CN102732614A - Detection kit for c.437G>A mutation of PJVK gene - Google Patents

Abstract

The invention provides a detection kit for c.437G>A mutation of the PJVK gene. Causes and types of sensorineural deafness and auditory neuropathy/auditory neuropathy spectrum disorders are diagnosed through detecting whether patients have the mutation gene or not. The mutation gene and a detection method mentioned above are beneficial for clinical screening of PJVK mutation for patients with sensorineural deafness and auditory neuropathy/auditory neuropathy spectrum disorders, providing bases for diagnosis and treatment of patients with sensorineural deafness and auditory neuropathy/auditory neuropathy spectrum disorders.

Description

Detect the PJVK gene c.437G＞test kit of A sudden change

Technical field

The present invention relates to the gene test field, be specifically related to be used to detect the test kit of PJVK transgenation gene; The invention still further relates to PJVK mutator gene and the application in diagnosis and/or treatment sensorineural deafness and the sick pedigree obstacle of auditory nerve disease/auditory nerve thereof.

Background technology

The sick pedigree obstacle of auditory nerve disease/auditory nerve (Auditory Neuropathy Spectrum Disorders; ANSD) be that a kind of cochlea external hair cell function is normal; But voice signal can not synchronously be transferred to the dysacousis property disease of brain from inner ear, is the unusual of auditory information transmission and processing process.It is master's hearing loss that the audiology inspection shows as with the low frequency dysacusis, and ABR can not be drawn, and how normal cochlear microphonic potential and otoacoustic emission be.Such disease is defined and is named in eighties of last century the nineties; It is a kind of dysaudia property disease of new knowledge; Sickness rate in high-risk infant is about 0.23%; Sickness rate is one of important dysaudia property disease that causes on infant and teenager's verbal communication obstacle then up to 11% in the unusual deaf sufferer youngster of the ABR due to a variety of causes.Yet its cause of disease, diseased region and pathogenesis are still very not clear and definite, cause pharmacological agent, wear osophone and artificial cochlea's implantation effect prediction and the assessment on have difficulty.In recent years, the discovery of the sick pedigree obstacle of a series of sensorineural deafnesses and auditory nerve disease/auditory nerve genes involved had disclosed the pathomechanism of the sick pedigree obstacle of auditory nerve from molecular level.Somatotype is carried out to the sick pedigree obstacle of auditory nerve in different lesions position according to different genes causes, and the clinical treatment that can be the sick pedigree obstacle of auditory nerve provides theoretical foundation.

Professors Starr etc. are divided into TYPE I type (diseased region is at auditory nerve) and TYPE II type (diseased region is in inner hair cell, tip dendron and cynapse) research both at home and abroad according to the difference of site of pathological change with it and show that the sick pedigree impaired patients of the auditory nerve that carries the OTOF transgenation is clinical and manyly show as the master with the TypeII type, are suitable for the artificial cochlea and implant and can obtain better effects.And the sick pedigree impaired patients of the auditory nerve that carries the PJVK transgenation clinical many be main with TYPE I type, its diseased region is at auditory nerve, the position exists deeply, artificial cochlea's implantation effect is not good enough.

The relevant gene of the sick pedigree obstacle of PJVK gene right and wrong syndrome and type recessive Hereditary Sensorineural Hearing Loss and auditory nerve disease/auditory nerve.Equaled to find first and named in 2006 by Del maghani, be also referred to as the DFNB59 gene, be positioned at karyomit(e) 2q31.1～2q31.3 zone, dna sequence dna total length 9800bp contains 7 exons, and wherein first exon is non-coding exon.The coding region sequence of this gene; Contain terminator shown in SEQ ID NO.1; The albumen pejvakin that coding is made up of 352 amino acid (shown in SEQ ID NO.2); Wherein the 249th～258 amino acids form the nuclear localization signal motif (nuclear localization signal, NLS), the 305th～331 amino acids form a kind of can with the interactional zinc fingers of DNA (zinc-binding domains).Pejvakin albumen mainly is expressed in the neuronal cell of cochlea Corti device, SGC and first three grade sense of hearing afferent pathway (cochlear nucleus, last olive complex body, inferior colliculus); Due to the diseased region of the sick pedigree obstacle of sensorineural deafness and auditory nerve disease/auditory nerve mainly be positioned at sense of hearing conduction path, the conduction and the intracellular matter that influences action potential exchange.Found first from 2006 and report the PJVK gene so far; Scholars in patients such as Iran, Morocco, Turkey and Holland, found respectively this gene 10 surplus kind sudden change, but the sudden change report of rare China's sensorineural deafness and this gene of the sick pedigree impaired patients of auditory nerve disease/auditory nerve.The applicant belongs to seminar has taken the lead in carrying out this gene to 150 sensorineural deafnesses and the sick pedigree impaired patients of auditory nerve disease/auditory nerve examination; And find that c.437G population of China distinctive>the A sudden change, and then researched and developed and detected this gene c.437G the test kit of A sudden change.This achievement helps in wider sensorineural deafness and the sick pedigree obstacle of auditory nerve disease/auditory nerve crowd, to carry out fast and efficiently the detection in mutational site; And then to its location somatotype, carrying out clinical intervention for the sick pedigree impaired patients of sensorineural deafness and auditory nerve disease/auditory nerve provides important directive significance.

Summary of the invention

One object of the present invention is to be provided for to detect the PJVK sudden change c.437G>test kit of A gene, its technical scheme is:

A kind of be used to detect relevant with sensorineural deafness and the sick pedigree obstacle of auditory nerve disease/auditory nerve be positioned at the PJVK gene c.437G test kit that A suddenlys change, said test kit comprises:

From testing sample, extract the reagent of DNA;

The PCR reaction reagent that is used for amplified sample DNA;

The reagent that pcr amplification product is checked order;

Wherein, the PCR reaction reagent of said amplified sample DNA comprises the PCR primer, and the target fragment of said PCR primer amplification comprises PJVK gene the 437th bit base.

Particularly, above-mentioned PCR primer is for being selected from:

PJVK-4F-1（SEQ?ID?NO.3）:5’-CAAATAGAGTCCCCTTCAACAG-3’，

PJVK-4R-1（SEQIDNO.4）:5’-CTCCAGTATGCACGTAATTTCA-3’；

PJVK-4F-2（SEQ?ID?NO.5）:5’-TGGCTCTACACACATTTGCT-3’，

PJVK-4R-2（SEQ?ID?NO.6）:5’-ACAAGCCTGACTAGAAATTCAA-3’；

PJVK-4F-3（SEQ?ID?NO.7）:5’-TACTTTGGCTCTACACACAT-3’，

PJVK-4R-3（SEQ?ID?NO.8）:5’-GCCTGACTAGAAATTCAATAC-3’；

PJVK-4F-4（SEQ?ID?NO.9）:5’-GCCTTGATTTACTATTAGGTG-3’，

PJVK-4R-4 (SEQ ID NO.10): 5 '-CTATACAACTGCAGCTCTTTC-3 '; In a pair of.

Another object of the present invention is to provide the mentioned reagent box to be used to detect and is positioned at the PJVK gene c.437G>method of A sudden change, may further 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 the PCR primer;

Wherein, the target fragment of said PCR primer amplification comprises PJVK gene the 437th bit base;

3) the PCR reaction product that obtains is directly checked order, and the sequence of sequencing result and PJVK normal gene is compared, determine whether to exist the PJVK gene c.437G the A mutational site.

Further, above-mentioned detection method is further comprising the steps of:

4) translate to confirm c.437G according to the normal reading framework A sudden change makes aminoacid sequence missense mutation take place from 146, makes l-arginine become Histidine (p.R146H).

Another purpose of the present invention provides the mentioned reagent box in the purposes that is used for detecting the sick pedigree obstacle of sensorineural deafness and auditory nerve disease/auditory nerve, in sensorineural deafness and the sick pedigree impaired patients of auditory nerve disease/auditory nerve, carrying out tumor susceptibility gene examination certain method of providing convenience; Simultaneously can guiding clinical treatment, and solid basis is provided for utilizing in the future this sudden change to carry out deaf gene therapy as target spot.

C.437G whether the detection method of using test kit of the present invention for existing the PJVK gene in the sample to be tested that comes from the patient through detection>the A sudden change, and judge this patient's sensorineural deafness and sick pedigree obstacle occurrence cause of auditory nerve disease/auditory nerve and type.Wherein, the PJVK gene is c.437G>the A sequence change causes in the amino acid cataloged procedure that at 437 missense taking place changes, the l-arginine that causes encoding becomes Histidine (p.R146H), and then influences the proteic normal function of pejvakin.

The sick pedigree obstacle of sensorineural deafness that the PJVK transgenation is relevant and auditory nerve disease/auditory nerve transmits with the mode of autosomal recessive inheritance.Examination is carried out in the method that the contriver uses the candidate gene examination and contrast that do not have family history normal to 150 routine non-syndrome type sensorineural deafnesses and the sick pedigree impaired patients of auditory nerve disease/auditory nerve and 99 routine hearing; Find that in the sick pedigree impaired patients of a routine auditory nerve PJVK gene is c.437G>the A sudden change; And this mutational site high conservative and this sudden change of discovery in 99 routine normal controls groups; Kind surplus the PJVK sudden change that report is relevant with sensorineural deafness and the sick pedigree obstacle of auditory nerve disease/auditory nerve in the world at present has 10; Still do not have c.437G the report of A sudden change, this sports newfound sensorineural deafness of Chinese population and the sick pedigree obstacle of auditory nerve disease/auditory nerve pathogenic mutation.

Fig. 1 provides the map of PJVK coding region amino acid and nucleic acid base; And mutational site (square frame mark) is shown; This sudden change makes the 437th the G base that is positioned at the PJVK gene coding region become the A base; Make 146 l-arginine become Histidine (p.R146H), the back takes place and influences the proteic normal function of genetic expression in sudden change.

Detect the method for any check point sudden change of this available this area that suddenlys change and carry out, for example PCR (polymerase chain reaction)-PCR sequencing PCR, adopt the PJVK gene DNA probe hybridization method of mark or with method of restriction fragment length polymorphism method or sequence specific primers or the like.

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 genomic dna;

2) be template with this DNA,, obtain pcr amplification product to carry out the PCR reaction near the PCR primer that designs PJVK gene or PJVK coding region the 437th bit base;

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

4) according to above result judge individuality to be measured whether be the PJVK transgenation c.437G the sick pedigree obstacle of auditory nerve that causes of A.

Further, this method can optionally comprise the steps:

5) translate to confirm whether the 146th amino acids exists the missense mutation (p.R146H) of l-arginine to Histidine by the normal reading frame.

In the experiment that the contriver carries out, collect various phonosensitive nerve deafness patients through deaf sick outpatient service and resource acquisition network, comprise the sick pedigree impaired patients of auditory nerve, set up resources bank.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 of phenol chloroform and extract genomic dna, quantitative back warehouse-in ,-20 ° of C preserve, 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 PJVK, Using P CR amplification.The PCR product directly checks order: sequencing primer is identical with the pcr amplification primer, and the ABI 3700DNA of company sequenator is used in forward and reverse order-checking.Sequence that obtains and the sequence among the Genbank (accession number: NC_000002.11) relatively confirm the PJVK mutational site.Translate to confirm the mutational site of PJVK by the normal reading frame.

Above-mentioned steps 2 resulting PCR reaction product can also detect with hybridization probe, and used hybridization probe can be and normal PJVK 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 ri, chromonic material or tagged, allele specific probe especially capable of using, and also whether the method examination of cutting of available constraints property enzyme exists the sudden change that has been determined.

Therefore, the reagent of detection pcr amplification product is selected from order-checking detection reagent, digestion with restriction enzyme detection reagent, restricted length polymorphism detection reagent, sequence specific primers detection reagent and probe hybridization detection reagent.The PCR primer that in aforesaid method, uses can be generally 15～30 bases according to known nucleotide sequence design, and GC content is about 45～50%, under suitable temperature, combines with terminal specificity, and it can utilize special computer programming.In a specific embodiment of the present invention, to use online primer-design software primer 3.0 and designed one couple of PCR primers, its sequence is:

Upstream primer: PJVK-4F-1:5 '-CAAATAGAGTCCCCTTCAACAG-3 ' (nt4328-nt4349)

Downstream primer: PJVK-4R-1:5 '-CTCCAGTATGCACGTAATTTCA-3 ' (nt4942-nt4963)

The standard sequence of PJVK normal gene can reference example such as Genbank:NC_000002.11.

Be used for detecting the PJVK gene c.437G the test kit of A sudden change can comprise following reagent:

Be used near the PCR primer of amplified sample DNA PJVK gene or PJVK gene coding region the 437th bit base; And following one or more combination of agents:

From testing sample, extract the reagent of DNA;

The PCR reaction reagent;

The reagent that pcr amplification product is directly checked order.

For example; Provide one in one embodiment of the invention and detect the PJVK gene c.437G the test kit of A sudden change; Be equipped with in the container in order to detect the PJVK gene c.437G the composition of 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 PJVK gene is c.437G in the test sample>test kit in A mutational site, can contain amplimer, dNTPs, be used for one or more of archaeal dna polymerase and damping fluid or the required reagent of sequencing reaction etc. of PCR reaction.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 PJVK-4F-1 and PJVK-4R-1 primer, the archaeal dna polymerase of the said PCR of being used for reaction is the enzyme that can be used in pcr amplification.

The method of use of said test kit mainly comprises the steps:

(1) DNA of extraction blood sample to be measured utilizes above-mentioned a pair of PJVK-4F-1 and PJVK-4R-1 primer, carries out the PCR reaction, obtains the PCR reaction product;

(2) 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;

Said step also can further comprise:

(3) translate to confirm whether the 146th amino acids exists the missense mutation (p.R146H) of l-arginine to Histidine by the normal reading frame.This test kit can detect the PJVK mutational site quickly and easily, thereby is applied in the detection and diagnosis or treat-ment of sensorineural deafness and the sick pedigree obstacle of auditory nerve disease/auditory nerve genes involved.

The present invention also provides the application of PJVK mutator gene in diagnosing or treat the sick pedigree obstacle of human sensorineural deafness and auditory nerve disease/auditory nerve.Through detecting from whether having the PJVK gene in patient's the sample to be tested c.437G>the A sudden change judges this patient's sensorineural deafness and sick pedigree obstacle occurrence cause of auditory nerve disease/auditory nerve and type, and then foundation is provided for clinical diagnosis and treatment; In addition; Aspect further clinical treatment, detecting to the PJVK gene having taken place c.437G after the A sudden change, can normal gene be imported cell and the expression therein that carry mutator gene; It can be recombinated with the endogenous mutator gene, thereby can carry out gene therapy.

The present invention proposes the reason and the detection method of type that whether exist the new sudden change of PJVK gene to diagnose sensorineural deafness and the sick pedigree obstacle of auditory nerve disease/auditory nerve to take place among the patient through detecting, this will help confirming the individualized treatment scheme for dissimilar sensorineural deafnesses and auditory nerve disease/auditory nerve disease pedigree impaired patients.

Below in conjunction with accompanying drawing,, describe in detail but do not limit the present invention through explanation to preferred embodiments of the present invention.

Description of drawings

Fig. 1 is PJVK gene coding region Nucleotide and amino acid whose contrast: sudden change is arranged in the G base of the 437th of PJVK gene; Enclosing what come with square frame is the base and corresponding amino acid of sudden change, and the base sequence after the sudden change makes the missense mutation (p.R146H) of the 146th amino acids generation l-arginine to Histidine.

Fig. 2 is a PCR reaction process synoptic diagram in the inventive method, shows temperature of reaction and time, and wherein * representes that each circulation reduces by 0.5 ℃.

Fig. 3 is in the inventive method, and the PCR product is carried out the quantitative agarose gel electrophoretogram of electrophoresis, and the fragment position of quantitative Marker has been shown among the figure.

Fig. 4 is PJVK gene sequencing result's in the inventive method a partial view, and square frame shows sensorineural deafness and position, the sick pedigree impaired patients mutational site of auditory nerve disease/auditory nerve; Wherein Fig. 4 A is a PJVK genetic heterozygosis sudden change sequencing result; Fig. 4 B is wild-type PJVK gene sequencing result.

Embodiment

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

[embodiment 1] blood sample to be measured extracts the pcr amplification with the PJVK gene coding region

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

1, research object

The hearing normal control that 150 examples is distributed non-syndrome type sensorineural deafness and the sick pedigree impaired patients of auditory nerve disease/auditory nerve and 99 no family histories carries out the examination of PJVK gene according to following method.Among the 150 routine patients, 1 routine patient shows as the unusual auditory function characteristic of the sick pedigree obstacle of typical auditory nerve, is diagnosed as the sick pedigree obstacle of non-syndrome type auditory nerve after the clinical observation of process and the inspection of the auditory function of system.To the PJVK gene test of the sick pedigree impaired patients of this routine auditory nerve find the patient be heterozygosis c.437G A suddenlys change.To not finding c.437G in 99 normal persons' of hearing the examination>A sudden change person.

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 ° of C～28 ° C) 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 nucleated cell layer in the middle of the careful sucking-off changes in the 5ml Ep pipe, and 5000 * g centrifugal 10 minutes, washes once with PBS then.5000 * g, centrifugal 10 minutes.

4) cell is suspended from 2ml TE damping fluid (10mM Tris.HCl; 1mM EDTA, pH8.0) in, add 10% SDS (dodecyl sulphate) to final concentration 0.5% (100 μ l); Protein kinase k 100～200 μ g/ml (10mg/ml), 50 ° of C water-baths 3～5 hours.

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) absolute ethyl alcohol of 2.5 times of volumes of adding.

10)-20 a ° C deposit D NA spends the night.

Second day

11) high speed centrifugation, 10000 * g, 10 minutes, 4 ° of C.

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, 400TE/10ml whole blood).

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

Two, the pcr amplification of PJVK gene coding region

1, primer sequence

Upstream primer: PJVK-4F-1:5 '-CAAATAGAGTCCCCTTCAACAG-3 ' (nt4328-nt4349)

Downstream primer: PJVK-4R-1:5 '-CTCCAGTATGCACGTAATTTCA-3 ' (nt4942-nt4963)

Annotate: PJVK gene order searching number: NC_000002.11

2, the foundation (table 1) of PCR reaction system

The PCR reaction system of table 1PJVK gene

Reagent name	Original liquid concentration	Application of sample amount (ul)
			Buffer	10×	5
The Taq enzyme	2U/ul	1
			dNTPs	2.5mmol?each	1

Pr?imer-L	20uM		1
				Primer-R	20uM		1
Additive	Betaine(5M)	10
			dd?H 2O		30
Total?Volume		50

Reagent and instrument: pcr amplification uses the Buffert of ancient cooking vessel state company, ancient cooking vessel state company's T aq enzyme, the dNTP of ancient cooking vessel state company, the Betaine of Sigma company, electrophoresis TaKaRa MarkerDL2000, dyestuff hundred dimension letter Genefinder.

Reaction conditions: reaction process (comprising temperature and time) is as shown in Figure 2.

The purifying of [embodiment 2] PJVK gene coding region pcr amplification product is with quantitative

One, the purifying of PCR product---96 well plate method

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 in the purifying plate, adds 50 μ l 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.

Two, electrophoresis is quantitative

1, sample is prepared

Get one 96 hole point templates, every hole adds kind 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: obturaging with adhesive plaster in dull and stereotyped two ends, all pours the 250ml glue 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: by the prescribed form application of sample, add the DNA standard substance with single rifle at last with the volley of rifle fire.

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, and is as shown in Figure 3, through behind the electrophoresis, has 6 bands visible, and fragment length is respectively 2000bp, 1000bp, and 750bp, 500bp, 250bp, 100bp, the total concn of DL2000 is 300ng/5 μ l.Get 5 μ l 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 PJVK gene coding region pcr amplification product of [embodiment 3] purifying

One, 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

Two, sequencing reaction

1, the required reagent of sequencing reaction should be fresh, need after autoclaved reagent must be sterilized, can use.The required equipment of sequencing reaction (first-class like 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 is as shown in table 2.

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

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

4, sample is put on the PCR appearance, and the process of the reaction of doing is seen table 3.

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

5, the sample that has reacted will in time take off from the PCR appearance, 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.

Three, the purifying of sequencing reaction thing and order-checking

1, in every hole, adds 20 μ l, 80% ethanol, 4, the centrifugal 30min of 000rpm; Sample panel is placed on the paper handkerchief of rolling well, in whizzer, gets rid of, speed can not surpass 1,000rpm 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 operation in the 2nd step;

4, repeat the operation in the 2nd step;

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, appearance is gone up in centrifugal back.

Sequencer map is as shown in Figure 4.

Deaf-related gene PJVK---c.437G>A mutational site test kit and application thereof are detected in [embodiment 4]

1, the composition of test kit

(1) amplification primers:

Upstream primer: PJVK-4F-1:5 '-CAAATAGAGTCCCCTTCAACAG-3 ' (nt4328-nt4349)

Downstream primer: PJVK-4R-1:5 '-CTCCAGTATGCACGTAATTTCA-3 ' (nt4942-nt4963)

Annotate: PJVK gene order searching number: NC_000002.11

(2) pcr amplification is with Taq enzyme 2U/ μ l

(3) 10 * damping fluids (containing 15ml MgCl2)

（4）dNTP?2.5mM

（5）Big-Dye?mix

2, method of use

Mainly comprise the steps:

1) pcr amplification

With the coding region of 3 pairs of PJVK genes of software Primer design PCR primer, reaction conditions is 94 ° of preparatory sex change of C 5 minutes, 94 ° of C sex change 30 seconds; 65-60 ° of C annealed 40 seconds, and 72 ° of C extended 10 circulations 40 seconds; 94 ° of C sex change 30 seconds, 60 ° of C annealing 40 seconds, 72 ° of C extended 40 seconds; 25 circulations, after reaction finishes again 72 ° of C extended 5 minutes, 4 ° of C preserve (Fig. 2).

2) PCR product purification

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

3) 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 3700DNA sequenator.Resulting order-checking collection of illustrative plates is analyzed, with the standard sequence comparison of NC_000002.11 to confirm whether the mutational site exists.

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

[embodiment 5] are following arbitrary to the pcr amplification primer with being selected from, and repeat the step of the foregoing description 1-4, all can reach identical detected result:

Upstream primer: PJVK-4F-2:5 '-TGGCTCTACACACATTTGCT-3 ',

Downstream primer: PJVK-4R-2:5 '-ACAAGCCTGACTAGAAATTCAA-3 ';

Upstream primer: PJVK-4F-3:5 '-TACTTTGGCTCTACACACAT-3 ',

Downstream primer: PJVK-4R-3:5 '-GCCTGACTAGAAATTCAATAC-3 ';

Upstream primer: PJVK-4F-4:5 '-GCCTTGATTTACTATTAGGTG-3 ',

Downstream primer: PJVK-4R-4:5 '-CTATACAACTGCAGCTCTTTC-3 '.

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 accompanying claims of the present invention.

Claims (5)

1. One kind be used to detect relevant with sensorineural deafness and the sick pedigree obstacle of auditory nerve disease/auditory nerve be positioned at the PJVK gene c.437G test kit that A suddenlys change, said test kit comprises:

   From testing sample, extract the reagent of DNA;

   The PCR reaction reagent that is used for amplified sample DNA;

   The reagent that pcr amplification product is checked order;

   Wherein, the PCR reaction reagent of said amplified sample DNA comprises the PCR primer, and the target fragment of said PCR primer amplification comprises PJVK gene the 437th bit base.
2. 2. test kit as claimed in claim 1, wherein said PCR primer are to be selected from a pair of in the following primer:

   PJVK-4F-1:5’-CAAATAGAGTCCCCTTCAACAG-3’，

   PJVK-4R-1:5’-CTCCAGTATGCACGTAATTTCA-3’；

   PJVK-4F-2:5’-TGGCTCTACACACATTTGCT-3’，

   PJVK-4R-2:5’-ACAAGCCTGACTAGAAATTCAA-3’；

   PJVK-4F-3:5’-TACTTTGGCTCTACACACAT-3’，

   PJVK-4R-3:5’-GCCTGACTAGAAATTCAATAC-3’；

   PJVK-4F-4:5’-GCCTTGATTTACTATTAGGTG-3’，

   PJVK-4R-4:5’-CTATACAACTGCAGCTCTTTC-3’。
3. 3. test kit according to claim 1 or claim 2, said test kit be used to detect be positioned at the PJVK gene c.437G the method for A sudden change, may further 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 the PCR primer;

   Wherein, the target fragment of said PCR primer amplification comprises PJVK gene the 437th bit base;

   3) the PCR reaction product that obtains is directly checked order, and the sequence of sequencing result and PJVK normal gene is compared, determine whether to exist the PJVK gene c.437G the A mutational site.
4. 4. like the said test kit of claim 3, wherein said method further comprises the steps:

   4) translate to confirm c.437G according to the normal reading framework A sudden change makes aminoacid sequence missense mutation take place from 146, makes l-arginine become Histidine (p.R146H).
5. 5. test kit as claimed in claim 1 is in the purposes that is used for detecting sensorineural deafness and the sick pedigree obstacle of auditory nerve disease/auditory nerve.

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