CN101230397B - Reagent case for detecting 281C >T mutation of large vestibular aqueduct related gene SLC26A4 - Google Patents

Abstract

The invention relates to a detecting method, which detects whether the SLC26A4 genetic mutation exists in the individual sample to be detected, and diagnoses the generation and the type of the large vestibular aqueduct in the individual to be detected; wherein, the SLC26A4 genetic mutation is 281C being larger than T heterozygous mutation on the exon 3 of SLC26A4 gene. The invention further relates to a reagent kit for detecting whether the SLC26A4 genetic mutation exists in the individual sample to be detected, and the application of the SLC26A4 gene in diagnosing and/or treating the large vestibular aqueduct related diseases. The genetic mutation and detecting method are favorable for the screening the SLC26A4 genetic mutation in the deaf people, and diagnose and treat the deaf people.

Description

Detect the test kit of 281C＞T sudden change of large vestibular aqueduct related gene SLC26A4

The application is dividing an application of Chinese patent application 200510132214.5.

Technical field

The present invention relates to be used to detect the test kit of SLC26A4 mutator gene.

Background technology

The SLC26A4 gene is positioned at 7q31, it is by location such as Everett at first, and find that this transgenation can cause a kind of recessive hereditary disease: Pendred syndrome, clinical manifestation is a thyrocele, and merges the phonosensitive nerve deafness of aquaductus vestibuli expansion or Mondini deformity (aquaductus vestibuli enlarges merging cochlea underdevelopment).Afterwards, Usami etc. showed to the examination result that the simple aquaductus vestibuli of 6 examples enlarges the SLC26A4 gene of family that the sudden change of this gene can also cause simple aquaductus vestibuli to enlarge, and its hereditary pattern also is a recessive inheritance.This shows that the SLC26A4 transgenation can cause aquaductus vestibuli to enlarge---simple property aquaductus vestibuli enlarges or merges cochlea odd-shaped aquaductus vestibuli and enlarges.It is the modal deformity of inner ear that aquaductus vestibuli enlarges, and it accounts for 1～8% in hereditary hearing impairment.Mainly showing as high frequency hearing loss clinically is main phonosensitive nerve deafness, and the deafness multilist now is that severe or utmost point severe are deaf.Morbidity is many in the Childhood, and its premorbid often has flu, fever, wound etc. to make the inducement of intracranial hypertension.

Enlarge relevant SLC26A4 gene mRNA total length 4930bp with aquaductus vestibuli, contain 21 exons, open reading frame 2343bp runs through exon 2 and exon 21.The albumen of this genes encoding---Pendrin is that a molecular weight is 86kD, contains 780 amino acid whose transmembrane proteins, belongs to ion transport Zijia family.Scott and Bidart etc. discover, Pendrin mainly is expressed in the teleblem of thyroid follicular cells and the chief cell of ductus endolymphaticus and endolymphytic sac, the transhipment of mediation iodide ion and chlorion plays a role keeping on parathyroid tissue and the endolymphatic ionic equilibrium.At Tiroidina, when the Pendrin dysfunction, it can not in time be transported to the folliculus colloid to iodide ion, makes iodide ion in the follicular cells inner accumulated, thereby can not effectively organise and combine, thereby cause generation strumous in the Pendred syndrome with thyroglobulin.Qvortrup etc. think, the similar thyroid follicle of the endolymphytic sac of rat has balance gelatinoid filling blister cavities, and the function class of endolymphytic sac chief cell is similar to thyroid follicular cells, can synthesize, secretion, absorption, digesting protein.The chlorion transit barrier changes the composition of endolymph fluid, thereby the damage sensory epithelial cell causes phonosensitive nerve deafness.And because the toxic mechanism that osmotic pressure changes and composition changes causes the membranous labyrinth structural modification.Because just stasi when ductus endolymphaticus and endolymphytic sac to 4 year old, so their expansion can cause the change of surrounding bone structure, for example change of aquaductus vestibuli or cochlea structure.

Enlarge the examination of patient SLC26A4 transgenation for aquaductus vestibuli, abroad extensively carry out, the pathogenic mutation site of report surpasses 100, comprises missense mutation, the sudden change of frameing shift, nonsense mutation and splice site sudden change, is distributed in each exon.The SLC26A4 gene has tangible heterogeneity, and not agnate its mutant form is incomplete same.Its encoded protein can not accurately navigate on the cytolemma after this transgenation, but is present in endoplasmic reticulum or the golgi body, influences ion transport.

Aquaductus vestibuli enlarges the discovery of patient SLC26A4 transgenation, not only can promote the development of the fundamental researchs such as pathogeny that this is sick, also will promote the gene diagnosis of aquaductus vestibuli expansion and carrying out of treatment greatly.General examination by antenatal diagnosis examination and newborn infant SLC26A4 transgenation, can reduce the natality that aquaductus vestibuli enlarges infant, and realize diagnosing before the disease, thereby instruct the behavior of the infant that carries Disease-causing gene, prophylactic generation, the quantity that this will significantly reduce deafness patient alleviates social pressures.Following research will be devoted to the gene therapy aspect, can predict, and gene therapy brings qualitative leap will for the treatment that comprises deaf various heredopathias.

Summary of the invention

One object of the present invention is to provide the diagnosis method that aquaductus vestibuli enlarges, whether it by detecting from existing the SLC26A4 mutator gene with mutant form of the present invention to judge that patient's aquaductus vestibuli enlarges the generation and the type of disease in patient's the sample to be tested, and then provide reference for clinical diagnosis and treatment.

Another object of the present invention is to be provided for detecting the test kit that whether has the SLC26A4 transgenation from the sample of individuality to be measured.

A further object of the present invention is to provide the application of SLC26A4 mutator gene in diagnosis and/or treatment aquaductus vestibuli expansion relative disease.

According to an aspect of the present invention, the invention provides detection method a kind of and aquaductus vestibuli expansion disease related gene.This detection method is by detecting from whether there being the SLC26A4 transgenation in the sample of individuality to be measured, and diagnose aquaductus vestibuli in this individuality to be measured to enlarge the generation and the type of disease, wherein said SLC26A4 transgenation is positioned at the 109G＞T heterozygous mutant of SLC26A4 gene extron 2, be positioned at the 334C＞T heterozygous mutant of SLC26A4 gene extron 4, be positioned at the 589G＞A heterozygous mutant of SLC26A4 gene extron 5, be positioned at the 387delC heterozygous mutant of SLC26A4 gene extron 4, be positioned at the 611G＞T heterozygous mutant of SLC26A4 gene extron 6, be positioned at the 812A＞G heterozygous mutant of SLC26A4 gene extron 7, be positioned at the 1175A＞G heterozygous mutant of SLC26A4 gene extron 10, be positioned at the 1540C＞T heterozygous mutant of SLC26A4 gene extron 13, be positioned at the 1746delG heterozygous mutant of SLC26A4 gene extron 16, be positioned at the 2054G＞T sudden change of SLC26A4 gene extron 18, be positioned at the 281C＞T heterozygous mutant of SLC26A4 gene extron 3, be positioned at the 1586T of SLC26A4 gene extron 14＞G sudden change or be positioned at the 227C＞T heterozygous mutant of SLC26A4 gene extron 3.

Described detection method can comprise 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 at described each the mutational site design of SLC26A4 gene;

3) the PCR product that obtains is carried out direct sequencing analysis, the sequence of resulting sequence and SLC26A4 normal gene is compared, determine whether to exist the described mutational site of SLC26A4 gene;

4) judge that according to above result the aquaductus vestibuli whether individuality to be measured causes for the SLC26A4 transgenation enlarges.

Described detection method can further comprise the steps:

5) translate to determine whether to exist following amino acid mutation site by the normal reading frame: E37X, P112S, G197R, X144, G204V, D271G, N392S, Q514X, X585, R685I, T94I, I529S or P76L.

Below, specify the detection method of SLC26A4 gene 281C＞T heterozygous mutant:

Carry out examination by the SLC26A4 gene that 89 routine aquaductus vestibulis is enlarged the control group member of family members and 80 normal good hearings, the infant of finding an aquaductus vestibuli expansion have the new mutational site of SLC26A4 gene (281C＞T, T94I).This mutational site is positioned at first of Pendrin and strides the film district, makes 94 the neutral Soviet Union of polarity acyl ammonia become nonpolar hydrophobic Isoleucine.The Study on Evolution result of homologous amino acid sequence shows that this mutational site has high conservative (Figure 12).Do not detect this sudden change in 80 normal peoples' the examination, illustrate that this mutational site enlarges relevant with aquaductus vestibuli.

Fig. 6 is the contrast figure of SLC26A4 coding region amino acid and nucleic acid base, and sudden change is positioned at the 281st bit base, becomes Isoleucine corresponding to the 94th amino acid by Threonine.The structural representation of Pendrin is seen Fig. 7, and the 94th amino acids is positioned at first of Pendrin and strides the film district.

In a specific embodiment of the present invention, detect the method that whether has SLC26A4 gene 281C＞T heterozygous mutant in the sample of individuality to be measured, comprise 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 following PCR primer;

PDS3-F：5’-GGCAAAAGCATGGTAAGCAC-3’

PDS3-R：5’-AGGGTAAGCAACCATCTGTCA-3’

3) the PCR reaction product that obtains is directly checked order, and the sequence of sequencing result and SLC26A4 normal gene is compared, determine whether to exist the 281C＞T mutational site of SLC26A4 gene; Or

4) adopt Sca I restriction enzyme to carry out endonuclease reaction the PCR reaction product that obtains, agarose electrophoresis detects, and determines whether to have 281C＞T mutational site;

5) judge according to above result whether this individuality to be measured exists SLC26A4 gene 281C＞T sudden change.

In another embodiment of the invention, adopt the endonuclease reaction of Sca I restriction enzyme to detect mutator gene, after above-mentioned steps 2 resulting PCR reaction product are carried out endonuclease reaction with Sca I, agarose electrophoresis detects, normal gene can be cut by Sca I, and mutator gene can not be cut by Sca I enzyme, determines whether to exist the mutational site thus; Judge according to detected result whether individuality to be measured is that the aquaductus vestibuli that SLC26A4 gene 281C＞the T sudden change causes enlarges.

In above-mentioned detection method at each mutational site of SLC26A4 gene, resulting PCR reaction product can also detect with methods such as sex change high performance liquid chromatography (DHPLC) or hybridization probes, used hybridization probe can be and normal SLC26A4 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.

According to a further aspect in the invention, the invention provides and be used to detect the test kit that whether has the SLC26A4 transgenation from the sample of individuality to be measured, 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 for the following mutational site of amplified sample DNA SLC26A4 gene:

Be positioned at the 109G＞T heterozygous mutant of SLC26A4 gene extron 2, be positioned at the 334C＞T heterozygous mutant of SLC26A4 gene extron 4, be positioned at the 589G＞A heterozygous mutant of SLC26A4 gene extron 5, be positioned at the 387delC heterozygous mutant of SLC26A4 gene extron 4, be positioned at the 611G＞T heterozygous mutant of SLC26A4 gene extron 6, be positioned at the 812A＞G heterozygous mutant of SLC26A4 gene extron 7, be positioned at the 1175A＞G heterozygous mutant of SLC26A4 gene extron 10, be positioned at the 1540C＞T heterozygous mutant of SLC26A4 gene extron 13, be positioned at the 1746delG heterozygous mutant of SLC26A4 gene extron 16, be positioned at the 2054G＞T sudden change of SLC26A4 gene extron 18, be positioned at the 281C＞T heterozygous mutant of SLC26A4 gene extron 3, be positioned at the 1586T of SLC26A4 gene extron 14＞G sudden change or be positioned at the 227C＞T heterozygous mutant of SLC26A4 gene extron 3.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 the described sudden change of SLC26A4 gene is provided in one embodiment of the invention, be equipped with in the container in order to detect the composition of the described sudden change of SLC26A4 gene, 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 the described mutational site of SLC26A4 gene 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 PDS-F and PDS-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 PDS-F and PDS-R primer, carries out pcr amplification reaction;

(2) the endonuclease reaction detection that suddenlys change; And/or

(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 described amino acid mutation site by the normal reading frame.

This test kit can detect the described mutational site of SLC26A4 gene quickly and easily, thereby is applied to sudden change detection and diagnoses and treatment thereof that aquaductus vestibuli enlarges case.

In accordance with a further aspect of the present invention, the invention provides the application of SLC26A4 mutator gene in diagnosis and/or the expansion of treatment aquaductus vestibuli, by detecting deaf occurrence cause and the type of judging this patient from the described sudden change that whether has the SLC26A4 gene in patient's the sample to be tested, and then provide reference for clinical diagnosis and treatment; In addition; aspect further clinical treatment, detected result be the described sudden change of SLC26A4 gene has taken place after, normal gene can be imported the cell that carry mutator gene and express therein; it can be recombinated with the endogenous mutator gene, thereby can carry out gene therapy.

The present invention provides the described 13 kinds of sudden changes that have the SLC26A4 gene in Chinese population first, and illustrates that this mutator gene enlarges relevant with aquaductus vestibuli.Simultaneously, whether the present invention proposes by detecting exists the SLC26A4 transgenation to judge the deaf reason that takes place and the detection method of type among the patient, this will help carrying out clinically the SLC26A4 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. 1～5 are the accompanying drawing about SLC26A4 gene 109G＞T heterozygous mutant:

Fig. 1 is the contrast of SLC26A4 coding region amino acid and nucleic acid base: sudden change is positioned at the 109th base, corresponding to the 37th amino acid become the translation termination signal (the grey mark be the sudden change base and amino acid), thereafter aminoacid sequence underscore mark, this section sequential amino acid deletion;

Fig. 2 is the structural representation of Pendrin, and asterisk illustrates the 37th amino acids position, the E37X sudden change takes place after, sequential amino acid deletion thereafter;

Fig. 3 shows temperature of reaction and time for PCR reaction process synoptic diagram in SLC26A4 gene 109G of the present invention＞T heterozygous mutant detection method;

Fig. 4 carries out the quantitative agarose gel electrophoretogram of electrophoresis in SLC26A4 gene 109G of the present invention＞T heterozygous mutant detection method to the PCR product, there is shown the fragment position of quantitative Marker;

Fig. 5 is the part sequencing result of the inventive method SLC26A4 gene extron 2, and the top is the normal control sequence, and the below is a mutant nucleotide sequence, the arrow indication be the mutational site (109G＞T, E37X);

Fig. 6～12 are the accompanying drawing about SLC26A4 gene 281C＞T heterozygous mutant:

Fig. 6 is the contrast of SLC26A4 coding region amino acid and nucleic acid base: sudden change is positioned at the 281st bit base, becomes Isoleucine (grey mark be base and the amino acid that suddenlys change) corresponding to the 94th amino acid by Threonine;

Fig. 7 is the structural representation of Pendrin, and asterisk illustrates the 94th amino acids position, and it is positioned at first of Pendrin and strides the film district;

Fig. 8 shows temperature of reaction and time for PCR reaction process synoptic diagram in SLC26A4 gene 281C of the present invention＞T heterozygous mutant detection method;

Fig. 9 carries out the quantitative agarose gel electrophoretogram of electrophoresis in SLC26A4 gene 281C of the present invention＞T heterozygous mutant detection method to the PCR product, there is shown the fragment position of quantitative Marker;

Figure 10 is the part sequencing result of the inventive method SLC26A4 gene extron 3, and the top is the normal control sequence, and the below is a mutant nucleotide sequence, the arrow indication be the mutational site (281C＞T, T94I);

Figure 11 is mutational site restriction enzyme digestion and electrophoresis figure in the T94I heterozygous mutant detection method in the inventive method, among the figure from left to right: first swimming lane be molecular weight standard, second for the restriction enzyme mapping of heterozygous mutant gene, the 3rd swimming lane be the restriction enzyme mapping of normal gene;

Restriction enzyme Sca I can cut at 279 and 280 bit bases, and make normal people PCR product fragment be cut into 280bp and two fragments of 120bp by original 400bp, and this restriction enzyme site disappears after the sudden change, can not cut, because this organizes this example for heterozygous mutant, so be shown as 3 bands on the running gel figure;

Figure 12 is the homologous amino acid sequence Study on Evolution, different plant species aminoacid sequence comparison (the arrow indication is the T94I site), and the aminoacid sequence of the different plant species of visible pendrin is Threonine (T) in this site, illustrate that the T94I sudden change is positioned at conservative region;

The embodiment of invention

The used test materials of the present invention if no special instructions, is commercially available purchase product.

Research process of the present invention: collect aquaductus vestibuli by deaf sick outpatient service and enlarge the patient, under the voluntary prerequisite of patient and family members thereof, the 5-10ml blood sample left and taken in signature informed consent postscript, set up the patient medical history database, incidence and contact method in detail record conditions of patients, the family.Then, use phenol chloroform method for extracting extraction genomic dna, quantitatively put in storage the back ,-20 ℃ of preservations, and every part of DNA sample is all accurately corresponding to the patient clinical data of registering.Then, describe the design primer according to the document of Coyle etc., part utilizes PrimerPremier 5.0 to design, and comprises SLC26A4 gene coding region and flanking sequence thereof, carries out pcr amplification.The PCR product is directly checked order: sequencing primer is identical with the pcr amplification primer, uses the ABI 3730DNA of company sequenator and carries out forward and reverse order-checking.The sequence and the standard sequence among the Genbank that obtain are compared, determine the SLC26A4 mutational site.At some mutational site of SLC26A4 gene, the PCR product is also optionally carried out endonuclease reaction: use specific 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.Translate to determine the mutational site of SLC26A4 gene by the normal reading frame.

The detection of SLC26A4 gene 109G＞T heterozygous mutant

The patient who enlarges with aquaductus vestibuli is as research object, by examination to each exon of coding region of the SLC26A4 gene of 89 example these patient and his family set members and 80 routine normal controls, find aquaductus vestibuli enlarge the patient on exon 2, have the new sudden change of SLC26A4 gene (109G＞T, E37X).This patient is a compound heterozygote, meets the pattern of autosomal recessive inheritance, and there is another sudden change in the allelotrope of its SLC26A4.This mutational site all is not found in 80 control groups, illustrate this mutational site and aquaductus vestibuli enlarge be divided into from.

The detection method of [embodiment 1] SLC26A4 gene 109G＞T heterozygous mutant

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

1, research object: patient's 89 examples that the aquaductus vestibuli that deaf sick outpatient service is collected enlarges, normal control 80 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 TE damping fluid dissolving (200 μ l TE/5ml whole bloods, 400 μ l TE/10ml whole bloods).

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

Two, the pcr amplification of SLC26A4 gene extron 4

1, primer sequence

Upstream primer:

PDS2-F：5’-CAGGACGCGGACCAGACT-3’(nt864-nt881)

Downstream primer:

PDS2-R：5’-CGAGACTGATGGAGCCACCCTC-3’(nt1350-nt1371)

Annotate: SLC26A4 gene order searching number: Gene ID:5172

2, the foundation of PCR reaction system

The composition of the PCR reaction system of SLC26A4 gene sees Table 1.

The PCR reaction system of table 1 SLC26A4 gene

Title	Original liquid concentration	Application of sample amount (μ l)	The system final concentration
				Damping fluid	10×	2.5	1×
dNTP	2.5mM	2.0	0.2mM
				Primer	10μM	0.6	0.24μM
The Taq enzyme	5units/μl	0.3	0.06units/μl
				Template (DNA that extracts in the step 1)	100ng/μl	1	4ng/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.

Three, 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 ℃ of refrigerators.

Four, 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 (1.0% agarose): take by weighing the 3.0g 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.

Five, the direct order-checking of SLC26A4 gene PCR amplified production

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

DNA purity: OD260/OD280=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 2 SLC26A4 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 3 SLC26A4 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 postposition-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.

[embodiment 2] mutational site Study on Evolution

Mutation analysis: the SeqmanTM software of using in DNAStar5.0 (Lasergene Inc.) software package 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 (109G＞T, E37X).

Study on Evolution: 109G＞T sudden change is positioned at the N-terminal of Pendrin, and it makes the amino acid coding end at 37, and 734 amino acid are thereafter lost.This sudden change must influence the 26S Proteasome Structure and Function of Pendrin.

The detection of SLC26A4 gene 281C＞T heterozygous mutant

The patient who enlarges with aquaductus vestibuli is as research object, by examination to each exon of coding region of the SLC26A4 gene of 89 example these patient and his family set members and 80 routine normal controls, find aquaductus vestibuli enlarge the patient on exon 3, have the new mutational site of SLC26A4 gene (281C＞T, T94I).This mutational site all is not found in 80 control groups, illustrate this mutational site and aquaductus vestibuli enlarge be divided into from.

The detection method of [embodiment 3] SLC26A4 gene 281C＞T heterozygous mutant

Basic skills and step are referring to embodiment 1.Pcr amplification primer sequence at SLC26A4 gene extron 3 is:

Upstream primer:

PDS3-F：5’-GGCAAAAGCATGGTAAGCAC-3’(nt2497-nt2516)

Downstream primer:

PDS3-R：5’-AGGGTAAGCAACCATCTGTCA-3’(nt2876-nt2896)

Annotate: SLC26A4 gene order searching number: Gene ID:5172

The PCR reaction process as shown in Figure 8; The PCR product is carried out the quantitative agarose gel electrophoretogram of electrophoresis see Fig. 9; Sequencer map such as Figure 10.

The mutational site endonuclease reaction detects

The composition of endonuclease reaction system sees Table 4, and reaction conditions is 37 ℃ of water-baths 3 hours.The application agarose electrophoresis detects, and method is quantitative referring to electrophoresis among the embodiment 1.The endonuclease reaction electrophorogram as shown in figure 11.

Restriction analysis is to have found a restriction enzyme ScaI restriction enzyme site in this mutational site, when not undergoing mutation, restriction enzyme ScaI can cut at 279 and 280 bit bases, and make PCR product fragment be cut into 280bp and two fragments of 120bp by original 400bp, and this restriction enzyme site disappears after the sudden change, can not cut.Because this example is a heterozygous mutant, so be shown as 3 bands on the running gel figure.

Table 4 endonuclease reaction system

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

ScaI restriction enzyme and 10 * L Buffer purchase in crystalline substance U.S. biotech firm.

[embodiment 4] 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 (281C＞T, T94I).

Study on Evolution: 281C＞T sudden change is positioned at first of Pendrin and strides the film district, makes polarity neutral Threonine become nonpolar hydrophobic Isoleucine, and homologous gene aminoacid sequence evolution Journal of Sex Research result shows that this sudden change is positioned at high conservative region (Figure 12).

Detect test kit and application thereof that aquaductus vestibuli enlarges relevant SLC26A4 transgenation

[embodiment 5]

The test kit that detects the SLC26A4 transgenation can be prepared into the independent test kit that detects each single mutational site as required, also can be prepared into the test kit that detects a plurality of or all SLC26A4 gene mutation sites, be that example describes with the test kit that detects 11 mutational sites of the present invention below:

1, test kit contains:

(1) amplification primers

Aforementioned is 11 pairs of primers that detect 13 mutational sites designs of SLC26A4 gene, perhaps other spendable primers at these 13 mutator genes designs;

Annotate: SLC26A4 gene order searching number: Gene ID:5172

Other reagent that are included in alternatively in the test kit comprise following reagent:

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

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

(4)dNTP 2.5mM

(5) Sca I restriction enzyme; The EcoRV restriction enzyme;

(6)10×L Buffer

(7)Big-Dye mix

2, using method

Mainly comprise the steps:

1) pcr amplification

Gather the sample (blood, body fluid, tissue etc.) of individuality to be measured, extract DNA, carry out pcr amplification respectively with above-mentioned primer at each mutator gene;

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

To carry out endonuclease reaction according to the endonuclease reaction system of embodiment 3 respectively at the amplified production in 281C＞T mutational site, judge whether to exist said mutation with reference to Figure 11.

4) sequencing reaction and checking

The PCR product at each mutational site of above-mentioned amplification is carried 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 corresponding amino acid mutation site by the normal reading frame.

Concrete grammar is referring to the detailed description among the embodiment of front.

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 claims of the present invention.

Claims (2)

1. test kit that is used to detect the 281C＞T heterozygous mutant that whether has SLC26A4 gene extron 3, described test kit comprises the PCR primer that is used for amplified sample DNA SLC26A4 gene the 281st bit base; And following one or more combination of agents:

From testing sample, extract the reagent of DNA;

Corresponding PCR reaction reagent;

The reagent that pcr amplification product is checked order; And/or

Pcr amplification product is carried out the reagent of digestion with restriction enzyme reaction.

2. test kit as claimed in claim 1, wherein said PCR primer is:

PDS3-F：5’-GGCAAAAGCATGGTAAGCAC-3’

PDS3-R：5’-AGGGTAAGCAACCATCTGTCA-3’。

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