207bp
			?4 ?8	Justice is arranged: CAGCCCTAAACACAGCATGG (61.6 ℃) antisense: TCTGGCAAAAGAGGCAACTT (60.0 ℃)	199bp
?4 ?9	Justice is arranged: CAGCCCTAAACACAGCATGG (61.6 ℃) antisense: AAATCTGGCAAAAGAGGCAA (59.8 ℃)	202bp
			?5 ?8	Justice is arranged: CCCTAAACACAGCATGGCAA (62.0 ℃) antisense: TCTGGCAAAAGAGGCAACTT (60.0 ℃)	196bp
?6 ?8	Justice is arranged: ACACAGCATGGCAACTCATC (59.7C) antisense: TCTGGCAAAAGAGGCAACTT (60.0 ℃)	190bp
			?6 ?9	Justice is arranged: ACACAGCATGGCAACTCATC (59.7 ℃) antisense: AAATCTGGCAAAAGAGGCAA (59.8 ℃)	193bp
?7 ?9	Justice is arranged: GCATGGCAACTCATCTACTCA (58.89 ℃) antisense: AAATCTGGCAAAAGAGGCAA (59.8 ℃)	188bp

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 " HumanPhysical Mapping 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 Version of ABI company 2.0 test kits, 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

D1?S463???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????????FAM????????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) collect the result, GENEHUNTER2.0 software (Massachusetts Institute Technology's hereditary system is write, from Http:// cmag.cit.nih.govOn can freely download) go up computing, calculate relevant site and chain P value, the Z value of phenotype.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 diabetes tumor susceptibility gene UTS2 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

Based on the result of embodiment 1, promptly karyomit(e) 1p36.33-36.23 zone is linked with diabetes B, utilize bioinformatics method from the NCBI snp database ( Www.ncbi.nlm.nih.gov/SNP) in select and be arranged in this zone or near 10 23 SNP sites it and be used for gene type and study with the glucose metabolism genes involved.Described 10 candidate genes are as follows:

(1) protein kinase C ζ subunit (PRKCZ) gene: be arranged in 1p36.33 district AL162271 clone.Protein kinase C is the special kinases of serine-threonine that phosphatide relies on, can be with the two phosphorylation, and this family comprises the member of 10 structurally associateds at least.

(2) 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.

(3) 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.

(4) PER3 (the proteic homologous protein 3 of [fruit bat] rhythm and pace of moving things) gene: be arranged in 1p36.23 district NT_002530.Locate to express at mammal brain, liver, skeletal muscle etc., be subjected to circadian rhythm and illumination effect, gene expression dose is periodic swinging.

(5) UTS2 (Urotensin II): be arranged in 1p36.23 district NT_002530.Its protein function is a kind of strong vasoconstrictor, and known this gene conservative property is very high.A g protein coupled receptor GPR14 can be used as its acceptor.

(6) DJ-1 (rna binding protein adjusting subunit): be arranged in 1p36.23 district NT_002163.

(7) 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.

(8) lysyl hydroxylase (PLOD) gene: be positioned among the 1p36.21 district dJ1077B9 (long 126525bp belongs to unfinished sequence).Lysyl hydroxylase is a heterodimeric protein, is incorporated on the after birth, but the hydroxylation of catalysis lysine residue, the hydroxylation Methionin group that is produced is the attachment sites of carbohydrate on collagen, this step is very important to the stability of intermolecular cross-linking.

(9) acetodehydrogenase 4 (ALDH4) gene: be arranged in 1p36 district AL080251 clone.Acetodehydrogenase is mitochondrial matrix NAD ⁺The dependent form desaturase, second step of catalysis proline(Pro) degradation step.

(10) 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.

Selected 23 SNP sites are as shown in table 6.

The SNP that table 6 1p36 district is selected by bioinformatics method

The SNP title	Gene title of living in and the position in gene		Contig of living in	The SNP type	Cytogenetic location	Physical distance (Mb) apart from the p end
							?rs262669	PRKCZ	Exon	NT_004350	C/T	1p36.33	1.215
?rs436045	PRKCZ	Intron	NT_004350	A/G	1p36.33	1.204
							?rs586965	SCNN1D	Intron	NT_025635	C/G	1p36.32	2.310
?rs609805	SCNN1D	Exon	NT_025635	A/G	1p36.32	2.311
							?ss91351	HBACH	Intron	NT_19265	C/T	1p36.23	5.931
?ss91350	HBACH	Intron	NT_019265	C/T	1p36.23	5.935
							?rs228669	PER3	Exon	NT_028054	C/T	1p36.23	7.548
?rs228691	PER3	Exon	NT_028054	A/G	1p36.23	7.559
							?rs170633	PER3	Intron	NT_028054	C/T	1p36.23	7.559
?rs228677	PER3	Exon	NT_028054	C/T	1p36.23	7.567
							?rs170629	UTS2	Intron	NT_028054	C/T	1p36.23	7.589
?rs228648	UTS2	Intron	NT_028054	C/T	1p36.23	7.592
							?rs161825	DJ-1	Exon	NT_028054	A/G	1p36.23	7.658
?rs15854	MTHFR	3 ' end	NT_004488	A/G	1p36.21	11.020
							?rs1801131	MTHFR	Exon	NT_004488	C/A	1p36.21	11.037
?rs1801133	MTHFR	Exon	NT_004488	C/T	1p36.21	11.039
							?rs15431	PLOD	Exon	NT_004488	T/G	1p36.21	11.218
?rs11740	ALDH4		NT_021902	A/G	1p36	14.900
							?rs14311	ALDH4		NT_021902	C/T	1p36	14.900
?rs9117	ALDH4		NT_021902	C/T	1p36	14.900
							?rs5259	CLCNKB	Exon	NT_004873	AA//C	1p36.13	19.302
?rs5251	CLCNKB	Exon	NT_004873	C/G	1p36.13	19.304
							?rs5254	CLCNKB	Exon	NT_004873	A/G	1p36.13	19.307

3, SNP locus gene phenotypic analysis

The main somatotype that adopts 3 kinds of methods to carry out SNP: length multiplicity single-basic extension (Length Multiplexed Single Base Extension, LM-SBE) reaction, 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 18-46 Nucleotide, 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-scalediscovery and genotyping of single-nucleotide polymorphisms in themouse.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.(Calf IntestineAlkaline Phosphatase CIP) can remove the phosphate group of dNTP 5 ' end calf intestinal alkaline phosphatase, makes it to form phosphodiester bond, thereby can not participate in the extension of DNA chain; (ExonucleaseI 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 1 μ l enzyme mixed solution, wherein contain CIP 1.5U/ μ l, ExoI 1U/ μ l, mixing is inserted 37 ℃ of water baths digestion 1.5 hours, and it is centrifugal to take out the back, and 90 ℃ of 15min deactivation CIP and ExoI 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?Secquenase????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) 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.

3) 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 normal control individuality of 48 picked at random, carry out earlier, the somatotype result is added up the back calculates each site gene frequency.Gene frequency is higher than 15% site and further in case group and control group, carries out somatotype again.The result shows that 1 (rs586965) do not amplify the PCR product in 23 candidate SNP locus; 3 (rs5251, rs15431 and rs15854) sites are detected in the individuality at all and are heterozygous; Having 7 (ss91350, ss91351, rs9117, rs5259, rs228691, rs228677 and rs170633) to be homozygous genotype of the same race in all the other 20 sites in detecting individuality, is not SNP; 4 (rs1801131, rs14311, rs5254 and rs609805) site gene frequencies are lower than 15%, do not carry out further somatotype; Carry out totally 8 in the SNP site (rs1801133, rs436045, rs228648, rs11740, rs262669, rs228669, rs170629 and rs161825) of somatotype at last, they lay respectively in 6 genes, and gene frequency is 26.3%-43.16%.

In case group and control group, the SNP site of above-mentioned 8 somatotypes all meets 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 rs228648 place of UTS2 gene intron, 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

Rs11740 case group 129 245 374 0.978

Control group 110 208 318

Amount to 239 453 692

Rs161825 case group 216 140 356 0.917

Control group 176 116 292

Amount to 392 256 648

Rs170629 case group 261 119 380 0.710

Control group 156 76 232

Amount to 417 195 612

Rs228648 case group 113 297 410 0.029

Control group 111 205 316

Amount to 224 502 726

Rs228669 case group 264 110 374 0.455

Control group 212 100 312

Amount to 476 210 686

Rs262669 case group 269 129 398 0.639

Control group 207 107 314

Amount to 476 236 712

Rs436045 case group 329 75 404 0.003

Control group 207 81 288

Amount to 536 156 692

Rs1801133 case group 224 156 380 0.607

Control group 133 101 234

Amount to 357 257 614

As above show as can be seen, there is significant difference in the gene frequency that is positioned at 1 SNP site rs228648 of UTS2 gene in case group and control group, therefore points out this site relevant with diabetes B.And then prompting UTS2 gene is a kind of new diabetes tumor susceptibility gene.

In addition, use the same method to 390 diabetes B patients, reach 118 normal control groups and equally the SNP site rs228648 of described UTS2 has been carried out somatotype separately, carry out statistical analysis, P＜0.05 has further proved above-mentioned experimental result, sees Table 8.

SNP site rs228648 allelotrope and genotype frequency and the statistical study of table 8 UTS2

Group	Number of individuals	Allelotrope	Genotype	Uncorrected	Gauged #
						Contrast case group 1 case group 2	? ?118 ? ?173 ? ?217 ?	G??????????A 122????????114 (0.517)????(0.483) 219????????315 (0.633) *?(0.367) 315????????119 (0.726) **(0.274)	The non-G/G OR 30 88 (0.254) (0.746) 63 110 1.68 (0.364) of G/G *(0.636) 111???????106???????3.07 (0.512) **(0.488)	95％CI?????P???????OR ? ? 1.01-2.88??0.049???2.07 ? 1.97-6.44??＜0.001?3.19	95％CI????P ? ? 1.16-3.72?0.014 ? 1.93-5.28?＜0.001

^*P＜0.05 compared with the control, ^*P＜0.001 compared with the control.# proofreaies and correct sex, age BMI

In the last table, the diabetic subject that 1 representative of case group is distributed, case group 2 is represented first disease person in each diabetic pedigree.Sample is carried out case-control group detect, and carry out somatotype, by statistical analysis as can be seen, case group 1 analytical results shows that P＜there were significant differences in 0.05 explanation.Case group 2 analytical resultss show P＜0.01, have illustrated than significant difference.By case group 1 and 2, results of statistical analysis to SNP site rs228648 (G-A) shows, compares with the normal control group, and there were significant differences, illustrate that rs228648 (G-A) is relevant with diabetes B, UTS2 (Urotensin II) gene is a tumor susceptibility gene of diabetes B.

The genome sequence at UTS2 place is shown in SEQ ID NO:11, total length is 10488bp, the SNP site (R) of being disclosed is between the 10087-10488 position of this gene, this regional nucleotide sequence is shown in Figure 1A, R is the SNP site among the figure, it represents base A/G polymorphism, and promptly this site can be A, also can be G.This regional complementary sequence is shown in Figure 1B, and Y represents the complementary base in described SNP site among the figure, and it represents base T/C.

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 only one.Length is between 18-46 the base, every one of 4 base design.Avoid containing too much G+C, the Tm value is between 55～80 ℃.Because the conversion hysteria person 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 '-GGCTTGGATATGAGTTGAAGTGGCCAAAGCAAAGAGAC-3 ' (SEQID NO:10), it is the same preface primer of this chain, promptly combines 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:1-9.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 table

＜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 susceptibility

<130>I20031212CB

<160>11

<170>PatentIn?version?3.1

<210>1

<211>20

<212>DNA

＜213〉artificial sequence

<400>1

tgtcattctt?cagggatggc???????????????????????????????????????????????20

<210>2

<211>20

<212>DNA

＜213〉artificial sequence

<400>2

tcattcttca?gggatggcag???????????????????????????????????????????????20

<210>3

<211>20

<212>DNA

＜213〉artificial sequence

<400>3

gatggcagcc?ctaaacacag???????????????????????????????????????????????20

<210>4

<211>20

<212>DNA

＜213〉artificial sequence

<400>4

cagccctaaa?cacagcatgg???????????????????????????????????????????????20

<210>5

<211>20

<212>DNA

＜213〉artificial sequence

<400>5

ccctaaacac?agcatggcaa???????????????????????????????????????????????20

<210>6

<211>20

<212>DNA

＜213〉artificial sequence

<400>6

acacagcatg?gcaactcatc????????????????????????????????????????????????20

<210>7

<211>21

<212>DNA

＜213〉artificial sequence

<400>7

gcatggcaac?tcatctactc?a??????????????????????????????????????????????21

<210>8

<211>20

<212>DNA

＜213〉artificial sequence

<400>8

tctggcaaaa??gaggcaactt???????????????????????????????????????????????20

<210>9

<211>20

<212>DNA

＜213〉artificial sequence

<400>9

aaatctggca?aaagaggcaa????????????????????????????????????????????????20

<210>10

<211>38

<212>DNA

＜213〉artificial sequence

<400>10

ggcttggata?tgagttgaag?tggccaaagc?aaagagac????????????????????????????38

<210>11

<211>10488

<212>DNA

<213>Homo?sapiens

<400>11

gagccccaaa?ccttagcctc?atttattttg?ttctgttaca?taagtcattt?tccccttaga????60

gtgcttgaag?aaatgccacc?tacaggttgt?gtacttttca?taatggtttc?catgaatgta????120

gtacgttcat?acaggcttca?ttcaacctgg?cgttcccctc?cataattaag?atgaaacatt????180

ccggttttct?cacaacacat?tagcacatac?tgtccattag?catatctggg?ataaccaggt????240

tttgggggtt?gagttttggc?cttcatcctt?gtagatccct?ttcctattga?tttcccacct????300

tccagtgaaa?ttctgaaagt?cttatcttaa?aaatcgatcc?gcttaccatg?ggcctattct????360

tgtaagtttc?agttagcatt?tgcatgtgta?atattaaaat?gaaagagctt?cttacccagt????420

gctgttgccc?ttttgagtat?ttttgttttt?aaaataatga?ttgtaaaatg?ttttacaagt????480

aatgtaaaag?ctagtatcat?tcttacatac?ttctgtgttt?aaattttcat?tcttaccaaa?????540

acagttaact?ctttctttcc?aatcaattta?tacaaaagag?gtcgctccag?ccctaccaca?????600

ggtctgactg?gcactgcctt?ttgtttgccc?ttgaacaggg?cagtgttgtg?gggactgcaa?????660

aagagaaaac?gtccaggcga?gcccagttgt?cctcgcccac?agggtcctgc?aggctccatc?????720

agtcaccgct?ttctatggcg?tttgtagttg?tgtcttttaa?gaagtgagtg?tgattgttta?????780

cttgataaat?cagctcactc?tctggtgctt?tttagagaag?tccctgattc?cttcttaaac?????840

ttggaatgat?agatgaaatt?cacacccctg?cagatcagaa?aaaacaaata?gaagaaaatg?????900

agggttacag?taacctgttg?tctttatata?acttgcaaca?aactaattta?tttttttttc?????960

ctttttttgt?ttttggtttt?ttatggtttt?ttaaggaaaa?tacttttctc?ctttgaagtt?????1020

ttacagcttt?ttgtaaatgc?gtcctgataa?tgattaggaa?aatcgacctt?ttcatccatg?????1080

atgaccatcc?tcatagctca?gatctccttt?caaagtagtg?gctttctgga?tggtaattcc?????1140

atcttaaggt?gtcagaacta?ttttcaaatg?ctgcctttga?cagttcttgg?aattttctga?????1200

tattaagcag?ttccatgcaa?atattcgtgt?tttataaata?gctctcatag?tctgctccat?????1260

cttgatagtt?aagtgatttc?tgaagcgttt?gtgtgtgtgt?tgatcaggtt?gtgtgatatt?????1320

tttgcttgat?agagaatcaa?atttgaaaca?attaaccagc?cagtagattg?tctgtcagtg?????1380

accttctgta?gtaataaagt?ttttgccact?gtaaataaaa?acagtatccg?tagctatcag?????1440

gatcattgcg?cactcatata?tgctaagcct?tctgttctct?aatagaagcc?tttcttttcc?????1500

attgtttctg?gatatttgta?ttatccaaat?gtgcttattt?ctttgcctta?gcacacgttt?????1560

tatggagtac?ttgttatact?aggtttgatt?tgaaactggt?gcttgtcgca?gaactgtcag?????1620

agcatgagga?gcgctcctcc?tgtgggtgga?cgcattcacg?cactcccagg?ttgcacctgc?????1680

tgctggcggt?gagcaggggg?ttcagcagct?tgaccgatgc?cccccgaggg?ggctctcccc?????1740

agcttaaact?ttgttgttta?aatttgttaa?ctttttatat?taatgactat?tgaaagtggt?????1800

aataaaaatt?tatattatag?gcttcaatgt?tttcatgaat?gttacccaaa?aagctgtgtt?????1860

ttctttggtc?agaggtcaaa?atttatgaaa?aacaaaatgc?tgtatgaatg?gaaatcattt?????1920

tgcaattgag?tgacacttca?ttgtaattca?cagtgtaaat?ttaatccaaa?ctgaaatttt?????1980

gtttcaactg?aatttgtaat?taactctgaa?tttgttttta?atcattagta?atatttcagt?????2040

tgggtatctt?tttaagtaaa?aacaacaaat?aaactctgta?catgtaaaac?gtgagaaatt?????2100

gtcctgccct?gtgtcaacaa?atctttgaat?gtgtattctg?tatcatttag?aagtgcttct?????2160

tatgatctat?tgaggggccc?tcatattagt?taaccttgtg?tttctcaggc?acattctgtg?????2220

tttgccttgg?ccaatgtgtc?tgagcaccta?ctatgtggag?gatgaagatg?caggcacttt?????2280

gaaatgtatt?ggcttattta?gcccttttac?acctagtgtt?ccattattgg?aacgctaagc?????2340

ttgtgggagc?tagggtgaac?ctgtgtacaa?gatctgactg?tgtaactggt?ggcaacaaaa????2400

tctagaaatt?aaaataggga?acaagatcct?gaagccaaca?gagcttttta?aagtttacac????2460

ctttcagacc?atttgcacct?ttcttttcta?taacaaacca?taataagagc?aggaagaaaa????2520

atagctaagt?atactttgcg?tgtatactac?tctgacaggt?gctggtctaa?gtgcttgatg????2580

tgaattattt?aatcctcaca?actgccctgt?gaaataagca?ttgtgttatt?gtcattttac????2640

agagaagtaa?actgaggcag?ggggagtcaa?gtaactggat?cagagactca?cagctggtgt????2700

gcactgggag?taggaatctg?accctagaaa?cctgatgcaa?agccccaatc?tgtatgaatc????2760

atcacctgat?acactaaaat?gagtagggac?ttcagaattc?tgcagtggaa?ttccactttt????2820

aagttatctc?tgatacagga?atagttttat?ggtgccaaca?ttgtgttcca?gagaactggt????2880

ttaatttcaa?cactgagaga?cacaccacag?tgttatcata?gctgtcctca?atgttagcat????2940

gtttctgagc?ttgcacattc?attccagttt?aaaacatctc?tcccttagat?ttgttcttga????3000

tgtctttttt?tttttttttt?tttttttgag?acggagcctc?gctctgttgc?caggctggag????3060

ggcagtggtg?caatctccgc?tcactgcaag?ctccacctcc?tgggttcacg?ctattctcct????3120

gcctcagcct?cccgagtagc?tgggactaca?agcccgccac?catgcccggc?taattttttt????3180

gtatttttag?tagagacgag?gtttcaccat?gttagccagg?atggtctcga?tctcctgacc????3240

tcgtgatcca?cccgcctcag?cctcccaaag?tgctgggatt?gcaggcttga?gccaccccac????3300

ccggccaagt?tcttgatgtc?tttagggtaa?acgctctgag?acctgtactc?tctaggaaac????3360

ttgtgatggt?cagtgacaag?attgacacaa?gtcgtgtgtt?gctctttggt?tgttgcagac????3420

ccctctggaa?tgtggtggga?tgtcagagct?caatgagaga?aacaagtttc?tcataaacat????3480

ccaggcctcc?cctatacatg?gttctgtgta?gaaccacaca?gaactctcag?gagttcacac????3540

tgtatattct?atacatacat?aaaccacatt?tatacagatg?tattcacgga?atagaagtca????3600

gtacttacac?acatagtttt?taaaaattcg?taagccaggc?tctttcttcc?acaatcttaa????3660

atcttgcctc?agctttcttc?tctctgcaag?agacatcaag?gtgtttttcc?cggtgtaaga????3720

aactgtgatc?cttgagctca?cagacgctga?gatcatacaa?gtttcaatgt?taaaagatta????3780

aaattgtcaa?gtgttcagga?ctttggtcag?ccaagagaaa?gaatctcagc?acatgaactt????3840

cccccacgaa?cttcagaaac?caaggagcat?cagacactcc?cgcccttcat?ctcagacacc????3900

cgtaactaga?gattttttcc?aagccaagat?actgttttct?ttttattttg?ttttatttta????3960

ttttttattt?tatttatttt?attgtttatt?ttcaagacac?tcttcctctg?ttgcccaggc????4020

tggagtgtag?tggtccgatc?tcggctcact?gcaagctcca?ccccaaaggt?ctgagcgatt????4080

ctcgtgcctc?agcctcctga?gcagttggga?ctacaggcac?ctgccaccat?gccctgctaa????4140

tttttgtatt?tttggtagag?acagggtttt?gtcatgttcc?caggatgatc?tcaaactcct????4200

ggtctcaagt?gatccaccca?tttcagcttc?ccaaagggct?aggattacag?gcgtgagcta????4260

ctgtgcctgg?ctgtgattta?ttttttaaaa?gtaatatgtt?gtctttttaa?aaggttgaga????4320

ggacatcata?gctaagataa?tgtagtttat?ttaaatgatt?tgccatttaa?caaaagttta????4380

acttttgtga?ctttttctta?gtgtttgctt?ctgttcattc?attagctttg?cttctgaaat????4440

gtcaaagaat?tttctaagga?taaatggaag?aaaagaataa?ttataaattt?tcaggacatg????4500

tttataggga?aaataaccac?tcatgatatg?tgcaaaacat?agaggattta?ttttccaggt????4560

aacaatgaac?agggtgtagt?ttgcctagtt?tttctccaca?ctgttttcaa?atcaagcatt????4620

gtgttatttt?tcatattcta?agatgggtgt?ttctgagctg?actaacagat?gcttatttca????4680

cttcagacac?agtatttcca?gaagcaatca?ggagtctcac?gtttcttgta?tggtttccag????4740

attctggcca?aaagatgact?cagtaaaatg?ttaggatctt?gtccagagaa?atcctgaaac????4800

taaaacaatc?caaacgaaca?acaacaacaa?aaaaaaacag?ataagttacc?aacgagtgac????4860

gattcctata?agaaaaagga?atcttgagtc?aagacactgg?acttcttaaa?agtatttgca????4920

tttttccctg?gactataatt?gtaacaagag?tgtcccgggc?ccgtggggct?cctgtgccca????4980

cctccctacc?tctaatgagt?gtccttccag?tctgttcagc?ccagcgattg?tcctgaattt????5040

tcctaaaccc?tcattgaccg?atttctattt?aaatcatttc?agcggctggg?caggtggctc????5100

atgcctataa?tcctagcact?ttaggaggct?gaggtgggca?gatcacttga?gcctaagagt????5160

ttgagaccag?cctgggcaac?atgatgaaac?ctcgtctcta?caaaaattag?ctgggtgtgg????5220

tggcgggctc?ctgtagtccc?agctactcgg?gaggctgagg?cattagaatt?gcttgaacct????5280

gggaggtgga?tgttgcagtg?agccaagatc?acaccactgc?actccagcct?gggtaacaga????5340

gcaagaccct?gtctcaataa?ataaataaat?aaatagtaac?atgttctgtg?catgtattgt????5400

cttctgtgta?ttgtatgtat?tgtgtatact?gtattgtatt?gtattgtatt?cttttgagat????5460

ggagttttgc?tctgttcccc?gggctggagt?gaagtagtgc?gatctcctct?cactgcaacc????5520

cccgcctcca?ggttcaagtg?atcctcctgc?accagcctcc?cgaatagctg?ggattacagg????5580

tgcatgccac?cacgcctggc?taagttttgt?attttttgta?gagacggggt?ttcgctgtgt????5640

tgcccaggct?ggtcttgaac?tcctggcctc?aagcaatccg?cctacctcgg?cctcccgtgc????5700

tgggatgaca?agtgtgagcc?accgcacccg?gccttctgta?catcgttcag?gtcaatgtct????5760

ggagcatggc?cttcctccat?tagatgttgt?cttgcagtgc?ccttcacact?ttcccatgca????5820

tgtgaatcac?ctggggatct?tgttaagatg?cagattttgc?ttccgcaggt?cctaggtggg????5880

cctaagatcc?tgcatccctc?actggcttcc?aggtgctccc?ggtgctgctg?gcggtgggcc????5940

gcatgttaga?tggtgaatga?gagcacaagt?ggatgccaga?ctggagagca?gggacctcac????6000

ccattttttt?cattctttca?caacttattt?gatttaccca?aaacaggcac?tgggtaggtt????6060

cctgttgcat?tcagatctta?tatcaatcaa?aatttggaaa?aattaatgct?ttgaaataat????6120

gctggccaat?tattattatt?attattttga?gatgaagtct?cattcttgtc?ccccaggctg????6180

gagtgcagtg?gcgcgatctc?agctcactgc?aacctctgcc?tcccgggttc?aagcgattct????6240

cctgcctcag?tctcccgagt?agctgggatt?acaggtgcca?ccacgcccag?ctaatttttg????6300

tatttttagt?agagacaggg?tttcaccatg?ttggccaggc?tggtctcgaa?cttctgacct????6360

caggtggtcc?gcccgcctcg?gcctcccaaa?gtgctgggat?cacaggcatg?agctaccgcg????6420

cctggtcatg?ctggccaatt?attttaaggt?gtggtttcca?ccaagaacac?tcctctagtt????6480

catgaattca?gagtcctgta?aaaccagtac?agaatgttca?cctttttaaa?cctaactcat????6540

aaatagagtc?acttaccttt?ctcaaatttc?ctcttgggtt?aaaaatgttg?gtacttgagt????6600

ctgaaaaaca?gttttgaagc?cagttcatca?gatctgttgt?ttctcttgta?tgcttgtttt????6660

aaaatataca?aaataatatt?actaaattat?gtctagtttc?tggtcttttt?ccacactgca????6720

gcaagctttt?cttgaatgca?gacagacagg?cactcctgtt?cttatctcga?attcacagaa????6780

gtcactgcat?tgtcctcagt?aacttcgcag?tccagtagct?cgtcaaagat?attttataac????6840

aaatgaactc?ctgtctatta?aacaatacct?atcttaaaag?gctcaaattt?tttttttttt????6900

ttttgagaca?gagtcttgct?ctgttgccca?ggctggagtg?cagtggtgag?atcttggctc????6960

accacaacct?ctgcctcctg?ggttcaagca?attctcctgc?ctcagcctcc?ctagtagctg????7020

ggattacagg?tgcccgccac?catgcccggc?tgatatttgt?atttttagtc?gagatgggtt????7080

ttcaccatgt?tggccaggct?ggtcctaaac?tcctgacttc?aagtgatccg?cccacctcag????7140

cctcccaaag?tgctgggatt?acaggtgtga?gccaccgcac?ccagcaaaag?gctcaatttt????7200

gtttccagaa?cttcctgagc?atatgtgtca?atgcttgggg?gaatgtggcc?acgggggaat????7260

aagctgacat?tttattaatg?actttctgtg?tgactcgagc?attttacttg?ccttgactca????7320

ttgactcttc?acaagttacc?ttctgaggtg?gaaacttaat?cattcctgat?tacagattaa????7380

gaaactggag?tacagaaaga?ttactttaag?ttttcagtaa?acgggaaact?aggattttaa????7440

attgcactca?ctggaaactg?gaataattac?caccaagctc?taccccgcaa?aaataaaagt????7500

ccacacagaa?agacaaaatg?ctgaggtacc?aaagagcaca?gtgcgagcaa?ggagctgctg????7560

gggaaaagaa?ggttgcacag?gccgggaggg?gagagtgttg?gtaattactt?attttatata????7620

gctcatcttt?atattccaaa?atagagcttt?acctcttcta?gatgaggaca?gacggtaagt????7680

tcagtagcaa?ttaaatcaga?cacgctataa?acatgagaag?cattttacct?gctttcctga????7740

gaatatcccc?tctttctgca?cccagcatct?ctggcagtat?ctgtagaagg?gaagctcttt????7800

ctagctcctc?cggagttaag?cgcgcgtctt?catgaggtgc?tacagagtaa?aaacagatac????7860

ttagaattgg?gttcatcctt?cagttagtta?tttctgttcc?ttgtgtcttt?ggcgagcacc????7920

agagataggg?agatgaacac?tagaaaaact?tccaacgctg?agttcatgac?gtgtcatcag????7980

tggtattcta?gaagaggtgg?aaatgctacc?agcccgtgtg?ccagccagca?gaatgggaag????8040

ccgggagacg?tggatctgac?acccgcgtga?tgagccaagc?ccctcggctg?cctcgaacaa????8100

gagctgaggg?ataagccaca?gcctttctca?aaaccatttt?ggttcaaaaa?gtctgactct????8160

ccctcatttc?tgcttatcta?ctgctgccta?ctgccttttt?gcttttagat?atttgggtgg????8220

tgacattgga?gaggaaaaag?aaagatcgtt?tacaatcttc?ctataaagcc?agtaggatgg????8280

tatcacctct?tcttttactg?aacttcaaga?tttcaataca?attattatac?ttagaattgg????8340

cttcaaaatc?atctgtaagg?aacttgattg?tggcatttcc?tatgtgaaat?gtggtcgacc????8400

agatggcata?tagaatgcat?ggtaagtgcg?cttaaggtgt?gtggaaaggg?cacaccggag????8460

tgtgaggcgt?ggtaagtgca?gacctgctgc?tgtgttgcga?ttcgaggctg?tgggagtcag????8520

ggtgggaggc?catgtgtggg?accagcgtgc?cacaggctta?caaatagctg?caaccttgat????8580

tggaggacag?tgagcagatc?atcataaagg?catcagagca?cattcacagc?acttctaaag????8640

actgttaatg?aaaatctata?aaatttcatc?atctgtaaat?gctgatagga?aagcacattt????8700

ctggagaaat?aaatgagaac?aaattctgcg?ataagctgcc?acttgtttcg?aatttgtgtg????8760

agatgctaca?ctcatttcat?ctatgtttct?taaattactt?gtaaaatttt?ttaagatgac????8820

agtagtagct?gagtattttc?acttatttca?tcttgagatg?tgtgctgttt?ctttgtaaat????8880

agcagagatt?cagtaaagtt?aatgccagtg?tttaactatt?agtaggacag?ccaatgatca????8940

tctcggtgac?tcgggaagtt?ggttggcagt?gcctgatgac?agtgcttgac?gatcatcaga????9000

aagctgcttg?ggatgaactt?ttagagacga?gaaactataa?aatccttggc?ttaaaagagg????9060

gtttgtttgt?tttttttctt?tatttaaaaa?aagcctattt?aaaaagtact?caagattaaa????9120

gataagatta?aatctagtta?agtttttgat?ggaacctaat?ccccaagcat?tagctttctc????9180

atttctctaa?ctaaatggaa?agataatggc?aaaatgagaa?actacttgac?tgatgtgtat????9240

ttctttaact?caacttagac?ttaaactgaa?ttaacataaa?tcaaaaggaa?acgatcaata????9300

tacttggaag?tctatgaact?ccctagttcc?ctagtttgtc?atttactaaa?aaatttttag????9360

taaacatttt?tgtttcgtag?aacacatatc?agatgatttt?tttaaacaaa?aggtgaaaca????9420

aataaatcca?ttaataatct?ataattagaa?caaccagtgc?tacattcaac?cagttcattt????9480

aaagtaaaaa?ctaaaagtaa?tgatgtttta?aatttcatgc?aatttttaaa?attggaaaaa????9540

tgtatatatt?acccaaaaca?agagtacaga?ggtaacatga?aacaagcaat?tatcctcttc????9600

tccttttaca?ttgtagagac?agaaaaaaaa?ctgtatccat?tactacatct?aaaatgtgca????9660

gtaaggatcc?tttaatattc?ttttcaaagc?tgggactatt?gagagaacaa?gactccagac????9720

tccttcgagc?aggaatccca?ttcttatctc?taacattgaa?tttaagacta?gtaaaggctc????9780

tttcaagact?aacataaggg?gaaaaaaaaa?atcttacctg?agagttgaaa?ggatatttcc????9840

ctggagtcaa?ggagaggaag?agataagaga?ggatttaaga?atcctatgaa?aagcaaacag????9900

caggaggcca?gcttatacat?gatcgccaca?agatagacgg?cttccttctt?ggcttctgtt??9960

gtagagaact?ttcaactgtc?tcctcattct?gcctgctcac?tgattatata?tatcatccta??10020

tgacctgaca?tcatcctctc?caaaaaaaaa?aaaaaaatga?atttattggc?tatggaggct??10080

aaaaggcaat?cactttgcat?tgatgtaatt?tttcaaggta?acaagtcata?gacaataaat??10140

taactgtctt?gttgcctagt?gtacaaagta?ggtattatat?aaagaacagt?gagtttataa??10200

atctggcaaa?agaggcaact?tacagcaata?aatcatgaat?taaaggaagg?cttggatatg??10260

agttgaagtg?gccaaagcaa?agagacgtgg?atttatgagt?ccgagcagaa?gtgacgcaga??10320

gcataagatg?aaatacgttg?gtttccattt?tttaatcttt?catgagtgag?tagatgagtt??10380

gccatgctgt?gtttagggct?gccatccctg?aagaatgaca?atctgtggaa?ttaaaacgta??10440

aaaccagcta?tttcctttaa?aacagtgaaa?agttaaaata?catagcaa???????????????10488

Claims (5)

1, be used to predict the primer of diabetes 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:1-10 composition.

3, primer as claimed in claim 2, it is the single-basic extension primer with the sequence shown in the SEQ ID NO:10.

4, be used to predict the test kit of diabetes susceptibility, it comprises each described primer as claim 1-3.

5, the UTS2 gene is used for predicting the application of the diagnostic reagent of diabetes susceptibility in preparation.

Images