CN101525620B - COPPOCK cataract disease-causing gene and detecting method thereof - Google Patents

Abstract

The invention relates to a COPPOCK cataract (granular nuclear cataract) disease-causing gene GJA3, and a G->A point mutation exists on the 427th basic group of a first exon of GJA3 gene of the COPPOCK cataract (granular nuclear cataract) patient. The invention also relates to the application of the mutant gene in the detection of diseases. The method provided by the invention can accurately diagnosticate congenital COPPOCK cataract (granular nuclear cataract) and distinguish the congenital COPPOCK cataract (granular nuclear cataract) from other congenital cataracts.

Description

COPPOCK cataract Disease-causing gene and detection method thereof

Technical field

What the present invention relates to is a kind of human variation's gene, specifically a kind of COPPOCK cataract Disease-causing gene GJA3.The invention still further relates to the application of this mutant gene in detecting disease.

Background technology

Congenital cataract (congenital cataract is that a kind of common eye is unusual CC), can cause tangible visual disorder or children blind, account for world children blind 1/10.Congenital cataract can isolate generation, can merge other eye heteroplasia, or the part of heredity multisystem syndromes.Approximately the congenital cataract of 1/3-1/4 is genetic, and the chain three kinds of modes of inheritance of autosomal dominant, autosomal recessive and X-all have report.Heredity congenital cataract is autosomal dominant mostly, and almost completely outer apparent still expressivity has nothing in common with each other.Although congenital cataract is the blind major causes of world children, its hereditary basis and pathogenesis are still not fully aware of.Location to the congenital cataract Disease-causing gene is an important step of understanding the molecular mechanism of its morbidity with the clone.

In recent years, along with the fast development of Protocols in Molecular Biology and information biology, increasing congenital cataract Disease-causing gene is positioned and clones.Up to now, individual surplus clear and definite localized site has 30 to autosomal dominant inheritance congenital cataract, individual surplus the Disease-causing gene that has found has 20.The Disease-causing gene of congenital cataract can be divided into 5 classes at present: (1) crystal protein gene comprises CRYAA, CRYAB, CRYBB1, CRYBB2, CRYBB3, CRYBA1/A3, CRYBA4, CRYGC, CRYGD, and CRYGS.(2) membrane protein gene comprises MIP, and LIM2. (3) slit connects protein gene, comprises GJA8, GJA3.(4) the cytoskeletal protein gene comprises BFSP1, BFSP2.(5) grow the regulatory factor gene, comprise PITX3, MAF, HSF4, FOXE3, EYA1, CHX10 and PAX6 etc.

Chinese patent application 200710144506.X discloses the relation of CRYGC genovariation and congenital nuclear cataract, the point mutation of a G-＞A of CRYGC gene the 3rd exon the 470th base, the codon of the 157th tryptophane that cause encoding has become terminator codon (W157X).And determine that this point mutation is the Disease-causing gene sudden change of congenital nuclear cataract.

Have many types because indication is detected in the clinical symptom of congenital cataract and laboratory, and studies show that, the different Clinical types transgenation with different respectively is relevant.Even same gene, the site difference of point mutation causes the symptom of congenital cataract just possible different thus.Therefore be necessary cataractous various Clinical types, especially also the Clinical types that does not have to find relevant molecular genetics basis is studied, to determine its accurate mutant gene title and site, and set up easily and fast and accurate test method according to this variation, be used for the diagnosis of congenital cataract.

Summary of the invention

The object of the present invention is to provide the COPPOCK cataract, be also referred to as the cataractous Disease-causing gene of nuclearity particulate state, orient the gene mutation site of this disease, and utilize this transgenation, the method that whether has this transgenation in the human body is provided.

The present invention is directed to a COPPOCK cataract family, Disease-causing gene is carried out chromosomal localization and candidate gene sudden change detection with the method for genescan and linkage analysis.After having got rid of the relevant chromosomal region of most of congenital cataract, with the Disease-causing gene Primary Location in No. 13 karyomit(e).Through accurately locating, Disease-causing gene is positioned karyomit(e) 13q11-12.Find a new point mutation by candidate gene GJA3 Gene Double to order-checking to congenital cataract in this locating area.By the normal member of family and 100 normal control GJA3 gene sequencing are not found this sudden change, single nucleotide polymorphisms (single nucleotide polymorphism, possibility SNP) have been got rid of.Thereby determine that the GJA3 transgenation is the cataractous Disease-causing gene sudden change of COPPOCK.Thus,

The invention provides a kind of human GJA3 gene of the variation shown in SEQ ID NO:1, it is characterized in that, the G of described GJA3 gene the 1st exon the 427th bit base sports A.Normal human GJA3 gene is shown in SEQ ID NO:2.

The present invention also provides a kind of above-mentioned Nucleotide of stating gene mutations that contains, and it is characterized in that, described Nucleotide contains from point mutation and extends more than 50 Nucleotide to length between 1000 bases at least to both sides or a side.

The present invention further provides described Nucleotide, it is characterized in that, described Nucleotide contains from point mutation and extends the Nucleotide of length between each and every one base of 300-600 at least to both sides or a side.

Whether deposit the method for described gene mutations in the biological specimen that the present invention also provides a kind of detection to derive from human body, it comprises step:

(1) from described sample, extracts and the purified genes material;

(2) described genetic material is carried out the polymerase chain amplified reaction, obtain product;

(3) described product is carried out nucleotide sequencing, to determine whether contain the GJA3 gene mutations in the described biological specimen.

Preferably, described biological specimen is a body fluid, and described body fluid includes but not limited to: blood, serum, amniotic fluid, lymph liquid etc.

Preferably described body fluid is blood.

Preferably, the primer that uses at described polymerase chain amplified reaction is to being: SEQ ID NO:6 and 7; Those skilled in the art can also design more kinds of primers according to the requirement of polymerase chain amplified reaction, to amplify the nucleotide sequence that comprises variant sites of the present invention.

The present invention also provides the application of above-mentioned Nucleotide in preparation congenital cataract disease gene diagnosing chip.Whether those skilled in the art are gene probe with this Nucleotide, according to the principle of gene recombination, can exist and this probe sequence complementary nucleotide sequence in the detection of biological sample.Therefore make also to detect whether exist gene mutation site of the present invention in the sample in this way.

Whether have the above-mentioned method of stating transgenation in the biological specimen that the present invention also provides a kind of detection to derive from human body, it comprises step:

(1) from described sample, extracts and the purified genes material;

(2) each described Nucleotide of genetic material and claim 1-3 that step (1) is obtained contacts.

(3) observe through step (2), whether described genetic material exists based on sequence-specific hybridization with described Nucleotide combines.

Preferably, described biological specimen is a blood.

Site according to GJA3 gene order variation provided by the invention, use the method for whether depositing described gene mutations in the biological specimen that detection provided by the invention derives from human body, a congenital COPPOCK cataract (nuclearity particulate state cataract) the normal Han nationality of family individuality is detected.The result shows that all cataract patients of family all have identical point mutation, and interior all the other normal individuals of family and 100 normal controls all do not have this sudden change.Prove that method provided by the invention can accurately diagnose congenital COPPOCK cataract (nuclearity particulate state cataract), and can distinguish mutually with other congenital cataract.

Description of drawings

Fig. 1 is mutant nucleotide sequence SEQ ID NO:1 and the normal sequence SEQ ID NO:2 contrast synoptic diagram in the mutational site.

Embodiment

Below in conjunction with embodiment the present invention is made a more detailed description; but following description only is used for the explanation of making an explanation property of the present invention; protection scope of the present invention is not carried out any restriction, protection scope of the present invention only is as the criterion with claims restricted portion.

The location of embodiment 1:GJA3 genovariation

(1) extraction of genomic dna: use the QIAGEN QIAamp DNA Blood Mini Kit of company test kit extracting blood sample genomic dna.

(2) genescan: divide preliminary scanning and two steps of fine scanning to carry out.At first the candidate region of having reported is tentatively scanned, in positive region, carried out fine scanning afterwards.

1, microsatellite marker: preliminary scanning uses genetic marker from Linkage mapping set version 2.5 (U.S. Applied Biosystems company); Fine scanning is selected from the Marshfield website with fluorescent marker, and sequence and relevant information are from the NCBI website, and be synthetic by American I nvitrogen company, 5 ' end FAM fluorescent mark.

2, multiplex PCR amplification: 5 μ L PCR reaction systems comprise: template DNA 1 μ L, 10 * PCR buffer0.5 μ L, dNTP 0.8 μ L, Mg ²⁺0.3 μ L, primer (forward and reverse 10pmol/ μ L) 0.05 μ L, HotStar TaqDNA polysaccharase 0.06 μ L, remaining volume is supplied by the sterilization distilled water.The PCR reaction conditions adopts " Touchdown " mode: pre-94 ℃ of sex change 30sec of sex change, the renaturation temperature is since 64 ℃, whenever carry out a thermal cycling renaturation temperature reduction and reduce to 57 ℃ for 0.5 ℃ always, the time of renaturation is 1min in the thermal cycling, extend to constant 72 ℃ of following 1.5min, 14 thermal cyclings altogether of this process.Through after this condition, reaction enters second and takes turns the promptly constant amplification phase.The renaturation temperature is set at 56 ℃, and other condition is constant, carries out 30 thermal cyclings.

3, gene type

PCR product 1 μ l mixes 95 ℃ of sex change 5min, ice baths immediately with MegaBACE1000TM ET400-R Size (diluting with 27: 100 with deionized water) 6 μ l.Go up electrophoresis 1h at MegaBACE1000 DNA analysis instrument (Britain AmershamPharmacia Biotech company), through software scans and collect graphic information, and, obtain the gene type data by GeneticProfiler 2.1 software analysis.

4, linkage analysis

After the gene type result manually proofreaded, adopt the Mlink linkage analysis software in the Linkage v5.1 software package to carry out the point-to-point transmission linkage analysis.Be 0.00,0.10,0.20,0.30 at recombination fraction (θ) respectively, 0.40 and calculated the score of logarithm advantage (log odds score, LOD) value at 0.50 o'clock.Judging criterion as a result: LOD value＞1 is chain for prompting, and＞2 is chain for supporting,＞3 for chain certainly, and＜-2 for negating chain.

(3) Fine Mapping

Near the positive region that obtains, selected more meticulous microsatellite marker.Genetic marker selects to come from marshfield and ncbi website.One 5 ' end was with FAM (blueness) fluorochrome label therein when the every pair of primer was synthetic, and the gene type of PCR condition, mark and the method for linkage analysis are all the same.

(4) haplotyping

Utilize Cyrillic software to produce a family collection of illustrative plates, can show the somatotype information of a plurality of genetic markers of each member in the collection of illustrative plates.Carrying out manual regulation for genetic affinity can arrange out each individual haplotype up and down to these information bases.According to the situation that haplotype is shared, can find the generation that exchanges and then the zone of one section minimum that can obtain finally sharing by all patients in individual patients.The zone of one section minimum that all patients of this section share promptly is the locating area of this family Disease-causing gene on karyomit(e).

By above-mentioned steps with the CRYAA assignment of genes gene mapping in being positioned at chromosomal 13q11-12 No. 13.

The chromosomal localization technology of gene is known technology in the molecular genetics field to those skilled in the art, and the present invention utilizes these known technology to remove to find to bring the mutant gene of disease to the mankind

Embodiment 2: the order-checking of mutant gene

(1) uses the QIAGEN QIAamp DNA Blood Mini Kit of company test kit extracting blood sample genomic dna;

(2) the sample DNA target fragment is carried out the PCR amplification in vitro, purpose is first exon (shown in the SEQ IN NO:1) that amplifies GJA3, shared 4 pairs of primer amplification first exons (table 1), the PCR reaction system is as shown in table 2, and the PCR response procedures is as shown in table 3.

Table 1COPPOCK cataract family GJA3 gene extron 1 sequencing primer

Table 2PCR reaction system

PCR is reflected on the 9700PCR instrument and carries out.

Table 3:PCR response procedures

PCR product electrophoresis detection

PCR reaction is got 2 * Loading Buffer that 2 μ l reaction product add 2 μ l after finishing, centrifugal 30 seconds of 4000rpm, last sample in 1.5% sepharose, electrophoresis 15min under the 100V voltage, gel video camera shot detection;

(3) the PCR product is carried out purifying

Adopt SAP (arctic shrimp alkaline phosphatase) the remaining dNTP that degrades, Exo (exonuclease) the remaining primer of degrading is pre-mixed the back with SAP and two kinds of enzymes of Exo and 10 * damping fluid and preserves, and contains each 0.5 unit of SAP and EXO in every microlitre mixed solution,

For each purification reaction, get PCR product 1.5-2 μ l and add SAP enzyme 1.5 μ l,

Response procedures is: SAP/Exo handled the PCR product 40 minutes at 37 ℃, and then 85 ℃ continue 20 minutes deactivation SAP/Exo enzymes, 4 ℃ of insulations then;

(4) the PCR product is checked order

The sequencing reaction system is 5 μ l, comprises the PCR product behind the purifying, sequencing primer 3.2pmol and BDT (BigDye Terminator 3.1, Applied Biosystems).0.5 μ l, remaining volume ddH ₂The O polishing,

The sequencing reaction program is as shown in table 4:

Table 4 sequencing reaction program

After sequencing reaction finishes, need to remove the small-molecule substance that sequencing reaction brings, avoid its interference to the product purifying (order-checking back purifying) once more that checks order to order-checking.Purification step is as follows:

1) every hole adds 25 μ l sodium-acetates, one ethanolic soln (3M sodium-acetate+100% ethanol, volume ratio are 1: 25) in 96 orifice plates of sequencing reaction;

4 ℃, centrifugal 45 minutes of 4000rpm; Be inverted, 600rpm dried in centrifugal 1 minute.

2) add 50 μ l, 70% ethanolic soln again; 4 ℃, centrifugal 10 minutes of 4000rpm; Be inverted 600rpm dried in centrifugal 1 minute; Repeat once.

3) room temperature leaves standstill and allows approximately its seasoning half an hour.Before order-checking, to make the sex change of DNA sample earlier.The method of sex change is to add 10 μ l HiDi (deionized formamide) in the sample behind purifying, and 95 ℃ keep making it in 5 minutes complete sex change on the PCR instrument.At once put into ice after the sex change and prevented its renaturation at least in 2 minutes.Then going up sample to ABI PRISM 3100 sequenators/somatotype instrument (Applied Biosystems) checks order.

(5) sequencing result analysis

Using SEQUENCE ANALYSIS software analyzes sequencing result.The blast program of application ncbi database ( Http:// www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi), mutant nucleotide sequence and the genome sequence of finding compared, sequencing result is compared.After getting rid of the possibility of SNP, all members of family and 100 normal controls are checked order, judge sudden change whether with family be divided into from.

Carry out pcr amplification by 4 pairs of primers shown in the table 1, show through sequencing analysis product, the 427th base of the 1st exon generation G-＞A point mutation of the present invention is positioned at the amplification scope of the 2nd pair of primer (being shown in SEQ ID NO:6 and 7), therefore, this can be used for detection to the transgenation of congenital COPPOCK cataract (nuclearity particulate state cataract) Disease-causing gene GJA3 to primer.

Embodiment 3: a congenital COPPOCK cataract family is carried out the detection of GJA3 genovariation

In order to verify effect of the present invention, we collect a congenital COPPOCK cataract family of Heilongjiang Province.General physical checkup is visited or done in family member to get rid of other eye disease and system exception through investigation.Family member is carried out detailed eye examination, comprised the fundoscopy behind eyesight, slit-lamp microscope, the mydriasis.To confirm the cataract type, take pictures, the cataract phenotype of record patient by slit-lamp microscope through the ophthalmology slit lamp examination for all patients.All are participated in investigation and are all understood the purpose and meaning of this research and signed Informed Consent Form by the family member of blood sample collection.This family is totally 4 generations, 150 family members, wherein male 78 people, women 72 people.Extract volunteer's peripheric venous blood 5ml, after doing anti-freezing and handle with EDTA ,-20 ℃ of refrigerators are preserved.Gather individual 100 people of normal Han nationality in addition, gather peripheric venous blood 5ml, the EDTA anti-freezing ,-20 ℃ of refrigerators are preserved group in contrast.The sequencing result of 100 normal controls and family normal people's sequencing result is in full accord.

Two-way sequencing result shows GJA3 gene the 1st exon the 427th base of congenital COPPOCK cataract family, the point mutation that a G-＞A is arranged is (shown in SEQ ID NO:1, the normal sequence of SEQ ID NO:2 for not morphing, see shown in Fig. 1 rectangular box), cause the 143rd glycine password GGG of polypeptid coding sequence to become arginine password AGG (G-＞R, perhaps Gly-＞Arg) (shown in SEQ ID NO:3, see shown in two solid lines of Fig. 1).All cataract patients of family all have identical point mutation, and interior all the other normal individuals of family and 100 normal controls all do not have this sudden change.Thereby having got rid of it is the possibility of rare SNP.Determine that the GJA3 transgenation is the Disease-causing gene sudden change of this family.

KLPI090072-2009-4-3

Sequence table

＜110〉Harbin Medical University

＜120〉COPPOCK cataract Disease-causing gene and detection method thereof

<130>

<160>11

<170>PatentIn?version?3.3

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<213>Homo?sapiens

<400>1

atgggcgact?ggagctttct?gggaagactc?ttagaaaatg?cacaggagca?ctccacggtc 60

atcggcaagg?tttggctgac?cgtgctgttc?atcttccgca?tcttggtgct?gggggccgcg 120

gcggaggacg?tgtggggcga?tgagcagtca?gacttcacct?gcaacaccca?gcagccgggc 180

tgcgagaacg?tctgctacga?cagggccttc?cccatctccc?acatccgctt?ctgggcgctg 240

cagatcatct?tcgtgtccac?gcccaccctc?atctacctgg?gccacgtgct?gcacatcgtg 300

cgcatggaag?agaagaagaa?agagagggag?gaggaggagc?agctgaagag?agagagcccc 360

agccccaagg?agccaccgca?ggacaatccc?tcgtcgcggg?acgaccgcgg?cagggtgcgc 420

atggccaggg?cgctgctgcg?gacctacgtc?ttcaacatca?tcttcaagac?gctgttcgag 480

gtgggcttca?tcgccggcca?gtactttctg?tacggcttcg?agctgaagcc?gctctaccgc 540

tgcgaccgct?ggccctgccc?caacacggtg?gactgcttca?tctccaggcc?cacggagaag 600

accatcttca?tcatcttcat?gctggcggtg?gcctgcgcgt?ccctgctgct?caacatgctg 660

gagatctacc?acctgggctg?gaagaagctc?aagcagggcg?tgaccagccg?cctcggcccg 720

gacgcctccg?aggccccgct?ggggacagcc?gatcccccgc?ccctgccccc?cagctcccgg 780

ccgcccgccg?ttgccatcgg?gttcccaccc?tactatgcgc?acaccgctgc?gcccctggga 840

caggcccgcg?ccgtgggcta?ccccggggcc?ccgccaccag?ccgcggactt?caaactgcta 900

gccctgaccg?aggcgcgcgg?aaagggccag?tccgccaagc?tctacaacgg?ccaccaccac 960

ctgctgatga?ctgagcagaa?ctgggccaac?caggcggccg?agcggcagcc?cccggcgctc 1020

aaggcttacc?cggcagcgtc?cacgcctgca?gcccccagcc?ccgtcggcag?cagctccccg 1080

ccactcgcgc?acgaggctga?ggcgggcgcg?gcgcccctgc?tgctggatgg?gagcggcagc 1140

agtctggagg?ggagcgccct?ggcagggacc?cccgaggagg?aggagcaggc?cgtgaccacc 1200

gcggcccaga?tgcaccagcc?gcccttgccc?ctcggagacc?caggtcgggc?cagcaaggcc 1260

agcagggcca?gcagcgggcg?ggccagaccg?gaggacttgg?ccatctag 1308

<210>2

<211>1308

<212>DNA

<213>Homo?sapiens

<400>2

atgggcgact?ggagctttct?gggaagactc?ttagaaaatg?cacaggagca?ctccacggtc 60

atcggcaagg?tttggctgac?cgtgctgttc?atcttccgca?tcttggtgct?gggggccgcg 120

gcggaggacg?tgtggggcga?tgagcagtca?gacttcacct?gcaacaccca?gcagccgggc 180

tgcgagaacg?tctgctacga?cagggccttc?cccatctccc?acatccgctt?ctgggcgctg 240

cagatcatct?tcgtgtccac?gcccaccctc?atctacctgg?gccacgtgct?gcacatcgtg 300

cgcatggaag?agaagaagaa?agagagggag?gaggaggagc?agctgaagag?agagagcccc 360

agccccaagg?agccaccgca?ggacaatccc?tcgtcgcggg?acgaccgcgg?cagggtgcgc 420

atggccgggg?cgctgctgcg?gacctacgtc?ttcaacatca?tcttcaagac?gctgttcgag 480

gtgggcttca?tcgccggcca?gtactttctg?tacggcttcg?agctgaagcc?gctctaccgc 540

tgcgaccgct?ggccctgccc?caacacggtg?gactgcttca?tctccaggcc?cacggagaag 600

accatcttca?tcatcttcat?gctggcggtg?gcctgcgcgt?ccctgctgct?caacatgctg 660

gagatctacc?acctgggctg?gaagaagctc?aagcagggcg?tgaccagccg?cctcggcccg 720

gacgcctccg?aggccccgct?ggggacagcc?gatcccccgc?ccctgccccc?cagctcccgg 780

ccgcccgccg?ttgccatcgg?gttcccaccc?tactatgcgc?acaccgctgc?gcccctggga 840

caggcccgcg?ccgtgggcta?ccccggggcc?ccgccaccag?ccgcggactt?caaactgcta 900

gccctgaccg?aggcgcgcgg?aaagggccag?tccgccaagc?tctacaacgg?ccaccaccac 960

ctgctgatga?ctgagcagaa?ctgggccaac?caggcggccg?agcggcagcc?cccggcgctc 1020

aaggcttacc?cggcagcgtc?cacgcctgca?gcccccagcc?ccgtcggcag?cagctccccg 1080

ccactcgcgc?acgaggctga?ggcgggcgcg?gcgcccctgc?tgctggatgg?gagcggcagc 1140

agtctggagg?ggagcgccct?ggcagggacc?cccgaggagg?aggagcaggc?cgtgaccacc 1200

gcggcccaga?tgcaccagcc?gcccttgccc?ctcggagacc?caggtcgggc?cagcaaggcc 1260

agcagggcca?gcagcgggcg?ggccagaccg?gaggacttgg?ccatctag 1308

<210>3

<211>435

<212>PRT

<213>Homo?sapiens

<400>3

Met?Gly?Asp?Trp?Ser?Phe?Leu?Gly?Arg?Leu?Leu?Glu?Asn?Ala?Gln?Glu

1 5 10 15

His?Ser?Thr?Val?Ile?Gly?Lys?Val?Trp?Leu?Thr?Val?Leu?Phe?Ile?Phe

20 25 30

Arg?Ile?Leu?Val?Leu?Gly?Ala?Ala?Ala?Glu?Asp?Val?Trp?Gly?Asp?Glu

35 40 45

Gln?Ser?Asp?Phe?Thr?Cys?Asn?Thr?Gln?Gln?Pro?Gly?Cys?Glu?Asn?Val

50 55 60

Cys?Tyr?Asp?Arg?Ala?Phe?Pro?Ile?Ser?His?Ile?Arg?Phe?Trp?Ala?Leu

65 70 75 80

Gln?Ile?Ile?Phe?Val?Ser?Thr?Pro?Thr?Leu?Ile?Tyr?Leu?Gly?His?Val

85 90 95

Leu?His?Ile?Val?Arg?Met?Glu?Glu?Lys?Lys?Lys?Glu?Arg?Glu?Glu?Glu

100 105 110

Glu?Gln?Leu?Lys?Arg?Glu?Ser?Pro?Ser?Pro?Lys?Glu?Pro?Pro?Gln?Asp

115 120 125

Asn?Pro?Ser?Ser?Arg?Asp?Asp?Arg?Gly?Arg?Val?Arg?Met?Ala?Arg?Ala

130 135 140

Leu?Leu?Arg?Thr?Tyr?Val?Phe?Asn?Ile?Ile?Phe?Lys?Thr?Leu?Phe?Glu

145 150 155 160

Val?Gly?Phe?Ile?Ala?Gly?Gln?Tyr?Phe?Leu?Tyr?Gly?Phe?Glu?Leu?Lys

165 170 175

Pro?Leu?Tyr?Arg?Cys?Asp?Arg?Trp?Pro?Cys?Pro?Asn?Thr?Val?Asp?Cys

180 185 190

Phe?Ile?Ser?Arg?Pro?Thr?Glu?Lys?Thr?Ile?Phe?Ile?Ile?Phe?Met?Leu

195 200 205

Ala?Val?Ala?Cys?Ala?Ser?Leu?Leu?Leu?Asn?Met?Leu?Glu?Ile?Tyr?His

210 215 220

Leu?Gly?Trp?Lys?Lys?Leu?Lys?Gln?Gly?Val?Thr?Ser?Arg?Leu?Gly?Pro

225 230 235 240

Asp?Ala?Ser?Glu?Ala?Pro?Leu?Gly?Thr?Ala?Asp?Pro?Pro?Pro?Leu?Pro

245 250 255

Pro?Ser?Ser?Arg?Pro?Pro?Ala?Val?Ala?Ile?Gly?Phe?Pro?Pro?Tyr?Tyr

260 265 270

Ala?His?Thr?Ala?Ala?Pro?Leu?Gly?Gln?Ala?Arg?Ala?Val?Gly?Tyr?Pro

275 280 285

Gly?Ala?Pro?Pro?Pro?Ala?Ala?Asp?Phe?Lys?Leu?Leu?Ala?Leu?Thr?Glu

290 295 300

Ala?Arg?Gly?Lys?Gly?Gln?Ser?Ala?Lys?Leu?Tyr?Asn?Gly?His?His?His

305 310 315 320

Leu?Leu?Met?Thr?Glu?Gln?Asn?Trp?Ala?Asn?Gln?Ala?Ala?Glu?Arg?Gln

325 330 335

Pro?Pro?Ala?Leu?Lys?Ala?Tyr?Pro?Ala?Ala?Ser?Thr?Pro?Ala?Ala?Pro

340 345 350

Ser?Pro?Val?Gly?Ser?Ser?Ser?Pro?Pro?Leu?Ala?His?Glu?Ala?Glu?Ala

355 360 365

Gly?Ala?Ala?Pro?Leu?Leu?Leu?Asp?Gly?Ser?Gly?Ser?Ser?Leu?Glu?Gly

370 375 380

Ser?Ala?Leu?Ala?Gly?Thr?Pro?Glu?Glu?Glu?Glu?Gln?Ala?Val?Thr?Thr

385 390 395 400

Ala?Ala?Gln?Met?His?Gln?Pro?Pro?Leu?Pro?Leu?Gly?Asp?Pro?Gly?Arg

405 410 415

Ala?Ser?Lys?Ala?Ser?Arg?Ala?Ser?Ser?Gly?Arg?Ala?Arg?Pro?Glu?Asp

420 425 430

Leu?Ala?Ile

435

<210>4

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>4

ccatcccagt?accatccag 19

<210>5

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>5

tctcttcagc?tgctcctcct 20

<210>6

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>6

agaacgtctg?ctacgacagg 20

<210>7

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>7

cctgcttgag?cttcttccag 20

<210>8

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>8

cgagctgaag?ccgctcta 18

<210>9

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>9

ctgccgggta?agccttga 18

<210>10

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>10

cgcggacttc?aaactgcta 19

<210>11

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>11

tctatctgct?ggtgggaagt?g 21

Claims (2)

1. the human GJA3 gene of a variation, it is characterized in that: its nucleotides sequence is classified as shown in the SEQ ID NO:1.

2. the application of the human GJA3 gene of the described variation of claim 1 in preparation congenital cataract disease gene diagnosing chip.

Images