CN1730655A - Human genetic deaf related GJB6 mutant gent and its uses in gennetic deaf diagnosis - Google Patents

Abstract

The invention relates to human genetic deaf related GJB6 mutant gene, wherein the mutation gene is related to human genetic deafness, the invention also provides a method for diagnosing the cause and type of the deaf occurrence through determining the existence of the mutation gene in the body of the patients, the mutation gene and the detection method can be used for GJB6 mutation screening for deaf patients clinically.

Description

GJB6 mutator gene relevant and the application in the diagnosing hereditary deafness thereof with human inheritance's induced deafness

Technical field

The present invention relates to new GJB6 mutator gene, relate in particular to the new GJB6 mutator gene relevant with human inheritance's induced deafness.

The invention still further relates to and use the detection method that new GJB6 mutator gene detects human inheritance's induced deafness.

The invention still further relates to the test kit that is used to detect new GJB6 mutator gene.

The invention still further relates to the new application of GJB6 mutator gene in the diagnosing human hereditary hearing impairment.

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 (＜1 kDa) can directly connect by the slit such as the second messenger, exchange between the tenuigenin of two cells, 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.

The GJB6 gene structurally is connected protein similar with other, be a member who connects in the protein family, the CX30 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.Can cause auditory dysesthesia in known sudden change, but compare with GJB2 that the sudden change of the GJB6 that is reported is less, the GJB6 transgenation not only can cause the phonosensitive nerve deafness of dominance or recessive form heredity, but also relevant with dermatosis.GJB6 and the GJB2 13p12 zone that coexists for the deaf family that is positioned at this zone, when GJB2 does not find to suddenly change, just should consider to also have the another one deaf gene in this zone.Grifa A. etc. are in an Italian family, a kind of missense mutation T5M that finds GJB6 (Cx30) causes the tardy induced deafness of autosomal dominant inheritance, but the point mutation of GJB6 is not a kind of deaf common reason that causes, in Israel and French family, found the large stretch of disappearance of GJB6, but all there is not to determine the concrete scope that lacks, Castillo etc. in 422 familys (364 Spain's familys and 58 Cuba's familys) have found a large fragment deletion (342Kb) that comprises GJB6, disappearance does not comprise in the GJB2 scope, in Spain's family of suffering from non-syndromes type prelingual, higher ratio is arranged, morbidity when suddenling change at homozygotic state or with GJB2.This large fragment deletion of GJB6 may be to cause a deaf common cause in infancy.But but do not find this disappearance in Australia and Chinese family, prompting GJB6 large fragment deletion may just exist in certain crowd.

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 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.

Summary of the invention

One object of the present invention is the new GJB6 mutator gene that provides relevant with human inheritance's induced deafness, and this GJB6 mutator gene has 233C → A heterozygous mutant, and its amino acids coding has the mutational site of A78D accordingly.

Another object of the present invention is to provide the method for diagnosing human hereditary hearing impairment, by detecting from whether existing GJB6 mutator gene 233C → A sudden change to judge deaf reason and the type that takes place of this patient in patient's the sample to be tested, and then provide reference for clinical diagnosis and treatment.

A further object of the present invention is to be provided for detecting the GJB6 sudden change (test kit of gene of 233C → A).

The GJB6 sudden change that another purpose of the present invention is the to provide new (233C → A) application of gene in diagnosis or treatment human inheritance induced deafness.

According to an aspect of the present invention, by in 141 various phonosensitive nerve deafness patient groups and 150 normal good hearing control groups, carrying out examination, find a congenital utmost point severe deafness infant and its father have the new mutational site of the GJB6 gene relevant with hereditary hearing impairment (233C → A, A78D).This mutational site all is not found in 150 control groups, illustrates that this mutational site is relevant with hereditary hearing impairment.Fig. 1 provides the contrast figure of GJB6 coding region amino acid and nucleic acid base, and the mutational site is shown.This mutational site is positioned at GJB6 expression of gene product---and connect second membrane-spanning domain (Fig. 2) of PROTEIN C x30, illustrate that this site is very important for Cx30.Show that by the result who difference is connected Argine Monohydrochloride sequence Study on Evolution this mutational site has high conservative.

According to a further aspect in the invention, the method for diagnosing human hereditary hearing impairment provided by the present invention is to come from deaf occurrence cause and the type that whether exists GJB6 gene 233C → A (A78D) sudden change to judge this patient in patient's the sample to be tested by detection.

Detect the method for any check point sudden change of this available this area that suddenlys change and carry out, the GJB6 gene DNA probe hybridization method of for example digestion with restriction enzyme reaction, PCR (polymerase chain reaction)-sequencing, employing mark or with the restriction fragment length polymorphism method etc.

In one embodiment of the invention, adopt pcr amplification-direct sequencing to detect sample, specifically comprise the steps:

1) sample of collection individuality to be measured, for example blood, body fluid or tissue extract DNA;

2) be template with this DNA, carry out the PCR reaction, obtain the PCR reaction product with PCR primer near the design of the coding region GJB6 gene or the 233rd base of GJB6 gene;

3) the PCR product that obtains is carried out direct sequencing analysis, the sequence of resulting sequence and GJB6 normal gene is compared, determine whether to exist the GJB6 mutational site.

4) judge according to above result whether individuality to be measured is the hereditary hearing impairment that GJB6 transgenation 233C → A causes.

Further, this method can optionally comprise the steps:

5) translate to determine whether to exist 78A → D Cx30 amino acid mutation site by the normal reading frame.

In another embodiment of the invention, adopt the endonuclease reaction of APaI restriction enzyme to detect mutator gene, after above-mentioned steps 2 resulting PCR reaction product are carried out endonuclease reaction with ApaI, agarose electrophoresis detects, normal gene can be cut by ApaI, and mutator gene can not be cut by the ApaI enzyme, determines whether to exist the mutational site thus; Judge according to detected result whether individuality to be measured is the hereditary hearing impairment that GJB6 gene 233C → the A sudden change causes.

Above-mentioned steps 2 resulting PCR reaction product can also detect with hybridization probe, and used hybridization probe can be and normal GJB6 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, especially can utilize the allele specific probe, whether have the sudden change that has been determined with examination.

The PCR primer that uses in aforesaid method can be generally 15-30 base according to known nucleotide sequence design, and GC content is about 45-50%, combines with terminal specificity under suitable temperature, and it can utilize special computer programming.In a specific embodiment of the present invention, to use primer-design software and designed one couple of PCR primers, its sequence is:

CX30-F：5’-TCAGGGATAAACCAGCGCAAT-3’(nt1916-nt1935)

CX30-R：5’-GTTGGTATTGCCTTCTGGAGAAGA-3’(nt1066-nt1935)

The standard sequence of GJB6 normal gene can reference example such as Genbank NT_009799.

In accordance with a further aspect of the present invention, the present invention further provides the test kit that is used to detect GJB6 gene 233C → A sudden change, can comprise one or more combination of following several reagent in the test kit:

From testing sample, extract the reagent of DNA;

The PCR primer and the corresponding PCR reaction reagent that are used near the coding region of the 233rd base of amplified sample DNA GJB6 gene or GJB6 gene;

Pcr amplification product is carried out the reagent of digestion with restriction enzyme reaction; And/or

The reagent that pcr amplification product is checked order.

For example, a test kit that detects GJB6 gene 233C → A sudden change is provided in one embodiment of the invention, be equipped with in the container in order to detect the composition of GJB6 gene 233C → 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, after adopting pcr amplification, the direct test kit in GJB6 gene 233C → A mutational site in the test sample can contain amplimer, dNTP, be used for one or more of the archaeal dna polymerase of PCR reaction and damping fluid, endonuclease reaction and/or the required reagent of sequencing reaction etc.It is known to those skilled in the art that above component only is that schematically for example, described primer can adopt above-mentioned a pair of CX30-F and CX30-R primer, the archaeal dna polymerase of the described PCR of being used for reaction is the enzyme that can be used in pcr amplification.The using method of described test kit mainly comprises the steps:

(1) DNA of extraction blood sample to be measured utilizes above-mentioned a pair of CX30-F and CX30-R primer, carries out the PCR reaction;

(2) the endonuclease reaction detection that suddenlys change;

(3) directly order-checking behind the 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 Genbank;

(4) translate to determine whether to exist Cx30 amino acid mutation site by the normal reading frame.

This test kit can detect GJB6 gene 233C → A mutational site quickly and easily, thereby is applied in the detection and deaf diagnosis or methods of treatment of deaf-related gene.

According to another aspect of the invention, the application of GJB6 mutator gene in diagnosis or treatment human inheritance induced deafness is provided, by detecting from whether existing GJB6 gene 233C → A sudden change to judge this patient's deaf occurrence cause and type in patient's the sample to be tested, and then provide reference for clinical diagnosis and treatment; In addition; aspect further clinical treatment,, normal gene can be imported cell and the expression therein that carry mutator gene detecting to after GJB6 gene 233C → A sudden change has taken place; it can be recombinated with the endogenous mutator gene, thereby can carry out gene therapy.

The present invention provides first and had GJB6 gene 233C → A sudden change in Chinese population, and illustrates that this mutator gene is relevant with hereditary hearing impairment.Also do not discover the GJB6 transgenation among the auditory dysesthesia patient at home at present, so this mutational site is significant for the diagnosis of deafness patient.Whether the present invention has proposed to exist the new sudden change of GJB6 gene to diagnose the deaf reason that takes place and the detection method of type among the patient by detecting simultaneously, this will help carrying out clinically the GJB6 Mutation Screening work of deafness patient, for deafness patient provides diagnosis and treatment service.

Below in conjunction with accompanying drawing,, describe in detail but do not limit the present invention by explanation to better embodiment of the present invention.

Brief description of drawings

Fig. 1 is the contrast of GJB6 coding region amino acid and nucleic acid base: sudden change is positioned at second of Cx30 and strides the film district, the 233rd base.Enclosing what come with square frame is the base and the amino acid of sudden change;

Fig. 2 is the CX30 structural representation,

The expression sudden change is positioned at second membrane-spanning domain (M2);

Fig. 3 is a PCR reaction process synoptic diagram in the inventive method, shows temperature of reaction and time;

Fig. 4 is in the inventive method, and the PCR product is carried out the quantitative agarose gel electrophoretogram of electrophoresis, there is shown the fragment position of quantitative Marker;

Fig. 5 is the inventive method GJB6 gene 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 (233C → A, A78D);

Fig. 6 is restriction enzyme digestion and electrophoresis figure in mutational site in the inventive method,

Among the figure from left to right: first swimming lane is that molecular weight standard, the second and the 3rd swimming lane are that restriction enzyme mapping, the 4th and the 5th swimming lane of normal gene is the restriction enzyme mapping of heterozygous mutant gene,

Restriction enzyme A paI can cut at 234 and 235 bit bases, and make normal people PCR product fragment be cut into 623bp and two fragments of 247bp by original 870bp, and this restriction enzyme site disappears after the sudden change, can not cut, because this is organized two examples and is heterozygous mutant, so be shown as 3 bands on the running gel figure;

Fig. 7 is the different Argine Monohydrochloride sequence Study on Evolution that connect,

By to multiple connection Argine Monohydrochloride sequence alignment, the arrow indication is the Ala78 site, and as seen all connect that this site is L-Ala (Ala) in albumen, are illustrated as high conservative region, and prompting 233C → A sudden change is positioned at the high conservative zone.

The embodiment of invention

Choose the GJB6 gene as research object, by in 141 various phonosensitive nerve deafness patient groups and 150 normal good hearing control groups, carrying out examination, find a congenital utmost point severe deafness infant and its father have a mutational site new with deaf relevant GJB6 gene (233C → A, A78D).This mutational site in 150 control groups all less than finding, illustrate this mutational site and deafness be divided into from.This mutational site is positioned at second membrane-spanning domain that connects PROTEIN C x30.

In the research that the present invention 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 GJB6, use pcr amplification.Can carry out endonuclease reaction to the PCR product: use the APaI restriction enzyme and normal people PCR product can be cut into two fragments, and after sudden change occurring, restriction enzyme site disappears, and can not cut.Also can directly check order to the PCR product: sequencing primer is identical with the pcr amplification primer, and the ABI 3730DNA of company sequenator is used in forward and reverse order-checking.The sequence and the sequence among the Genebank that obtain are relatively determined the GJB6 mutational site.Translate to determine the mutational site of Cx30 by the normal reading frame.

The pcr amplification of the extraction of blood sample to be measured and GJB6 gene coding region

[embodiment 1]

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

1, research object: various sensorineural deafness patient 141 examples that deaf sick outpatient service is collected, normal control 150 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

1) anticoagulation is done 1 times of dilution with PBS.

2) the lymph parting liquid (18 ℃-28 ℃) of 2 times of volumes of adding 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) dehydrated alcohol of 2.5 times of volumes of adding.

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 GJB6 gene coding region

1, primer sequence

Upstream primer:

CX30-F：5’-TCAGGGATAAACCAGCGCAAT-3′(nt1916-nt1935)

Downstream primer:

CX30-R：5’-GTTGGTATTGCCTTCTGGAGAAGA-3′(nt1066-nt1935)

Annotate: GJB6 gene order searching number: NT 009799

2, the foundation of PCR reaction system

The composition of the PCR reaction system of GJB6 gene sees Table 1.

The PCR reaction system of table 1GJB6 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 (DNA that extracts in the step 1)	100ng/μl	1					10ng/μl
			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 3.

The purifying of GJB6 gene coding region pcr amplification product and quantitative

[embodiment 2]

One, the purifying of PCR product

1, in 96 orifice plates that the PCR product is housed, adds 50 μ l sterilized waters, 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 50 μ l 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 20 μ l, static 15 minutes, shook again 15 minutes, be drawn onto then in new 96 orifice plates.

5, be kept in-20 degree refrigerators.

Two, electrophoresis is quantitative

1, sample is prepared

PCR product (2 μ l)+sample-loading buffer (6 μ l) is totally 8 μ l * 96

Get one 96 hole point templates, every hole adds sample damping fluid 6 μ l earlier, and from the chamber plate that the PCR product is housed, PCR product (2 μ l) 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 10 μ l 10mg/ml), shake 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 DL 2000, as shown in Figure 4, 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/5 μ l.Get 5ul DL2000 during electrophoresis, therefore the content of every band is 50ng.Get 3 μ l (PCR product)+5 μ l (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.

The direct order-checking of the GJB6 gene coding region pcr amplification product of purifying

[embodiment 3]

1, the purity of PCR 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 can use after autoclaved reagent must be sterilized.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 5 μ l, and all ingredients add-on sees Table 2.

The sequencing reaction system of table 2GJB6 gene PCR amplified production

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 *			0.25μl
5 * damping fluid (Tris-HCl pH9.0, MgCl 2)			0.875μl(Kit)
				Primer			3.2pmol
Mend to 5 μ l 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 on the PCR instrument, and the process of the reaction of doing sees Table 3.

The sequencing reaction process of table 3GJB6 gene PCR amplified production

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) sample that has reacted will in time take off from the PCR instrument, and 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 l, 80% ethanol, the centrifugal 30min of 4000rpm; Sample panel is placed on the paper handkerchief of rolling well, gets rid of in whizzer, speed can not surpass 1000rpm when getting rid of;

(2) in every hole, add 30 μ l, 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 5 μ l methane amides, 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 5.

The mutational site endonuclease reaction detects

[embodiment 4]

The composition of endonuclease reaction system sees Table 4, and reaction conditions is 37 ℃ of water-baths 2 hours.The application agarose electrophoresis detects, and method is quantitative referring to electrophoresis among the embodiment 2.The endonuclease reaction electrophorogram as shown in Figure 6.

Restriction analysis is to have found a restriction enzyme A paI restriction enzyme site in this mutational site, when not undergoing mutation, restriction enzyme A paI can cut at 234 and 235 bit bases, and make PCR product fragment be cut into 623bp and two fragments of 247bp by original 870bp, and this restriction enzyme site disappears after the sudden change, can not cut.Because this is organized two examples and is heterozygous mutant, so be shown as 3 bands on the running gel figure.

Table 4 endonuclease reaction system

Reagent	Sample size
		ApaI	1μl
10×L Buffer	2μl
		The PCR product	5μl
ddH 2O	Up to 20μl

ApaI restriction enzyme and 10 * L Buffer purchase in precious biotech firm, and the PCR product is the pcr amplification product among the embodiment 2.

The mutational site Study on Evolution

[embodiment 5]

Mutation analysis: use the Seqman in DNAStar5.O (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 (233C → A, A78D).

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 Ala78 site is positioned at second and strides the film district, conservative at connection protein family camber, referring to Fig. 7.

Detect deaf-related gene GJB6 gene 233C → A mutational site test kit and application thereof

[embodiment 6]

1, test kit contains:

(1) amplification primers

CX30-F：5’-TCAGGGATAAACCAGCGCAAT-3’(nt1916-nt1935)

CX30-R：5’-GTTGGTATTGCCTTCTGGAGAAGA-3’(nt1066-nt1935)

Annotate: GJB6 gene order searching number: NT_009799

(2) pcr amplification Taq enzyme 5U/ μ l

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

(4)dNTP 2mM

(5) ApaI restriction enzyme

(6)10×L Buffer

(7)Big-Dye mix

2, using method

Mainly comprise the steps:

1) pcr amplification

With the coding region of 3 pairs of GJB6 genes of software Primer design PCR primer, reaction conditions is 94 ℃ of pre-sex change 4 minutes, 94 ℃ of sex change 1 minute, annealing temperature 60 ℃ minutes, 72 ℃ were extended 2 minutes, 35 circulations, after reaction finishes again 72 ℃ extended 4 ℃ of preservations 5 minutes;

2) 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;

3) endonuclease reaction

Use APaI restriction enzyme 1 μ l, 10 * L Buffer, 2 μ l, PCR product 5 μ l add deionized water to 20 μ l, 37 ℃ of water-baths 2 hours, normal people PCR product is cut into two fragments, and after sudden change occurring, restriction enzyme site disappears, and can not cut;

4) 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 Genbank;

5) translate to determine whether to exist Cx30 amino acid mutation site by the normal reading frame.

Concrete grammar is referring to embodiment 1,2,3,4.

Above detailed description of the present invention does not limit the present invention, and those skilled in the art can make various changes and distortion according to the present invention, only otherwise break away from spirit of the present invention, all should belong to the defined scope of the appended claim of the present invention.

Claims (9)

1, GJB6 mutator gene is characterized in that it has 233C → A heterozygous mutant, and described sudden change is relevant with human inheritance's induced deafness.

2, the described GJB6 mutator gene of claim 1 is characterized in that described mutator gene amino acids coding has the mutational site of A78D.

3, a kind of detection method is characterized in that deriving from the sample of individuality to be measured whether have the described GJB6 transgenation point of claim 1 by detection, and diagnoses the generation and the type of idiogenetics induced deafness to be measured.

4, the described detection method of claim 3 is characterized in that comprising the steps:

1) blood, body fluid or the tissue samples of collection individuality to be measured extract DNA;

2) be template with this DNA, carry out the PCR reaction, obtain the PCR reaction product with PCR primer near the design of the coding region GJB6 gene or the 233rd base of GJB6 gene;

3) the PCR product that obtains is carried out direct sequencing analysis, the sequence of resulting sequence and GJB6 normal gene is compared, determine whether to exist the GJB6 mutational site;

4) judge according to above result whether individuality to be measured is the hereditary hearing impairment that GJB6 gene 233C → the A sudden change causes.

5, the described detection method of claim 4 is characterized in that described detection method further comprises the steps:

5) translate to determine whether to exist A78D amino acid mutation site by the normal reading frame.

6, the described detection method of claim 3 is characterized in that comprising the steps:

1) blood, body fluid or the tissue samples of collection individuality to be measured extract DNA;

2) be template with this DNA, carry out the PCR reaction, obtain the PCR reaction product with PCR primer near the design of the coding region GJB6 gene or the 233rd base of GJB6 gene;

3) adopt APa I restriction enzyme to carry out endonuclease reaction the PCR reaction product that obtains, agarose electrophoresis detects, and determines whether to have the GJB6 mutational site;

4) judge according to above result whether individuality to be measured is the hereditary hearing impairment that GJB6 gene 233C → the A sudden change causes.

7, claim 4 or 6 described detection methods is characterized in that described PCR primer sequence is:

CX30-F：5’-TCAGGGATAAACCAGCGCAAT-3’

CX30-R：5’-GTTGGTATTGCCTTCTGGAGAAGA-3’

8, a kind of detection kit is used for the diagnosing human hereditary hearing impairment, comprises following combination of agents:

From testing sample, extract the reagent of DNA;

The PCR primer and the corresponding PCR reaction reagent that are used near the coding region of the 233rd base of amplified sample DNA GJB6 gene or GJB6 gene;

Pcr amplification product is carried out the reagent of digestion with restriction enzyme reaction; And/or

The reagent that pcr amplification product is checked order.

9, the application of the described GJB6 mutator gene of claim 1 in diagnosing human hereditary hearing impairment disease.

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