CN101054412A - Pathogenic gene for far end arthrosis bend, detection method and application thereof - Google Patents

Abstract

The invention relates to TNNI2 gene, more specificly a far end arthrogryposis pathogenic gene and its detection method and uses. The gene possesses the amino acid sequence of SEQ ID No:1. The pathogenic gene mutation of genetic 2B type far end arthrogryposis is firstly discovered in seven generation family line in which troponin subunit I2 (TNNI2) the eigth exon 175 point codon AAG for coding lysine is deleted. The present invention also relates to disease diagnosis kit which is convenient and quick , highly effective.

Description

A kind of pathogenic gene for far end arthrosis bend and detection method thereof and application

Technical field

The present invention relates to the TNNI2 gene, specifically a kind of pathogenic gene for far end arthrosis bend and detection method and application.

Background technology

The function of TNNI2 gene coded protein: human myofilament is mainly by actotropomyosin (Tropomysin, TMP), troponin (Troponin, TNN) and Actin muscle (Actin) form, TPM-TNN-Actin mixture and actomyosin (Myosin) coordinative role is controlled the contraction and the diastole of muscle.(document 1.Sung SS, Brassington AME, Grannatt K, Rutherford A, WhitbyFG., Krakowiak PA, Jorde LB, Carey JC, Bamshad M.Mutations in genesencoding fast-twitch contractile proteins cause distal arthrogryposis syndromes.Am.J.Hum.Genet, 2003,72:681-690.), the troponin (Troponin of human body, TNN) spherical in shape, by TNNI, TNNT, three subunits of TNNC form, and TNNC has the binding site of the two negative charges of some bands, thereby the Ca to occurring in the sarcoplasm ²⁺Very big avidity is arranged, and the effect of TNNT is that whole troponin molecule is incorporated into TPM, TNNI with TNNC in conjunction with Ca ²⁺Information pass to TPM, cause the conformational change of TPM, remove it to the inhibition that muscle fibrin and cross-bridges mutually combine, make Muscle contraction.The mixture of TNN is Ca in the skeletal muscle ²⁺Transmitter and setter, TNNI stops Muscle contraction by stoping the moving actomyosin ATP enzyme of flesh to activate, therefore the carboxy moiety of amino-acid residue has determined Ca at least ²⁺Susceptibility.When the aminoacid deletion of forming TNNI2, replacement, changed Ca ²⁺Susceptibility, influence the contraction of muscle.(document 2.Stefancsik R, Randall JD, Mao C, Sarkar S.Structure and sequence of the human fast skeletaltroponin T (TNNT3) gene:insight into the evolution of the gene and the origin ofthe developmentally regulated isoforms.Comp.Funct.Genomics, 2003,4 (6): 609-625.)

The TNNI2 gene is crossed over 4KB, is made up of 8 exons, and exon length does not wait from 4～222bp, the long 701bp of mRNA, 182 amino acid of encoding.

8 exons, 422-425,1067-1096,1241-1247,1795-1836,1920-2048,2211-2300,2423-2599,2845-3066; 7 introns, 426-1065,1070-1240,1248-1794,1837-1919,2049-2210,2301-2422,2560-2844.

The TNNI2 structure of far end arthrosis bend family pathogenic mutation gene (underscore partly is an exon):

GCTGACCTCGGGTGAGATGGGCTGAGGCGAGGTGTGGATACTCGCAGCTACCCCTCAGCTGACCCGAGCTGTGTG

CCCGGCTGAGGCCACAGGCAAAGCCAGGCACACTGTCCTCAGGCTCCTTACGAGAACGACAGAGGCATCTCCAGC

GCGTCACCGAGCCCTAAATAGAGTAGCCCAGCCACGGCACCCCCCACCAAGACTTCTTGGACTGGGCGGCAGCAC

GCGGCCAGGCCAGGCGGCCGGACAGGTGGGGAGGTCTCTGTGGCTCTCCACGCCCCCATTGGTCTGAGGAGGACT

CTATGCCCTTTCTGAGCAGGGGCCCAGCCTGGGGGAGGCCATTTATACCCCTCCCCCTGGGCCCACCAGCCCAAC

TCGCCGCTGCCGGCCTGACCTCGCTCCCAGCCCTGCTGCCCAGATT CTAGGTGAGGCCCAGCCCGGCCCGCCGAG

GCCGGGGGACAGGGCGTGGCTCGAGCTGGTTTGAGGGAGGACTTCCTGGGGCGGGGGTCTGGGGGCCTGGGGGAT

GCCACCAGCAGCCCCCTTTGGGGCCTGCACGGGTCCGGGAGTGAGAAGGAGAGGGTTTGGGGAGGCAGGTGTAGC

GAGAGGCAGGTATTAGACACTCCCTGACCACCAGGCTTCACCTCTGGGGACCCTGGAAGGAGAGTTTAGGGGTCA

AGAAGCCCCAGAACCTGAGCCCCCAGGCCAAACTGAGGAGGGATTTCTTCCATGCACTTAGGCCCAAAGCCAAAG

AGAGAGGGTTAAAAATAACAGCGTCACTCAGGCGGCCACCTGCGCTGGCCCATCCCACCCTCCCTCGGGGACAGC

TGCAGCTCCTCAGGCTATGCCTGGGACATTTTGGGAACACTTTCTCCTCTTACTTCTCACCCTGGGGAATTCCAA

GACATTGTCCTTGAAGGAGGTGAGAGTAGGGGGAGGAGGTGAGAGTAGGGGGTGGGCGGGAGGGGGCTGTCATCA

GGAGCCCTGAACCCCTCACCACCTACCTGATGGGCACAGGCATCACGGTGGCAAGGGCCTGGCCAACACCTCTGT

CTTCCTCTCCCCACAG GCTCCAAGCTCAGGACCTCAGGATGGGAGAGTAAGTGGTACCCCTGTACCCCCATACAG

TGACCCTGCCCACCTCCTGCCCTGTCCACCCCATCACACACTCCGACCCCGCCAGCCATGGCCCTAACCTCTGTC

TTCTCTCCCCGTCCTGCCTGCGTCCTCCCTCCCTGGACAG TGAGGAGGTAAGTAGTTGCTGGGGGCTCAAAACAT

GACGAGGAGGGGGCTCCCCCAACTCAGCATAGGTCATAGGTCACAGCCTCAAGGCCCTAAGGCCCAGAGAAGATG

GCCTGGCCCTCCCCGCTGGCAAAAGTGCCCCACCCAACCACCAAGACGCTGCTGAAAGAGAGGAAGTGGCTGCCC

CAAGCCTTCTGGGGCAGGGGAAGTGTGGCTGTGGCCTGGTCACAGGGGAATCACTTCTTCTTTCTGATTCCTGCA

CTTCCCGCCCCCACCTCACCGGCCGACCTGCCCCCAACTGGCCCTCCTCTCCCCCAGCCTCTGGCCTCCCAGCAC

CATGTGCCACCTGGCAAAGTGTCACACACCACACAGCACCATGTCCCAGTGCAAGGTCTGGGGCCAGGGAGGCCC

AGCCTGCCCATCATGGCCTCAGGAGCCCCCAGGCTCTGCTGGTCCCGGAGCCGGAGCCCCTGACCTGGCCAGGGA

GCGTGGAAGGGGGTGGGGGTGCTCCAGGCCTGGAGGCCCTGACTCGACCCCCTGTCCCCTGCCCTGCAG AAGCGG

AACAGGGCCATCACGGCCCGCAGGCAGCACCTGAAGGTAGGTGTGGGCTCCCGGGGGGGTGGCCCAGGTGGGTCT

GCAGGGGAGCTGGCTGCAGCCCCTCACCGCCTGCCCCACCGCAG AGCGTGATGCTGCAGATAGCGGCCACGGAGC

TGGAGAAGGAGGAGAGCCGCCGTGAGGCAGAGAAGCAGAACTACCTGGCGGAGCACTGCCCGCCGCTGCATATCC

CGGGCTCCATGTCTGAAGTGCAGGTACCAGCCCCTCCCCGGCCACCCCGCCTCCCCAGCAGCAGGCCTGCCTGCT

AACTCAGTTTCCCCACTTGTCAAGAGGGCCAGTGGGGTGGTCAGGGCAGGGGCAGGTGACCGTGGCTGCCAAGTG

TCAGGACGGCCGCCCGCCCCCACACCCACCCCTAG GAGCTCTGCAAACAGCTGCACGCCAAGATCGATGCGGCTG

AAGAGGAGAAGTACGACATGGAGGTGAGGGTGCAGAAGACCAGCAAGGAGGTGAGTGGTGGCGGCGGGCCGGCGG

CAGGTGGGTAGGCGGTGGCCCAGCGGGCAGGCGGGGCGGGCCGGGGAGGCCGAGACCACCGGGACCTCGGGCTCC

CACCCGGCTCCCCTGCCCACAG CTGGAGGACATGAACCAGAAGCTATTTGATCTGCGGGGCAAGTTCAAGCGGCC

CCCACTGCGGAGGGTGCGCATGTCGGCCGATGCCATGCTCAAGGCCCTGCTGGGCTCGAAGCACAAGGTGTGCAT

GGACCTGAGGGCCAACCTGAAGCAGGTCAAGAAGGAGGACACAGAGAAGGTGCGTGCCACGGGGGGAGCACCACC

ACACCTACCCTGCCGGGGAAGCACCTCCCACACCTGCCCCGCCTGGGACCACCTCCCACACCTGCCTGCCGGGGC

CCTGTACCACTGCCCCTCCAGGGTGCCATGCAGGGGACACTCCACTGCCCAATGCAGAGGGTAAACTGAGGCTGA

AGGTGGTGTGGACCAGGGTGCGTGCATAAGTGGGTGAGCCTGAGCTCTCTCCTGCCCTCTCCTCCACAG GAGCGG

GACCTGCGAGACGTGGGTGACTGGAGGAAGAACATCGAGGAGAAGTCTGGCATGGAGGGCCGGAAGAAGATGTTT

GAGTCCGAGTCCTAGGCCACTCGCTGCCCCTACGCCTGCCCCGGTGCCCGGCTCCCAGCAGAACATACTAGGGAG

ATGCACCCAGAGCCTGCCAGGGAGGGCTGGCCTCACCACCACCGTCAATAAAGGATTTGAATCCCCATGGCTGGT

CTGGTCTGGCTCTCCCCAGCCTCTTGGGCCATGCTCTGGGCCCCCGCATCGGTGGCCTGCAGTGTGGTCAGTGGC

CAGCGGGGAAAGCCTGGGAGGAGGCCACACGGGGCCTGGGATTTCAGGTTGGGAGGGAAGCTGCTTCCAGCAAGG

AGGTGAGCCTGGGAAGGCCCCTACAGGGGAATCCACCCCAGGCTGCACGGGGCTGTTTGGTGAGGGCCCTGATAG

TGCCCCAGGCGTTCCTGGGGCTCGGCCTGGGCACATCCAACATGCAGGGCTGGGGATGGAGGCTGTGGGACCGAG

CCAGGTCTGGGTGGGAGTTCCTGAGATAAGTGGGTCTCACATTTGCACAGCCACGCAAAGGGCCTCCCGGGGCTC

CAGAGAGATCCACACATGTGACACACGTGTGGCCCCCGCAGTGCTGGGGGCAGCCCCCTATGCCTCCCAAGCTGC

CCCTCCTCAGGGAGCTCATCTGAGGCAGGAAGCCAAGGGCCACATTAGACCCCAGGCTGCTGTCCACGCGTGCCC

ACCCCCAGATCCTCCTGCTCATCCCCCTCCCACATCACTTTGGGACAGACCCCCAGCCCCGGGGCCTCCCTGCTT

TTCCTATCTGCCCCCTGCCCTGAGCACTGAGGCTGGGCACGGGGAGCGTGGAGGGGCGAGCCTGCACGGTGGGCC

ATGACCAGGCCAAATTGGCAGTCTCCGGGCTGGGGACCAGCATGGTGCTGGGGGTTGGCCAGCCTGGTCTCTGGT

CCTTACTGCCACCCTGAAAGGCAGGCTGTGTCACTCCTCACCATGCCAGGGGACCAAGAGACCTGTGCCCATCAG

GCAGGGGCTAGGCCTGGCCCTGACCTGGCTCCAGTGTGGCTCCCAAGTCTCGTTTCCACTCAGCCCCAAGACCCA

GCCCCATCTGGCCACCAAGGACAAGGCAGCTGACAGGTTCTGACAAGGCCGGTGACAGCAAGGCCACGTTATCAG

CCCTGCTCTTTGAAGGCTGACAGAGCAGGCACGCCAAGGTCCCTGTGCTCAGATCTGCGCTGGGGAGGCTGCCAG

CCTGCGCTGTGCCCTTGCCCCCGGGAGGTCCCATCGAT

Amino acid sequence coded (underscore partly is the 8th exons coding amino acid): MGDEEKRNRAITARRQHLKSVMLQIAATELEKEESRREAEKQNYLAEHCPPLHIPG SMSEVQELCKQLHAKIDAAEEEKYDMEVRVQKTSKELEDMNQKLFDLRGKFKRPPL RRVRMSADAMLKALLGSKHKVCMDLRANLKQVKKEDTE KERDLRDVGDWRKNIEEKSGMEGRKKMFESES

Summary of the invention

The object of the present invention is to provide a kind of pathogenic gene for far end arthrosis bend and detection method thereof and application.The applicant has found morbific transgenation first in the big family of 2B type far end arthrosis bend of one 7 generation heredity of China, 75 codings of troponin subunit I 2 (TNNI2) the 8th exons 1 Methionin codon AAG disappearance, and this sports the world and finds first.Simultaneously at such disease development diagnostic kit, this kit method is easy, quick, price is cheap, efficient, is suitable for promoting in scientific research and medical institutions.

For achieving the above object, the technical solution used in the present invention is:

A kind of pathogenic gene for far end arthrosis bend has aminoacid sequence among the sequence table SEQ ID No:1.

The detection method of described pathogenic gene for far end arthrosis bend, use nested PCR method and detect:

1) extracts person under inspection's blood template DNA;

2) design of primers and reaction conditions:

First round PCR:

Forward primer: TNNI2-1F:5 '-GGTGACCGTGGCTGCCAAGTGTC-3 '

Reverse primer: TNNI2-1R:5 '-AGCTCAGGCTCACCCACTTATGCA-3 ';

Reaction system: be total to 20ul, person under inspection's template DNA 1ng, 10 * buffer (damping fluid) 2ul/ pipe, Taqase (heat-resisting polymerase): 1U/ pipe, 2.5mMdNTP:2ul/ pipe, sterilized water: 14ul/ pipe;

With the aforesaid liquid mixing, moment is centrifugal, increases on the pcr amplification instrument, and amplification condition is: 95 ℃, and 4min; 94 ℃ of 40sec, 61 ℃ of 1min, 72 ℃ of 45sec, 35cycles, 72 ℃ of 8min; Separate the purpose band with the PAGE gel electrophoresis on electrophoresis apparatus, the purpose band is 885bp or 883bp, and normal people is 885bp sheet segment length, is 883bp sheet segment length unusually;

Second takes turns PCR: primer sequence: 5 '-TGGGTGACTGGAGGAAGAACAT-3 '

5’-AGCTCAGGCTCACCCACTTATGCA-3’

Reaction system: be total to 20ul, person under inspection first round PCR product is template DNA 10ng, 10 * buffer2ul/ pipe, Taqase:1U/ pipe, 2.5mMdNTP:2ul/ pipe, sterilized water: 14ul/ pipe;

With the aforesaid liquid mixing, moment is centrifugal, increases on the pcr amplification instrument, and amplification condition is: 95 ℃, and 4min; 94 ℃ of 40sec, 60 ℃ of 40sec, 72 ℃ of 45sec, 35cycles, 72 ℃ of 8min; On electrophoresis apparatus, separate the purpose band with the PAGE gel electrophoresis, the normal artificial 143bp band of purpose strap section, the patient is two band 140/143bp.

Described pathogenic gene for far end arthrosis bend is used to prepare the reagent or the test kit of diagnosis far end arthrosis bend 2B type, DAS, the crooked deformity of brothers, equinvoarus or talipes equinovalgus disease.

1. the applicant has found morbific transgenation first in the big family of 2B type far end arthrosis bend of one 7 generation heredity of China, 75 codings of troponin subunit I 2 (TNNI2) the 8th exons 1 Methionin codon AAG disappearance, this sports the world and finds first.

2. the applicant develops diagnostic kit at such disease, and this kit method is easy, quick, price is cheap, efficient, is widely used, and is suitable for promoting in scientific research and medical institutions.Can diagnose brothers' deformity disease easily and accurately.

3. brothers' deformity sickness rate height in the crowd, many so far causes of disease do not have clear and definite diagnosis.The applicant is in the far end arthrosis bend disease that China finds first, will definitely raise the secret of brothers' deformity disease, for the research of this disease provides methodological help.

Embodiment

Embodiment 1

Normal the 8th exon sequence:

GAGCGGGACCTGCGAGACGTGGGTGACTGGAGGAAGAACATCGAGGAGAAGTCTGG CATGGAGGGCCGG AAGAAGATGTTTGAGTCCGAGTCCTAG wherein is with underscore partly to be deletion segment;

A kind of pathogenic gene for far end arthrosis bend has aminoacid sequence among the sequence table SEQ ID No:1.

The 8th exon SEQ ID No:1 sequence that the present invention has suddenlyd change is:

GAGCGGGACCTGCGAGACGTGGGTGACTGGAGGAAGAACATCGAGGAGAAGTCTGGCATGGAGGGCCGGAAGATGTTTGAGTCCGAGTCCTAG

(1) information of SEQ ID No:1 (referring to sequence table)

(A) sequence signature

* length: 93 base pairs

* type: nucleic acid

* chain: two strands

* topological framework: linearity

(b) molecule type: DNA

(C) suppose: not

(d) antisense: not

(e) initial source: troponin subunit I 2 (TNNI2) the 8th exon

(f) characteristic key words relevant: be " mutation " with nucleotide sequence

(2) preparation of gene:

Forward primer: 5 '-CTACAAGGAAAGCTGGGTC-3 ',

Reverse primer: 5 '-AAGGGCTGGAAGAGGTGG-3 '

Reaction system: be total to 20ul, person under inspection's template DNA 1ng, 10 * buffer 2ul/ pipe, Taqase:1U/ pipe, 2.5mMdNTP:2ul/ pipe, sterilized water: 14ul/ pipe;

Amplification condition: 95 ℃, 4min; 94 ℃ of 40sec, 61 ℃ of 1min, 72 ℃ of 45sec, 35cycles, 72 ℃ of 8min;

Embodiment 2

The 8th exon detection method that the present invention has suddenlyd change: use nested PCR method and detect,

(1) DNA extraction is adopted 5 milliliters of venous blood, and the Sodium Citrate anti-freezing is with conventional phenol chloroform extraction DNA, spectrophotometric instrumentation dna content, 10 sodium gram/microlitres;

(2) design of primers and reaction conditions:

First round PCR:

Primer sequence:

Forward primer: TNNI2-1F:5 '-GGTGACCGTGGCTGCCAAGTGTC-3 '

Reverse primer: TNNI2-1R:5 '-AGCTCAGGCTCACCCACTTATGCA-3 '

Reaction system: be total to 20ul

1. person under inspection's template DNA 10ng (1ul)

2. 10 * buffer 2ul/ manages

3. Taqase:1U/ manages (1ul)

4. 2.5mMdNTP:2ul/ manages

5. sterilized water: 14ul/ manages

With the aforesaid liquid mixing, moment is centrifugal, increases on the pcr amplification instrument, and amplification condition is: 95 ℃, and 4min; 94 ℃ of 40sec, 61 ℃ of 1min, 72 ℃ of 45sec, 35cycles, 72 ℃ of 8min.

Separate the purpose band with the PAGE gel electrophoresis on electrophoresis apparatus, the purpose band is 885bp or 883bp, and normal people is 885bp sheet segment length, is 883bp sheet segment length unusually.

Second takes turns PCR:

Primer sequence: 5 '-TGGGTGACTGGAGGAAGAACAT-3 '

5’-AGCTCAGGCTCACCCACTTATGCA-3’

Reaction system: be total to 20ul

1. person under inspection first round PCR product is template DNA 1ul

2. 10 * buffer 2ul/ manages

3. Taqase:1U/ manages

4. 2.5mMdNTP:2ul/ manages

5. sterilized water: 14ul/ manages

With the aforesaid liquid mixing, moment is centrifugal, increases on the pcr amplification instrument, and amplification condition is: 95 ℃, and 4min; 94 ℃ of 40sec, 60 ℃ of 40sec, 72 ℃ of 45sec, 35cycles, 72 ℃ of 8min.

On electrophoresis apparatus, separate the purpose band with the PAGE gel electrophoresis, the normal artificial 143bp band of purpose strap section, the patient is two band 140/143bp.

Embodiment 3

Test kit is formed:

(1) primer pipe

	Forward primer	Reverse primer
			First round primer	5’-CTACAAGGAAAGCTGGGTC-3’	5’-AAGGGCTGGAAGAGGTGG-3’
Second takes turns primer	5’-TGGGTGACTGGAGGAAGAACAT-3’	5’-GTGCATCTCCCTAGTATGTTCTGC-3’

First round amplifing reagent is formed:

Pipe 1 (18ul system): 2.5mMdNTP, 10 * buffer, 10mM first round primer;

Manage 2 whiterusss and Taqase mixed solution 4ul; (Taqase1U/ pipe)

First round amplification method: will manage 1 and 2 mix, add 1ul person under inspection template DNA with pipe, mixing, moment is centrifugal, increases on the pcr amplification instrument.Amplification condition is: 95 ℃, and 4min; 94 ℃ of 40sec, 61 ℃ of 1min, 72 ℃ of 45sec, 35cycles, 72 ℃ of 8min.

The product of amplification can be directly used in dna sequencing, also can be used for second and take turns pcr template.Order-checking can detect this segmental any sudden change.

Second takes turns amplifing reagent forms:

Pipe 3 (18ul systems): 2.5mMdNTP, 10 * buffer, 10mM second take turns primer;

Manage 4 whiterusss and Taqase mixed solution 4ul; (Taqase1U/ pipe)

Second takes turns amplification method: will manage 3 and 4 mix, add 1ul first round pcr amplification product with pipe, and mixing, moment is centrifugal, increases on the pcr amplification instrument.Amplification condition is: 95 ℃, and 4min; 94 ℃ of 40sec, 60 ℃ of 40sec, 72 ℃ of 45sec, 35cycles, 72 ℃ of 8min.

Carry out denaturing gel electrophoresis at the vertical slab electrophoresis instrument.8% gel, 7mol urea, 40% deionized formamide, 40 watts of electrophoresis 4～5 hours.Normal people is 143bp one band, and the patient is for being 140/143bp two bands.

The scope that test kit is used: to being diagnosed as the patient of far end arthrosis bend 2B type; To being diagnosed as the patient of DAS; To the crooked odd-shaped patient of brothers; Patient to equinvoarus or talipes equinovalgus.

Arthrogryposis

SEQUENCE LISTING

＜110〉Liaoning Research Institute of Family Planning

＜120〉a kind of pathogenic gene for far end arthrosis bend and detection method thereof and application

<130>

<160>9

<170>PatentIn version 3.1

<210>1

<211>93

<212>DNA

＜213〉TNNI2 the 8th exon

<220>

<221>mutation

<222>(1)..(93)

<223>

<400>1

gagcgggacc tgcgagacgt gggtgactgg aggaagaaca tcgaggagaa gtctggcatg 60

gagggccgga agatgtttga gtccgagtcc tag 93

<210>2

<211>4163

<212>DNA

<213>TNNI2

<220>

<221>CDS

<222>(1067)..(1096)

<223>

<220>

<221>CDS

<222>(1795)..(1836)

Arthrogryposis

<223>

<220>

<221>CDS

<222>(1920)..(2048)

<223>

<220>

<221>CDS

<222>(2211)..(2300)

<223>

<220>

<221>CDS

<222>(2423)..(2599)

<223>

<220>

<221>CDS

<222>(2845)..(3066)

<223>

<400>2

gctgacctcg ggtgagatgg gctgaggcga ggtgtggata ctcgcagcta cccctcagct 60

gacccgagct gtgtgcccgg ctgaggccac aggcaaagcc aggcacactg tcctcaggct 120

ccttacgaga acgacagagg catctccagc gcgtcaccga gccctaaata gagtagccca 180

gccacggcac cccccaccaa gacttcttgg actgggcggc agcacgcggc caggccaggc 240

ggccggacag gtggggaggt ctctgtggct ctccacgccc ccattggtct gaggaggact 300

ctatgccctt tctgagcagg ggcccagcct gggggaggcc atttataccc ctccccctgg 360

gcccaccagc ccaactcgcc gctgccggcc tgacctcgct cccagccctg ctgcccagat 420

tctaggtgag gcccagcccg gcccgccgag gccgggggac agggcgtggc tcgagctggt 480

ttgagggagg acttcctggg gcgggggtct gggggcctgg gggatgccac cagcagcccc 540

ctttggggcc tgcacgggtc cgggagtgag aaggagaggc tttggggagg cagctgtagc 600

gagaggcagg tattagacac tccctgacca ccaggcttca cctctgggga ccctggaagg 660

agagtttagg ggtcaagaag ccccagaacc tgagccccca ggccaaactg aggagggatt 720

tcttccatgc acttaggccc aaagccaaag agagagggtt aaaaataaca gcgtcactca 780

ggcggccacc tgcgctggcc catcccaccc tccctcgggg acagctgcag ctcctcaggc 840

tatgcctggg acattttggg aacactttct cctcttactt ctcaccctgg ggaattccaa 900

Arthrogryposis

gacattgtcc ttgaaggagg tgagagtagg gggaggaggt gagagtaggg ggtgggcggg 960

agggggctgt catcaggagc cctgaacccc tcaccaccta cctgatgggc acaggcatca 1020

cggtggcaag ggcctggcca acacctctgt cttcctctcc ccacag gct cca agc 1075

Ala Pro Ser

1

tca gga cct cag gat ggg aga gtaagtggta cccctgtacc cccatacagt 1126

Ser Gly Pro Gln Asp Gly Arg

5 10

gaccctgccc acctcctgcc ctgtccaccc catcacacac tccgaccccg ccagccatgg 1186

ccctaacctc tgtcttctct ccccgtcctg cctgcgtcct ccctccctgg acagtgagga 1246

ggtaagtagt tgctgggggc tcaaaacatg acgaggaggg ggctccccca actcagcata 1306

ggtcataggt cacagcctca aggccctaag gcccagagaa gatggcctgg ccctccccgc 1366

tggcaaaagt gccccaccca accaccaaga cgctgctgaa agagaggaag tggctgcccc 1426

aagccttctg gggcagggga agtgtggctg tggcctggtc acaggggaat cacttcttct 1486

ttctgattcc tgcacttccc gcccccacct caccggccga cctgccccca actggccctc 1546

ctctccccca gcctctggcc tcccagcacc atgtgccacc tggcaaagtg tcacacacca 1606

cacagcacca tgtcccagtg caaggtctgg ggccagggag gcccagcctg cccatcatgg 1666

cctcaggagc ccccaggctc tgctggtccc ggagccggag cccctgacct ggccagggag 1726

cgtggaaggg ggtgggggtg ctccaggcct ggaggccctg actcgacccc ctgtcccctg 1786

ccctgcag aag cgg aac agg gcc atc acg gcc cgc agg cag cac ctg aag 1836

Lys Arg Asn Arg Ala Ile Thr Ala Arg Arg Gln His Leu Lys

15 20

gtaggtgtgg gctcccgggg gggtggccca ggtgggtctg caggggagct ggctgcagcc 1896

cctcaccgcc tgccccaccg cag agc gtg atg ctg cag ata gcg gcc acg gag 1949

Ser Val Met Leu Gln Ile Ala Ala Thr Glu

25 30

ctg gag aag gag gag agc cgc cgt gag gca gag aag cag aac tac ctg 1997

Leu Glu Lys Glu Glu Ser Arg Arg Glu Ala Glu Lys Gln Asn Tyr Leu

35 40 45 50

gcg gag cac tgc ccg ccg ctg cat atc ccg ggc tcc atg tct gaa gtg 2045

Ala Glu His Cys Pro Pro Leu His Ile Pro Gly Ser Met Ser Glu Val

55 60 65

cag gtaccagccc ctccccggcc accccgcctc cccagcagca ggcctgcctg 2098

Gln

ctaactcagt ttccccactt gtcaagaggg ccagtggggt ggtcagggca ggggcaggtg 2158

accgtggctg ccaagtgtca ggacggccgc ccgcccccac acccacccct ag gag ctc 2216

Glu Leu

tgc aaa cag ctg cac gcc aag atc gat gcg gct gaa gag gag aag tac 2264

Cys Lys Gln Leu His Ala Lys Ile Asp Ala Ala Glu Glu Glu Lys Tyr

70 75 80 85

gac atg gag gtg agg gtg cag aag acc agc aag gag gtgagtggtg 2310

Asp Met Glu Val Arg Val Gln Lys Thr Ser Lys Glu

90 95

gcggcgggcc ggcggcaggt gggtaggcgg tggcccagcg ggcaggcggg gcgggccggg 2370

gaggccgaga ccaccgggac ctcgggctcc cacccggctc ccctgcccac ag ctg gag 2428

Leu Glu

Arthrogryposis

gac atg aac cag aag cta ttt gat ctg cgg ggc aag ttc aag cgg ccc 2476

Asp Met Asn G1n Lys Leu Phe Asp Leu Arg Gly Lys Phe Lys Arg Pro

100 105 110 115

cca ctg cgg agg gtg cgc atg tcg gcc gat gcc atg ctc aag gcc ctg 2524

Pro Leu Arg Arg Val Arg Met Ser Ala Asp Ala Met Leu Lys Ala Leu

120 125 130

ctg ggc tcg aag cac aag gtg tgc atg gac ctg agg gcc aac ctg aag 2572

Leu Gly Ser Lys His Lys Val Cys Met Asp Leu Arg Ala Asn Leu Lys

135 140 145

cag gtc aag aag gag gac aca gag aag gtgcgtgcca cggggggagc 2619

Gln Val Lys Lys Glu Asp Thr Glu Lys

150 155

accaccacac ctaccctgcc ggggaagcac ctcccacacc tgccccgcct gggaccacct 2679

cccacacctg cctgccgggg ccctgtacca ctgcccctcc agggtgccat gcaggggaca 2739

ctccactgcc caatgcagag ggtaaactga ggctgaaggt ggtgtggacc agggtgcgtg 2799

cataagtggg tgagcctgag ctctctcctg ccctctcctc cacag gag cgg gac ctg 2856

Glu Arg Asp Leu

160

cga gac gtg ggt gac tgg agg aag aac atc gag gag aag tct ggc atg 2904

Arg Asp Val Gly Asp Trp Arg Lys Asn Ile Glu Glu Lys Ser Gly Met

165 170 175

gag ggc cgg aag aag atg ttt gag tcc gag tcc tag gcc act cgc tgc 2952

Glu Gly Arg Lys Lys Met Phe Glu Ser Glu Ser Ala Thr Arg Cys

180 185 190

ccc tac gcc tgc ccc ggt gcc cgg ctc cca gca gaa cat act agg gag 3000

Pro Tyr Ala Cys Pro Gly Ala Arg Leu Pro Ala Glu His Thr Arg Glu

195 200 205

atg cac cca gag cct gcc agg gag ggc tgg cct cac cac cac cgt caa 3048

Met His Pro Glu Pro Ala Arg Glu Gly Trp Pro His His His Arg Gln

210 215 220

taa agg att tga atc ccc atggctggtc tggtctggct ctccccagcc 3096

Arg Ile Ile Pro

225

tcttgggcca tgctctgggc ccccgcatcg gtggcctgca gtgtggtcag tggccagcgg 3156

ggaaagcctg ggaggaggcc acacggggcc tgggatttca ggttgggagg gaagctgctt 3216

ccagcaagga ggtgagcctg ggaaggcccc tacaggggaa tccaccccag gctgcacggg 3276

gctgtttggt gagggccctg atagtgcccc aggcgttcct ggggctcggc ctgggcacat 3336

ccaacatgca gggctgggga tggaggctgt gggaccgagc caggtctggg tgggagttcc 3396

tgagataagt gggtctcaca tttgcacagc cacgcaaagg gcctcccggg gctccagaga 3456

gatccacaca tgtgacacac gtgtggcccc cgcagtgctg ggggcagccc cctatgcctc 3516

ccaagctgcc cctcctcagg gagctcatct gaggcaggaa gccaagggcc acattagacc 3576

ccaggctgct gtccacgcgt gcccaccccc agatcctcct gctcatcccc ctcccacatc 3636

actttgggac agacccccag ccccggggcc tccctgcttt tcctatctgc cccctgccct 3696

gagcactgag gctgggcacg gggagcgtgg aggggcgagc ctgcacggtg ggccatgacc 3756

aggccaaatt ggcagtctcc gggctgggga ccagcatggt gctgggggtt ggccagcctg 3816

gtctctggtc cttactgcca ccctgaaagg caggctgtgt cactcctcac catgccaggg 3876

gaccaagaga cctgtgccca tcaggcaggg gctaggcctg gccctgacct ggctccagtg 3936

Arthrogryposis

tggctcccaa gtctcgtttc cactcagccc caagacccag ccccatctgg ccaccaagga 3996

caaggcagct gacaggttct gacaaggccg gtgacagcaa ggccacgtta tcagccctgc 4056

tctttgaagg ctgacagagc aggcacgcca aggtccctgt gctcagatct gcgctgggga 4116

ggctgccagc ctgcgctgtg cccttgcccc cgggaggtcc catcgat 4163

<210>3

<211>10

<212>PRT

<213>TNNI2

<400>3

Ala Pro Ser Ser Gly Pro Gln Asp Gly Arg

1 5 10

<210>4

<211>14

<212>PRT

<213>TNNI2

<400>4

Lys Arg Asn Arg Ala Ile Thr Ala Arg Arg Gln His Leu Lys

1 5 10

<210>5

<211>43

<212>PRT

<213>TNNI2

<400>5

Ser Val Met Leu Gln Ile Ala Ala Thr Glu Leu Glu Lys Glu Glu Ser

1 5 10 15

Arg Arg Glu Ala Glu Lys Gln Asn Tyr Le uAla Glu His Cys Pro Pro

20 25 30

Leu His Ile Pro Gly Ser Met Ser Glu Val Gln

35 40

<210>6

<211>30

<212>PRT

<213>TNNI2

Arthrogryposis

<400>6

Glu Leu Cys Lys Gln Leu His Ala Lys Ile Asp Ala Ala Glu Glu Glu

1 5 10 15

Lys Tyr Asp Met Glu Val Arg Val Gln Lys Thr Ser Lys Glu

20 25 30

<210>7

<211>59

<212>PRT

<213>TNNI2

<400>7

Leu Glu Asp Met Asn Gln Lys Leu Phe Asp Leu Arg Gly Lys Phe Lys

1 5 10 15

Arg Pro Pro Leu Arg Arg Val Arg Met Ser Ala Asp Ala Met Leu Lys

20 25 30

Ala Leu Leu Gly Ser Lys His Lys Val Cys Met Asp Leu Arg Ala Asn

35 40 45

Leu Lys Gln Val Lys Lys Glu Asp Thr Glu Lys

50 55

<210>8

<211>31

<212>PRT

<213>TNNI2

<400>8

Glu Arg Asp Leu Arg AspVal Gly Asp Trp Arg Lys Asn Ile Glu Glu

1 5 10 15

Lys Ser Gly Met Glu Gly Arg Lys Lys Met Phe Glu Ser Glu Ser

20 25 30

<210>9

<211>36

<212>PRT

<213>TNNI2

<400>9

Ala Thr Arg Cys Pro Tyr Ala Cys Pro Gly Ala Arg Leu Pro Ala Glu

1 5 10 15

Arthrogryposis

His Thr Arg Glu Met His Pro Glu Pro Ala Arg Glu Gly Trp Pro His

20 25 30

His His Arg Gln

35

Claims (4)

1. a pathogenic gene for far end arthrosis bend is characterized in that: have aminoacid sequence among the sequence table SEQ ID No:1.

2. the detection method of the described pathogenic gene for far end arthrosis bend of claim 1 is characterized in that: use nested PCR method and detect:

1) extracts person under inspection's blood template DNA;

2) design of primers and reaction conditions:

First round PCR:

Forward primer: TNNI2-1F:5 '-GGTGACCGTGGCTGCCAAGTGTC-3 '

Reverse primer: TNNI2-1R:5 '-AGCTCAGGCTCACCCACTTATGCA-3 ';

Reaction system: be total to 20ul, person under inspection's template DNA 10ng ul, 10 * buffer 2ul/ pipe, Taqase:1U/ pipe, 2.5mMdNTP:2ul/ pipe, sterilized water: 14ul/ pipe;

With the aforesaid liquid mixing, moment is centrifugal, increases on the pcr amplification instrument, and amplification condition is: 95 ℃, and 4min; 94 ℃ of 40sec, 61 ℃ of 1min, 72 ℃ of 45sec, 35cycles, 72 ℃ of 8min; Separate the purpose band with the PAGE gel electrophoresis on electrophoresis apparatus, the purpose band is 885bp or 883bp, and normal people is 885bp sheet segment length, is 883bp sheet segment length unusually;

Second takes turns PCR: primer sequence: 5 '-TGGGTGACTGGAGGAAGAACAT-3 '

5’-AGCTCAGGCTCACCCACTTATGCA-3’

Reaction system: be total to 20ul, person under inspection first round PCR product is template DNA 10ng, 10 * buffer2ul/ pipe, Taqase:1U/ pipe, 2.5mMdNTP:2ul/ pipe, sterilized water: 14ul/ pipe;

With the aforesaid liquid mixing, moment is centrifugal, increases on the pcr amplification instrument, and amplification condition is: 95 ℃, and 4min; 94 ℃ of 40sec, 60 ℃ of 40sec, 72 ℃ of 45sec, 35cycles, 72 ℃ of 8min; On electrophoresis apparatus, separate the purpose band with the PAGE gel electrophoresis, the normal artificial 143bp band of purpose strap section, the patient is two band 140/143bp.

3. the application of the described pathogenic gene for far end arthrosis bend of claim 1 is characterized in that: described pathogenic gene for far end arthrosis bend is used to prepare the reagent or the test kit of diagnosis far end arthrosis bend 2B type, DAS, the crooked deformity of brothers, equinvoarus or talipes equinovalgus disease.

4. according to the application of the described pathogenic gene for far end arthrosis bend of claim 3, it is characterized in that: described reagent or test kit adopt

First round primer:

Forward 5 '-CTACAAGGAAAGCTGGGTC-3 ',

Reverse 5 '-AAGGGCTGGAAGAGGTGG-3 ';

Second takes turns primer:

Forward 5 '-TGGGTGACTGGAGGAAGAACAT-3 ',

Reverse 5 '-GTGCATCTCCCTAGTATGTTCTGC-3 '.