CN1690222A - Method for detecting deafness-related gene mutation - Google Patents

Abstract

The invention relates to a detection method for deaf-correlated gene, which includes detecting the variation of the 250-sited base of GJB3 gene or the variation of expression product resulting from the base. The invention farther concerns the reagent box detecting the point of discontinuity of the gene and its application. The invention also relates to the application of the detection method.

Description

The detection method of deaf-related gene mutation

Technical field

The present invention relates to the detection method of deaf-related gene mutation.More particularly, the present invention relates to detect the method for the GJB3 gene 250G relevant → A sudden change with human inheritance's induced deafness.

Background technology

Connecting albumen is to form the albumen unit that the slit connects, play keying action in the communication between vertebrate cells, recent years, studies show that connection albumen and human diseases are in close relations, connect the many tissue systems of protein gene sudden change influence, and same transgenation has relation with different disease, can cause auditory system, the pathology of peripheral nerve and skin with the patient of β-connection protein mutation.

The slit connects the zone that contacts between cell, extensively distribute in different tissues.Ion and other small molecules (＜1kDa), can directly connect by the slit such as the second messenger, between the tenuigenin of two cells, exchange, so that the slit is connected in the cell-cell communication is extremely important.They comprise from tens to thousands of film hemichannels of striding, and also claim to connect corpusculum, these connect corpusculums and flanking cell same be connected corpusculum in conjunction with the formation hydrophilic pathway, make cell be in contact with one another.Connecting corpusculum is formed by six transmembrane proteins (promptly connecting albumen).20 kinds of dissimilar connection albumen are arranged,, can be divided into α, β, γ and non-classified according to different molecular weight and homology.Great majority connect protein gene and exist in groups, formed just as GJB2, GJB6 and GJA3 on No. 13 karyomit(e)s are long-armed, α and β-be connected protein gene group structure are closely similar, and two exons are arranged, first is a non-coding region, and second exon comprises whole coding region.Connect proteic subunit and can form identical or different connection corpusculum.Therefore connecting corpusculum can be formed by identical connection albumen, also can be formed by different connection protein subunits.In addition, two connection corpusculums that connect cell can be identical (passages of the same type), also can be different (heterozygosis channel type).

Connecting albumen has four to stride the film district, connects proteic carboxyl and aminoterminal and second, third ring of striding between the film district all is positioned at tenuigenin.Electron crystallography is discovered recently, and two concentric rings of film section formation are striden in six proteic four of connections in the connection corpusculum.Interior annular pore-forming, the lipid layer of outer ring surface cell membrane.The 3rd strides film district and two cell outskirts formation hole walls, and the former makes passage have the water permeability, and the latter participates in the interaction that two cells connect corpusculums.Interaction between the connection albumen different zones is vital for the permeability of slit connecting passage.The main mutability that connects protein sequence is positioned at the tenuigenin inner compartment, participates in the adjusting of channel activity.

The slit connects the selectivity that shows molecular size and electric charge, has specificity for forming the connection protein subunit that connects corpusculum.The opening of slit connecting passage can be regulated by several factors, and some connect albumen by phosphorylation, and voltage, acidifying or several other factors are regulated.As if the adjusting that the slit connects permeability has three kinds of different forms (quick, medium and long-term), the influence that adjusting factor for a long time most is vulnerable to suddenly change.

Slit connection in the inner ear is positioned between the sustenticular cell of Corti ' s device, and discovery is also arranged in the cell of cochlea sidewall.Having detected Cx26 in all in the ear cell expresses.Slit in the inner ear connects and is divided into two systems: epithelial cell slit connected system and reticular tissue slit connected system.Two kinds of systems are to keeping K in the endolymph ⁺The ion high density plays an important role, and this is most important for the normal function of keeping auditory system.After Corti ' s device hair cell is subjected to sonic stimulation, K ⁺Ion flows in the hair cell, connects by the slit then to turn back in the endolymph, keeps endolymphatic potential.

For the difference sudden change that connects protein gene is how to cause deafness, still not really clear now.Although for the case of recessive inheritance, the great majority sudden change causes lacking certain connection albumen, and some sudden change has changed the amino acid at specific position, the dominant effect may occur.Connect protein mutation and can influence the normal mode that cochlea is connected with Corti ' s device slit, may cause in the slit connects, occurring dissimilar connection corpusculum (forming slit of the same type connection), and unusual slit connection mode (may be owing to lack the connection albumen of a certain type or have unusual connection albumen) can influence K in the cochlea owing to Cx30 may will exist ⁺Ion circulation approach, this will influence endolymphatic potential, finally causes hair cell to lose function.GJB3 (Cx30) expresses in cochlea spiral limbus and spiral ligament, and in auditory nerve expression is arranged also, so except the dysfunction of cochlea, also may influence the nerve conduction of acoustic information.

GJB3 structure and function: structurally be connected protein similar with other, the GJB3 gene is a member who connects in the protein family, the CX31 composition that the coding slit connects, six connection protein moleculars are assembled into hemichannel or connect corpusculum, form a complete cell interior passageway with the corresponding part of flanking cell.Connect the sequence conservation that protein family has height, four membrane-spanning domains are arranged and link together by ring and two extracellular loop in the tenuigenin.In known sudden change, can cause deafness and dermatosis, but compare with GJB2, the sudden change of the GJB3 that is reported is less, and the GJB3 transgenation not only can cause the phonosensitive nerve deafness of dominance or recessive form heredity, but also relevant with dermatosis and peripheral neurophaty.

1998, China Xia Jiahui professor has reported two GJB3 sudden changes that cause dominant inheritance form high frequency auditory dysesthesia the earliest, the method that they use homology EST search and nest-type PRC clones the GJB3 gene, and with the GJB3 assignment of genes gene mapping at 1p33-p35, in the family of 42 heredopathias, carry out examination, in the deaf family in a Zhejiang, found a missense mutation, the 547th base that is positioned at the GJB3 gene coding region is mutated into A by G, make No. 183 amino acid that connects PROTEIN C x31 become Methionin by L-glutamic acid, they have also found a nonsense mutation in a Hunan family simultaneously, the 538th base of GJB3 becomes T by C, cause stopping at No. 180 amino acid position coding, they also use the method for RT-PCR and find to have in the murine inner ear tissue gjb3 to express, and this has verified that further GJB3 is relevant with the phonosensitive nerve deafness.2000, Liu etc. have reported two GJB3 sudden changes that cause the recessive inheritance deafness, they have screened 25 Chinese Sichuan familys of suffering from the recessive inheritance deafness, two GJB3 sudden changes have been found in two familys therein, one is in three base deletions of 423-425 (423-425delATT), causes the 141st coded amino acid-Isoleucine disappearance; Another is that the 423rd base becomes G by A, makes No. 141 amino acid become Xie Ansuan by Isoleucine.141 Isoleucine is positioned at and connects proteic the 3rd the conservative film district of striding, and the formation that connects hole wall for the slit is very crucial.Calendar year 2001, Lopez-Bigas has found a sudden change in a family of suffering from peripheral neurophaty and sensorineural deafness, at 196-198 three base deletions (GACdel) are arranged, and causes 66 aspartic acids disappearances.They also find have gjb3 to express in mouse cochlea and auditory nerve.

A wonderful frontier has been opened up in the discovery that connects the protein gene sudden change, for deafness patient is opened the new diagnosis of a fan, the gate of treatment.To connect proteic multi-functional characteristic consistent with the slit, connects the disease that proteic different sudden change can cause different tissues with a kind of.In addition, the proteic sudden change of different connections can cause same or similar disease.Though most of prelinguals all are that the deafness of the morbidity of growing up then mainly is because in several results with connection albumen missense mutation of the negative effect of dominance owing to lack due to certain specific connection albumen (Cx26 and Cx30).Severally cause that the sudden change of autosomal dominant inheritance induced deafness obviously is to have influence on amino-acid residue, these amino-acid residues are all most important for connecting albumen correct assembling or gate polarity (gating polarity), the function that connects the albumen passage for the wild-type restraining effect that has superiority.Wherein there are some amino acid changes on their caused disease phenotypes, to show very big variability, this may be owing to is connected albumen or other factors results of interaction with other, and these other connection albumen or factor has the effect that compensates deleterious effect.The pathologic, physiologic of these diseases can be better understood in the development of mouse model and the research that takes place in the sudden change that the mankind find.To be used as diagnostic tool now to deaf patient and kinsfolk thereof for connecting the suddenly change understanding of caused disease molecular basis of protein gene.Following research will concentrate on the development of treatment, can predict, and will make prevention and treatment measure to several years former some chronic diseases that also do not have to solve.

At present, only found 5 GJB3 gene mutation sites, helped further being familiar with the GJB3 gene for normal hearing physiological function meaning by finding new mutational site by domestic and international colleague's research, more deep understanding its with deaf relation.In former 5 mutational sites finding, wherein 4 mutational sites are found in Chinese population, and we this time find the new mutational site of GJB3 gene once more in Chinese population, this prompting may be at Chinese population GJB3 sudden change sickness rate than other crowd's height, and might exist mutantional hotspot, this will help carrying out clinically in the future deaf patient's GJB3 Mutation Screening work, for deafness patient diagnosis and treatment provide service.

Summary of the invention

The contriver chooses the GJB3 gene as research object, by carrying out examination 89 various phonosensitive nerve deafness patients and 103 normal good hearing control groups, find a mutational site new with deaf relevant GJB3 gene (250G → A, V84I).This mutational site all less than finding, this mutational site is described with deaf relevant, and this mutational site is positioned at second membrane-spanning domain (as shown in Figure 3) of Cx31 in 103 control groups.The sudden change in same position all has the report relevant with disease to other member Cx26 with Cx32 in the connection protein family, illustrates that this site is very important for Cx31.Based on this discovery, finished the present invention.

Therefore, according to the present invention, provide:

1, a kind of detection method of deaf-related gene, this method comprise the change that detects the 250th base of GJB3 gene or the change of the expression product that caused by this sequence change.

2, according to above the 1st detection method, the G that changes into of wherein said the 250th base sports A.

3, according to above the 1st detection method, wherein said expression product is for connecting PROTEIN C x31.

4, according to above the 1st detection method, proteic the 84th amino acids of changing into this GJB3 genes encoding of wherein said expression product is changed into Isoleucine by Xie Ansuan.

5, according to above the 1st detection method, wherein this method is undertaken by pcr amplification-direct sequencing.

6, according to above the 5th detection method, it is characterized in that comprising the steps: A: the coding region design primer for the GJB3 gene, carry out pcr amplification, perhaps, carry out pcr amplification for designing primer near the 250th base of GJB3 gene; The directly order-checking of B:PCR reaction back is relatively determined the GJB3 mutational site with the sequence among resulting sequence and the Genbank.

7, according to above the 6th detection method, it is characterized in that further comprising the steps: C: translate to determine the GJB3 gene mutation site by the normal reading frame.

8, according to above the 1st detection method, wherein the probe by the GJB3 gene with hybridize the change that detects described base from the vitro samples separated DNA.

9,, wherein detect the change of described base by restriction fragment length polymorphism according to above the 1st detection method.

10, a kind of test kit that detects GJB3 gene 250G → A mutational site, it comprises: amplification primers, dNTP is used for archaeal dna polymerase and damping fluid thereof that PCR reacts.

11, according to the application of above the 1st detection method in the diagnosis of deafness or treatment.

The base sequence of normal GJB3 gene is shown in sequence in the sequence table 1, and the proteic aminoacid sequence of the Cx31 of its coding is shown in sequence 2, and the base sequence of the GJB3 gene of sudden change is shown in sequence in the sequence table 3, and its aminoacid sequence is shown in sequence 4.As shown in sequence table, the sudden change of 250G → A has caused codon to become ATC by GTC, and the 84th Xie Ansuan of its coding becomes Isoleucine.

Fig. 1 has provided the contrast figure of GJB3 coding region amino acid and nucleic acid base, and shows the mutational site.Detect the method for any check point sudden change of this available this area that suddenlys change and carry out, for example the PCR-sequencing with the dna probe of mark, or is used the restriction fragment length polymorphism method.Therefore, used sequencing is exemplary in this paper embodiment, is not restrictive.

PCR (polymerase chain reaction) is a known method.In the experiment that the contriver carries out, collect the deaf inheritance resource, set up the hereditary hearing impairment resources bank.Collect various phonosensitive nerve deafness patients by deaf sick outpatient service, under the voluntary prerequisite of patient, signature informed consent postscript is left and taken the 5-10ml blood sample, and is set up the patient medical history database, incidence in detail record conditions of patients, the family and contact method.Then, use the extractive method extraction of phenol chloroform genomic dna, quantitatively put in storage the back ,-20 ℃ of preservations, and every part of DNA sample is all in detail corresponding to the patient clinical data of registering.Then, use online primer-design software Primer3 design primer, comprise the whole coding region of GJB3, use pcr amplification.The PCR product directly checks order: sequencing primer is identical with the pcr amplification primer, and the ABI 3730DNA of company sequenator (the order-checking collection of illustrative plates as shown in Figure 2) is used in forward and reverse order-checking.Sequence that obtains and the sequence among the Genebank are relatively determined the GJB3 mutational site.Translate to determine the mutational site of Cx31 by the normal reading frame.

The present invention also provides a kind of test kit that detects deaf-related gene GJB3-250G → A mutational site, its contain be contained in one or be divided in a plurality of containers in order to detect the composition of GJB3 gene 250G → A sudden change, what provide simultaneously with it can be through manufacturing, use and the marketing information of the audit of medication management mechanism of government, relevant medicine or biological products.For example, adopt pcr amplification after, directly the test kit in GJB3 gene 250G → A mutational site in the test sample can contain amplimer, dNTP is used for one or more of the archaeal dna polymerase of PCR reaction and damping fluid thereof etc.Those skilled in the art are known, above component only is schematic, for example, described primer can be according to known nucleotide sequence design, be generally 15-30 base, GC content is about 45-50%, combines with terminal specificity under suitable temperature, it can utilize special computer programming, and the archaeal dna polymerase of the described PCR of being used for reaction is the enzyme that can be used in pcr amplification.Using method is as follows:

Pcr amplification

To the coding region of GJB3 gene design PCR primer, reaction conditions is 94 ℃ of pre-sex change 5 minutes with software Primer3,94 ℃ of sex change 30 seconds, 58 ℃ of annealing temperatures 30 seconds, 72 ℃ were extended 40 seconds, 35 circulations, after reaction finishes again 72 ℃ extended 4 ℃ of preservations 7 minutes.

The PCR product purification

The MultiScreen-PCR plate that will contain the PCR product vacuumizes, and adds deionized water, leaves standstill, and the MultiScreen-PCR plate is placed on the mixing tank shake subsequently, is transferred in 96 orifice plates of another cleaning after the PCR product behind the purifying dissolves again.

Sequencing reaction and checking

Carry out sequencing reaction with the PCR primer as sequencing primer, on the ABI9700 thermal cycler, carry out sequencing reaction.After reaction finished, extension products was splined on ABI PRISM 3730DNA sequenator.Resulting order-checking collection of illustrative plates is analyzed, relatively can be found the mutational site with the standard sequence among the Genebank.This test kit can detect GJB3-250G → A mutational site quickly and easily, thereby is applied in the detection and deaf methods of treatment of deaf-related gene.

Said mutation also can detect with hybridization probe, and used hybridization probe can be and normal GJB3 nucleotide sequence hybridization, or with the nucleotide sequence hybridization of sudden change, or with their probe of complementary sequence hybridization.These probes can be used radio isotope, chromonic material or fluorescent substance mark.Before carrying out probe in detecting, use pcr amplification.Especially, utilize the allele specific probe, whether the sudden change that can examination be determined exists.

The be found to be deaf diagnosis and the treatment in this mutational site provide the foundation.Therefore the present invention also relates to described detection method in the diagnosis of deafness or the application in the treatment.For example, detecting to after 250G → A sudden change has taken place, normal gene can be imported the cell and the expression therein of carrying mutator gene, it can be recombinated with the endogenous mutator gene, thereby can carry out gene therapy.And the contriver shows that to the result that difference connects Argine Monohydrochloride sequence Study on Evolution this mutational site has high conservative, and therefore discovery of the present invention is significant.

The accompanying drawing summary

Fig. 1 is the contrast of GJB3 coding region amino acid and nucleic acid base: sudden change is positioned at second of GJB3 and strides the film district, the 250th base.Enclosing what come with square frame is the base and the amino acid of sudden change.

Fig. 2 is the GJB3 sequencing result, and the top is the normal control sequence, and the below is a mutant nucleotide sequence, the arrow indication be the mutational site (250G → A, V84I).

Fig. 3 is the CX31 structural representation,

The expression sudden change is positioned at second and strides film threshold (M2).

Fig. 4 is the different Argine Monohydrochloride sequence Study on Evolution (the prompting sudden change is positioned at the high conservative zone) that connect: multiple connection Argine Monohydrochloride sequence nucleotide sequence comparison, the arrow indication is the Val84 site, as seen this site is Xie Ansuan (Val) in all connection albumen, is illustrated as high conservative region.

Fig. 5 is a PCR reaction process synoptic diagram, shows temperature of reaction and time.

Fig. 6 is electrophoretic band figure among the embodiment 2.

Embodiment 1

Blood sample to be measured extracts the pcr amplification with the GJB3 gene coding region

One, the preparation of object blood sample DNA to be measured

1, research object: various sensorineural deafness patient 89 examples that deaf sick outpatient service is collected, wherein deaf 83 examples of non-syndrome type wherein have family history person's 10 examples, distribute 73 examples, deaf 6 examples.Normal control 103 examples.To all its medical history of participator's probe and family history, and it is carried out a medical examination, the otology inspection comprises otoscopy, audiological evaluation.Everyone blood sample collection 5-10ml after the signature Informed Consent Form.

2, extracting genome DNA: adopt the phenol chloroform extraction method.

First day

L, anticoagulation are done 1 times of dilution with PBS.

2, the lymph parting liquid (18 ℃-28 ℃) that adds 2 times of volumes in centrifuge tube is spread the blood that 1 times of volume of one deck has diluted, room temperature 1000 * g, centrifugal 20 minutes above.

3, supernatant is abandoned in suction, and karyocyte layer in the middle of the careful sucking-off changes in the 5mlEp pipe, and 5000 * g centrifugal 10 minutes, washes once with PBS then.5000 * g, centrifugal 10 minutes.

4, cell is suspended from the 2mlTE damping fluid, adds 10% SDS to final concentration 0.5% (100 μ l), protein kinase k 100-200 μ g/ml (10mg/ml), 50 ℃ water-bath 3-5 hour.

5, use the phenol chloroform extraction.With isopyknic saturated phenol, add mixing, 5000 * g, centrifugal 10 minutes.

6, suct clear liquid to new centrifuge tube, abandon lower sediment, add equal-volume phenol: chloroform mixture, mixing, 5000 * g, centrifugal 10 minutes.

7, suct clear liquid to new centrifuge tube, abandon lower sediment, add the equal-volume chloroform: iso pentane alcohol mixture, mixing, 5000 * g, centrifugal 10 minutes.

8, suct clear liquid to new centrifuge tube, abandon lower sediment, add the sodium acetate of 1/10 volume, mixing.

9, the dehydrated alcohol that adds 2.5 times of volumes.

10 ,-20 a ℃ deposit D NA spends the night.

Second day

11, high speed centrifugation, 10000 * g, 10 minutes, 4 ℃.

12, abandon supernatant, add 75% ethanol 2ml, high speed centrifugation 10000 * g, 5 minutes

13, abandon supernatant, dry up.

14, with the dissolving of TE damping fluid, (200 μ l TE/5ml whole bloods, 400TE/10ml whole blood)

15, packing.1% agarose electrophoresis and spectrophotometer are quantitative.

Two, the pcr amplification of GJB3 gene coding region

1. primer sequence:

Upstream primer: CX31-F:5 '-TTCATTCATACGATGGTTTTTCC-3 ' (nt3513-nt3535)

Downstream primer: CX31-R:5 '-TCACTCAGCCCCTGTAGGAC-3 ' (nt4477-nt4458)

Annotate: GJB3 gene order searching number: NT004511

2.PCR the foundation of reaction system (subordinate list 1):

The PCR reaction system of subordinate list 1:GJB3 gene

Title	Original liquid concentration	Application of sample amount (μ l)	The system final concentration
				Damping fluid	10×	?2.5	??1×
dNTP	2.5mM	?3.0	??400μM
				Primer	10μM	?1	??7.5pmol
The Taq enzyme	5units/μl	?1	??1U/μl
				Template (is carried in the step 1	100ng/μl	?1	??10ng/μl

The DNA that gets)
				The total reaction system	ddH 2O polishing to 25 μ l

Wherein damping fluid, dNTP, Taq enzyme are buied from precious biotech firm.Primer is given birth to worker company by Shanghai and is synthesized.

Reaction conditions: PCR is reflected on ABI company 9700 thermal cyclers and carries out, and reaction process (comprising temperature and time) as shown in Figure 5.

Embodiment 2

The purifying of GJB3 gene coding region pcr amplification product and quantitative

The purifying of PCR product---96 well plate method

1. in 96 orifice plates that the PCR product is housed, add 50ul sterilized water, mixing.

2. it is transferred in the Millipore purifying plate, be put on the vacuum pump suction filtration about 3 minutes, see in the purifying plate not having water to get final product.

3. the deionized water that adds 50ul in the purifying plate once more continues suction filtration, in the purifying plate, do not have water till.

4. the purifying plate is taken off from vacuum pump, in plate, add the deionized water of 20ul, static 15 minutes, shook again 15 minutes, be drawn onto then in new 96 orifice plates.

5. be kept in-20 degree refrigerators.

Electrophoresis is quantitative

1, sample is prepared

PCR product (2ul)+sample-loading buffer (6ul) is 8ul * 96 altogether

Get one 96 hole point templates, every hole adds sample damping fluid 6ul earlier, and from the chamber plate that the PCR product is housed, PCR product (2ul) is shifted out with the volley of rifle fire in every hole, transfers on the point template, mixing, centrifugal (chamber plate hole number corresponding one by one with 96 hole point templates).

2, flow process

1) joins glue (0.8% agarose): take by weighing the 2.4g agarose, be suspended in (500ml Erlenmeyer flask) among 300ml 0.5 * TBE.

2) colloidal sol: high fire is heated to and boils in the microwave oven, and constantly boiling number minute is noted not boiling, and takes out mixing therebetween.

3) cool glue: treat that glue dissolves fully, from microwave oven, take out that cool to about 60 ℃, (about 10ul 10mg/ml), shakes up to add 1 EB.

4) shop glue: obturage with adhesive plaster in dull and stereotyped two ends, the 250ml glue is all poured into flat board, inserts comb scale.

5) gluing: flat board is put in the electrophoresis chamber that fills electrophoresis liquid (0.5 * TBE, liquid level apart from glue face 1 to 2mm), pulled up comb scale.

6) application of sample:, add the DNA standard substance with single rifle at last with volley of rifle fire form application of sample in accordance with regulations.

7) walk glue: cover the electrophoresis chamber lid, check positive and negative level, open electrophoresis apparatus, regulate electrophoretic voltage.

8) quantitative: as to walk from well 1.5 to 2cm places when tetrabromophenol sulfonphthalein, close electrophoresis apparatus, carefully get glue, put into pickup camera and take a picture, carry out quantitatively.Quantitatively marker is DL2000, as shown in Figure 6, through behind the electrophoresis, 6 bands is arranged as seen, and fragment length is respectively 2000bp, 1000bp, and 750bp, 500bp, 250bp, 100bp, the total concn of DL2000 is 300ng/5ul.Get 5ul DL2000 during electrophoresis, therefore the content of every band is 50ng.Get 3ul (PCR product)+5ul (sample-loading buffer) during PCR product electrophoresis and carry out electrophoresis.The content of relatively judging the PCR product according to the gray-scale value of gray-scale value behind the PCR product electrophoresis and DL2000.

Embodiment 3

The direct order-checking of the GJB3 gene coding region pcr amplification product of purifying

1.PCR the purity of product D NA template and consumption requirement

DNA purity: OD ₂₆₀/ OD ₂₈₀=1.6～2.0.

DNA concentration: PCR product 10ng/ μ L.

The DNA consumption:

The PCR product

100-200bp??????1-3ng

200-500bp??????3-10ng

500-1000bp?????5-20ng

1000-2000bp????10-40ng

＞2000bp???????40-100ng

2. sequencing reaction

(1), the required reagent of sequencing reaction should be fresh preparation, need after autoclaved reagent must be sterilized, can use.The required equipment of sequencing reaction (first-class as 384 orifice plates, tip) should be cleaning sterile equally.

(2), in order to guarantee the fresh of sample and reaction reagent that check order, should be during application of sample in operation on ice.

(3), present reaction system is 5ul, all ingredients add-on is as follows:

Template	The PCR product of purifying (preparation among the embodiment 2)	??200-500bp	????3-10ng
				??500-1000bp	????5-20ng
		??1000-2000bp	????40-100ng
				????BigDye?v3.1*			????0.25ul
5 * damping fluid (Tris-HCl pH9.0, MgCl2)			????0.875ul(Kit)
				Primer			????3.2pmol
Mend to 5ul with sterilized water

* BigDye 3.1 is a kind of fluorescence dye that is used for sequencing reaction of u.s.a. applied biosystem company (ABI) production.

(4), sample is put in and does following reaction on the PCR instrument:

Step	Effect
		????1	96℃，2min.
????2	Repeat 30 circulations of following process: ● 96 ℃, 10sec; ● 50 ℃, 5sec; ● 60 ℃, 4min.
		????3	Remain on 4 ℃, up to purifying

(5), the sample that reacted will in time take off from the PCR instrument, the sample that will carry out purifying in the short period of time is positioned in 4 ℃ of refrigerators, surpass more than one day could purifying sample to be positioned over-20 ℃ of refrigerators freezing.

3. the purifying of sequencing reaction thing and order-checking

(1) in every hole, adds 20 μ L80% ethanol, 4, the centrifugal 30min of 000rpm; Sample panel is placed on the paper handkerchief of rolling well, gets rid of in whizzer, speed can not surpass 1,000rpm when getting rid of;

(2) in every hole, add 30ul 70% ethanol, 4, the centrifugal 10min of 000rpm gets rid of;

(3) repeat the 2nd operation that goes on foot;

(4) repeat the 2nd operation that goes on foot;

(5) sample panel is put in the clean drawer the dry 30min of lucifuge;

(6) add the 5ul methane amide, the envelope film is in the centrifugal being placed on-20 ℃ refrigerator;

(7) go up the preceding 95 ℃ of sex change of machine 5 minutes, placed 2 minutes on ice, sample is gone up in centrifugal back.Sequencer map as shown in Figure 2.

Embodiment 4

The mutational site Study on Evolution

Mutation analysis: use the Seqman in DNAStar5.0 (Lasergene inc.) software package ^TMSoftware carries out the sequence comparative analysis.The standard sequence that sequence that order-checking is obtained and NCBI retrieve is compared, and finds out mutant nucleotide sequence, found the mutational site (250G → A, V84I).

Study on Evolution: in database, find out various 28 kinds and connect the Argine Monohydrochloride sequence, use ClustalX software and compare.By discovering that the V84 site is positioned at second and strides the film district, conservative at connection protein family camber, referring to Fig. 4.

Embodiment 5

Detect deaf-related gene GJB3-250G → A mutational site test kit and application thereof

1, test kit contains:

(1) amplification primers:

Upstream primer: CX31-F:5 '-TTCATTCATACGATGGTTTTTCC-3 ' (nt3513-nt3535)

Downstream primer: CX31-R:5 '-TCACTCAGCCCCTGTAGGAC-3 ' (nt4477-nt4458)

Annotate: GJB3 gene order searching number: NT004511

(2) pcr amplification Taq enzyme 5U/ul

(3) 10 * damping fluids (contain 15ml MgCl ₂)

(4)dNTP????????2mM

(5)Big-Dye?mix

2, using method:

Mainly comprise the steps:

A: extract the DNA of blood sample to be measured, utilize above-mentioned primer, carry out the PCR reaction;

Directly order-checking behind the B:PCR reaction product purifying is with the sequence of gained and the existence in the relatively more definite mutational site of the standard sequence among the Genebank.

C: translate to determine Cx31 amino acid mutation site by the normal reading frame.

Concrete grammar is referring to embodiment 1,2,3.

It should be understood that, after having read foregoing of the present invention, those of ordinary skills can do various changes to the present invention under the situation that does not depart from scope of the present invention, therefore this change or its equivalents drop in the application's letter of authorization institute restricted portion equally.

Sequence table

SEQUENCE?LISTING

＜110〉PLA General Hospital, Sinogenomax Co., Ltd.

＜120〉detection method of deaf-related gene mutation

<130>

<160>4

<170>PatentIn?version?3.1

<210>1

<211>813

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(1)..(810)

<223>

<400>1

atg?gac?tgg?aag?aca?ctc?cag?gcc?cta?ctg?agc?ggt?gtg?aac?aag?tac?????48

Met?Asp?Trp?Lys?Thr?Leu?Gln?Ala?Leu?Leu?Ser?Gly?Val?Asn?Lys?Tyr

1???????????????5???????????????????10??????????????????15

tcc?aca?gcg?ttc?ggg?cgc?atc?tgg?ctg?tcc?gtg?gtg?ttc?gtc?ttc?cgg?????96

Ser?Thr?Ala?Phe?Gly?Arg?Ile?Trp?Leu?Ser?Val?Val?Phe?Val?Phe?Arg

20??????????????????25??????????????????30

gtg?ctg?gta?tac?gtg?gtg?gct?gca?gag?cgc?gtg?tgg?ggg?gat?gag?cag????144

Val?Leu?Val?Tyr?Val?Val?Ala?Ala?Glu?Arg?Val?Trp?Gly?Asp?Glu?Gln

35??????????????????40??????????????????45

aag?gac?ttt?gac?tgc?aac?acc?aag?cag?ccc?ggc?tgc?acc?aac?gtc?tgc????192

Lys?Asp?Phe?Asp?Cys?Asn?Thr?Lys?Gln?Pro?Gly?Cys?Thr?Asn?Val?Cys

50??????????????????55??????????????????60

tac?gac?aac?tac?ttc?ccc?atc?tcc?aac?atc?cgc?ctc?tgg?gcc?ctg?cag????240

Tyr?Asp?Asn?Tyr?Phe?Pro?Ile?Ser?Asn?Ile?Arg?Leu?Trp?Ala?Leu?Gln

65??????????????????70??????????????????75??????????????????80

ctc?atc?ttc?gtc?aca?tgc?ccc?tcg?ctg?ctg?gtc?atc?ctg?cac?gtg?gcc????288

Leu?Ile?Phe?Val?Thr?Cys?Pro?Ser?Leu?Leu?Val?Ile?Leu?His?Val?Ala

85??????????????????90??????????????????95

tac?cgt?gag?gag?cgg?gag?cgc?cgg?cac?cgc?cag?aaa?cac?ggg?gac?cag????336

Tyr?Arg?Glu?Glu?Arg?Glu?Arg?Arg?His?Arg?Gln?Lys?His?Gly?Asp?Gln

100?????????????????105?????????????????110

tgc?gcc?aag?ctg?tac?gac?aac?gca?ggc?aag?aag?cac?gga?ggc?ctg?tgg????384

Cys?Ala?Lys?Leu?Tyr?Asp?Asn?Ala?Gly?Lys?Lys?His?Gly?Gly?Leu?Trp

115?????????????????120?????????????????125

tgg?acc?tac?ctg?ttc?agc?ctc?atc?ttc?aag?ctc?atc?att?gag?ttc?ctc????432

Sequence table

Trp?Thr?Tyr?Leu?Phe?Ser?Leu?Ile?Phe?Lys?Leu?Ile?Ile?Glu?Phe?Leu

130?????????????????135?????????????????140

ttc?ctc?tac?ctg?ctg?cac?act?ctc?tgg?cat?ggc?ttc?aat?atg?ccg?cgc????480

Phe?Leu?Tyr?Leu?Leu?His?Thr?Leu?Trp?His?Gly?Phe?Asn?Met?Pro?Arg

145?????????????????150?????????????????155?????????????????160

ctg?gtg?cag?tgt?gcc?aac?gtg?gcc?ccc?tgc?ccc?aac?atc?gtg?gac?tgc????528

Leu?Val?Gln?Cys?Ala?Asn?Val?Ala?Pro?Cys?Pro?Asn?Ile?Val?Asp?Cys

165?????????????????170?????????????????175

tac?att?gcc?cga?cct?acc?gag?aag?aaa?atc?ttc?acc?tac?ttc?atg?gtg????576

Tyr?Ile?Ala?Arg?Pro?Thr?Glu?Lys?Lys?Ile?Phe?Thr?Tyr?Phe?Met?Val

180?????????????????185?????????????????190

ggc?gcc?tcc?gcc?gtc?tgc?atc?gta?ctc?acc?atc?tgt?gag?ctc?tgc?tac????624

Gly?Ala?Ser?Ala?Val?Cys?Ile?Val?Leu?Thr?Ile?Cys?Glu?Leu?Cys?Tyr

195?????????????????200?????????????????205

ctc?atc?tgc?cac?agg?gtc?ctg?cga?ggc?ctg?cac?aag?gac?aag?cct?cga????672

Leu?Ile?Cys?His?Arg?Val?Leu?Arg?Gly?Leu?His?Lys?Asp?Lys?Pro?Arg

210?????????????????215?????????????????220

ggg?ggt?tgc?agc?ccc?tcg?tcc?tcc?gcc?agc?cga?gct?tcc?acc?tgc?cgc????720

Gly?Gly?Cys?Ser?Pro?Ser?Ser?Ser?Ala?Ser?Arg?Ala?Ser?Thr?Cys?Arg

225?????????????????230?????????????????235?????????????????240

tgc?cac?cac?aag?ctg?gtg?gag?gct?ggg?gag?gtg?gat?cca?gac?cca?ggc????768

Cys?His?His?Lys?Leu?Val?Glu?Ala?Gly?Glu?Val?Asp?Pro?Asp?Pro?Gly

245?????????????????250?????????????????255

aat?aac?aag?ctg?cag?gct?tca?gca?ccc?aac?ctg?acc?ccc?atc?tga????????813

Asn?Asn?Lys?Leu?Gln?Ala?Ser?Ala?Pro?Asn?Leu?Thr?Pro?Ile

260?????????????????265?????????????????270

<210>2

<211>270

<212>PRT

＜213〉people

<400>2

Met?Asp?Trp?Lys?Thr?Leu?Gln?Ala?Leu?Leu?Ser?Gly?Val?Asn?Lys?Tyr

1???????????????5???????????????????10??????????????????15

Ser?Thr?Ala?Phe?Gly?Arg?Ile?Trp?Leu?Ser?Val?Val?Phe?Val?Phe?Arg

20??????????????????25??????????????????30

Val?Leu?Val?Tyr?Val?Val?Ala?Ala?Glu?Arg?Val?Trp?Gly?Asp?Glu?Gln

35??????????????????40??????????????????45

Lys?Asp?Phe?Asp?Cys?Asn?Thr?Lys?Gln?Pro?Gly?Cys?Thr?Asn?Val?Cys

Sequence table

50??????????????????55??????????????????60

Tyr?Asp?Asn?Tyr?Phe?Pro?Ile?Ser?Asn?Ile?Arg?Leu?Trp?Ala?Leu?Gln

65??????????????????70??????????????????75??????????????????80

Leu?Ile?Phe?Val?Thr?Cys?Pro?Ser?Leu?Leu?Val?Ile?Leu?His?Val?Ala

85??????????????????90??????????????????95

Tyr?Arg?Glu?Glu?Arg?Glu?Arg?Arg?His?Arg?Gln?Lys?His?Gly?Asp?Gln

100?????????????????105?????????????????110

Cys?Ala?Lys?Leu?Tyr?Asp?Asn?Ala?Gly?Lys?Lys?His?Gly?Gly?Leu?Trp

115?????????????????120?????????????????125

Trp?Thr?Tyr?Leu?Phe?Ser?Leu?Ile?Phe?Lys?Leu?Ile?Ile?Glu?Phe?Leu

130?????????????????135?????????????????140

Phe?Leu?Tyr?Leu?Leu?His?Thr?Leu?Trp?His?Gly?Phe?Asn?Met?Pro?Arg

145?????????????????150?????????????????155?????????????????160

Leu?Val?Gln?Cys?Ala?Asn?Val?Ala?Pro?Cys?Pro?Asn?Ile?Val?Asp?Cys

165?????????????????170?????????????????175

Tyr?Ile?Ala?Arg?Pro?Thr?Glu?Lys?Lys?Ile?Phe?Thr?Tyr?Phe?Met?Val

180?????????????????185?????????????????190

Gly?Ala?Ser?Ala?Val?Cys?Ile?Val?Leu?Thr?Ile?Cys?Glu?Leu?Cys?Tyr

195?????????????????200?????????????????205

Leu?Ile?Cys?His?Arg?Val?Leu?Arg?Gly?Leu?His?Lys?Asp?Lys?Pro?Arg

210?????????????????215?????????????????220

Gly?Gly?Cys?Ser?Pro?Ser?Ser?Ser?Ala?Ser?Arg?Ala?Ser?Thr?Cys?Arg

225?????????????????230?????????????????235?????????????????240

Cys?His?His?Lys?Leu?Val?Glu?Ala?Gly?Glu?Val?Asp?Pro?Asp?Pro?Gly

245?????????????????250?????????????????255

Asn?Asn?Lys?Leu?Gln?Ala?Ser?Ala?Pro?Asn?Leu?Thr?Pro?Ile

260?????????????????265?????????????????270

<210>3

Sequence table

<211>813

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(1)..(810)

<223>

<400>3

atg?gac?tgg?aag?aca?ctc?cag?gcc?cta?ctg?agc?ggt?gtg?aac?aag?tac?????48

Met?Asp?Trp?Lys?Thr?Leu?Gln?Ala?Leu?Leu?Ser?Gly?Val?Asn?Lys?Tyr

1???????????????5???????????????????10??????????????????15

tcc?aca?gcg?ttc?ggg?cgc?atc?tgg?ctg?tcc?gtg?gtg?ttc?gtc?ttc?cgg?????96

Ser?Thr?Ala?Phe?Gly?Arg?Ile?Trp?Leu?Ser?Val?Val?Phe?Val?Phe?Arg

20??????????????????25??????????????????30

gtg?ctg?gta?tac?gtg?gtg?gct?gca?gag?cgc?gtg?tgg?ggg?gat?gag?cag????144

Val?Leu?Val?Tyr?Val?Val?Ala?Ala?Glu?Arg?Val?Trp?Gly?Asp?Glu?Gln

35??????????????????40??????????????????45

aag?gac?ttt?gac?tgc?aac?acc?aag?cag?ccc?ggc?tgc?acc?aac?gtc?tgc????192

Lys?Asp?Phe?Asp?Cys?Asn?Thr?Lys?Gln?Pro?Gly?Cys?Thr?Asn?Val?Cys

50??????????????????55??????????????????60

tac?gac?aac?tac?ttc?ccc?atc?tcc?aac?atc?cgc?ctc?tgg?gcc?ctg?cag????240

Tyr?Asp?Asn?Tyr?Phe?Pro?Ile?Ser?Asn?Ile?Arg?Leu?Trp?Ala?Leu?Gln

65??????????????????70??????????????????75??????????????????80

ctc?atc?ttc?atc?aca?tgc?ccc?tcg?ctg?ctg?gtc?atc?ctg?cac?gtg?gcc????288

Leu?Ile?Phe?Ile?Thr?Cys?Pro?Ser?Leu?Leu?Val?Ile?Leu?His?Val?Ala

85??????????????????90??????????????????95

tac?cgt?gag?gag?cgg?gag?cgc?egg?cac?cgc?cag?aaa?cac?ggg?gac?cag????336

Tyr?Arg?Glu?Glu?Arg?Glu?Arg?Arg?His?Arg?Gln?Lys?His?Gly?Asp?Gln

100?????????????????105?????????????????110

tgc?gcc?aag?ctg?tac?gac?aac?gca?ggc?aag?aag?cac?gga?ggc?ctg?tgg????384

Cys?Ala?Lys?Leu?Tyr?Asp?Asn?Ala?Gly?Lys?Lys?His?Gly?Gly?Leu?Trp

115?????????????????120?????????????????125

tgg?acc?tac?ctg?ttc?agc?ctc?atc?ttc?aag?ctc?atc?att?gag?ttc?ctc????432

Trp?Thr?Tyr?Leu?Phe?Ser?Leu?Ile?Phe?Lys?Leu?Ile?Ile?Glu?Phe?Leu

130?????????????????135?????????????????140

ttc?ctc?tac?ctg?ctg?cac?act?ctc?tgg?cat?ggc?ttc?aat?atg?ccg?cgc????480

Phe?Leu?Tyr?Leu?Leu?His?Thr?Leu?Trp?His?Gly?Phe?Asn?Met?Pro?Arg

145?????????????????150?????????????????155?????????????????160

ctg?gtg?cag?tgt?gcc?aac?gtg?gcc?ccc?tgc?ccc?aac?atc?gtg?gac?tgc????528

Leu?Val?Gln?Cys?Ala?Asn?Val?Ala?Pro?Cys?Pro?Asn?Ile?Val?Asp?Cys

165?????????????????170?????????????????175

tac?att?gcc?cga?cct?acc?gag?aag?aaa?atc?ttc?acc?tac?ttc?atg?gtg????576

Tyr?Ile?Ala?Arg?Pro?Thr?Glu?Lys?Lys?Ile?Phe?Thr?Tyr?Phe?Met?Val

Sequence table

180?????????????????185?????????????????190

ggc?gcc?tcc?gcc?gtc?tgc?atc?gta?ctc?acc?atc?tgt?gag?ctc?tgc?tac????624

Gly?Ala?Ser?Ala?Val?Cys?Ile?Val?Leu?Thr?Ile?Cys?Glu?Leu?Cys?Tyr

195?????????????????200?????????????????205

ctc?atc?tgc?cac?agg?gtc?ctg?cga?ggc?ctg?cae?aag?gac?aag?cct?cga????672

Leu?Ile?Cys?His?Arg?Val?Leu?Arg?Gly?Leu?His?Lys?Asp?Lys?Pro?Arg

210?????????????????215?????????????????220

ggg?ggt?tgc?agc?ccc?tcg?tcc?tcc?gcc?agc?cga?gct?tcc?acc?tgc?cgc????720

Gly?Gly?Cys?Ser?Pro?Ser?Ser?Ser?Ala?Ser?Arg?Ala?Ser?Thr?Cys?Arg

225?????????????????230?????????????????235?????????????????240

tgc?cac?cac?aag?ctg?gtg?gag?gct?ggg?gag?gtg?gat?cca?gac?cca?ggc????768

Cys?His?His?Lys?Leu?Val?Glu?Ala?Gly?Glu?Val?Asp?Pro?Asp?Pro?Gly

245?????????????????250?????????????????255

aat?aac?aag?ctg?cag?gct?tca?gca?ccc?aac?ctg?acc?ccc?atc?tga????????813

Asn?Asn?Lys?Leu?Gln?Ala?Ser?Ala?Pro?Asn?Leu?Thr?Pro?Ile

260?????????????????265?????????????????270

<210>4

<211>270

<212>PRT

＜213〉people

<400>4

Met?Asp?Trp?Lys?Thr?Leu?Gln?Ala?Leu?Leu?Ser?Gly?Val?Asn?Lys?Tyr

1???????????????5???????????????????10??????????????????15

Ser?Thr?Ala?Phe?Gly?Arg?Ile?Trp?Leu?Ser?Val?Val?Phe?Val?Phe?Arg

20??????????????????25??????????????????30

Val?Leu?Val?Tyr?Val?Val?Ala?Ala?Glu?Arg?Val?Trp?Gly?Asp?Glu?Gln

35??????????????????40??????????????????45

Lys?Asp?Phe?Asp?Cys?Asn?Thr?Lys?Gln?Pro?Gly?Cys?Thr?Asn?Val?Cys

50??????????????????55??????????????????60

Tyr?Asp?Asn?Tyr?Phe?Pro?Ile?Ser?Asn?Ile?Arg?Leu?Trp?Ala?Leu?Gln

65??????????????????70??????????????????75??????????????????80

Leu?Ile?Phe?Ile?Thr?Cys?Pro?Ser?Leu?Leu?Val?Ile?Leu?His?Val?Ala

85??????????????????90??????????????????95

Tyr?Arg?Glu?Glu?Arg?Glu?Arg?Arg?His?Arg?Gln?Lys?His?Gly?Asp?Gln

100?????????????????105?????????????????110

Sequence table

Cys?Ala?Lys?Leu?Tyr?Asp?Asn?Ala?Gly?Lys?Lys?His?Gly?Gly?Leu?Trp

115?????????????????120?????????????????125

Trp?Thr?Tyr?Leu?Phe?Ser?Leu?Ile?Phe?Lys?Leu?Ile?Ile?Glu?Phe?Leu

130?????????????????135?????????????????140

Phe?Leu?Tyr?Leu?Leu?His?Thr?Leu?Trp?His?Gly?Phe?Asn?Met?Pro?Arg

145?????????????????150?????????????????155?????????????????160

Leu?Val?Gln?Cys?Ala?Asn?Val?Ala?Pro?Cys?Pro?Asn?Ile?Val?Asp?Cys

165?????????????????170?????????????????175

Tyr?Ile?Ala?Arg?Pro?Thr?Glu?Lys?Lys?Ile?Phe?Thr?Tyr?Phe?Met?Val

180?????????????????185?????????????????190

Gly?Ala?Ser?Ala?Val?Cys?Ile?Val?Leu?Thr?Ile?Cys?Glu?Leu?Cys?Tyr

195?????????????????200?????????????????205

Leu?Ile?Cys?His?Arg?Val?Leu?Arg?Gly?Leu?His?Lys?Asp?Lys?Pro?Arg

210?????????????????215?????????????????220

Gly?Gly?Cys?Ser?Pro?Ser?Ser?Ser?Ala?Ser?Arg?Ala?Ser?Thr?Cys?Arg

225?????????????????230?????????????????235?????????????????240

Cys?His?His?Lys?Leu?Val?Glu?Ala?Gly?Glu?Val?Asp?Pro?Asp?Pro?Gly

245?????????????????250?????????????????255

Asn?Asn?Lys?Leu?Gln?Ala?Ser?Ala?Pro?Asn?Leu?Thr?Pro?Ile

260?????????????????265?????????????????270

Claims (11)

1, a kind of detection method of deaf-related gene, this method comprise the change that detects the 250th base of GJB3 gene or the change of the expression product that caused by this sequence change.

2, according to the detection method of claim 1, the G that changes into of wherein said the 250th base sports A.

3, according to the detection method of claim 1, wherein said expression product is for connecting PROTEIN C x31.

4, according to the detection method of claim 1, proteic the 84th amino acids of changing into this GJB3 genes encoding of wherein said expression product is changed into Isoleucine by Xie Ansuan.

5, according to the detection method of claim 1, wherein this method is undertaken by pcr amplification-direct sequencing.

6, according to the detection method of claim 5, it is characterized in that comprising the steps: A: for the coding region of GJB3 gene design primer, carry out pcr amplification, perhaps, carry out pcr amplification for designing primer near the 250 base of GJB3 gene; The directly order-checking of B:PCR reaction back is relatively determined the GJB3 mutational site with the sequence among resulting sequence and the Genbank.

7, according to the detection method of claim 6, it is characterized in that further comprising the steps: C: translate to determine the GJB3 gene mutation site by the normal reading frame.

8, according to the detection method of claim 1, wherein the probe by the GJB3 gene with hybridize the change that detects described base from the vitro samples separated DNA.

9, according to the detection method of claim 1, wherein detect the change of described base by restriction fragment length polymorphism.

10, a kind of test kit that detects GJB3 gene 250G → A mutational site, it comprises: amplification primers, dNTP is used for archaeal dna polymerase and damping fluid thereof that PCR reacts.

11, according to the detection method of claim 1 in the diagnosis of deafness or the application in the treatment.

Images