CN110964808A - Detection kit for mutation of Wadenberg syndrome type I pathogenic gene PAX3 - Google Patents

Abstract

The invention discloses a detection kit for mutation of Warderberg syndrome type I pathogenic gene PAX 3. The kit comprises a reagent for extracting DNA from a sample to be detected, a PCR reaction reagent for amplifying the sample DNA and a reagent for sequencing a PCR amplification product; the PCR reaction reagent for amplifying the sample DNA comprises PCR primers. The kit is used for detecting whether the patient has PAX3 gene c.773T > C mutation or not, so that the cause of Wadenberg syndrome I is diagnosed, the kit is favorable for clinically carrying out the PAX3 mutation screening work of the Wadenberg syndrome I patient, and a basis is provided for the diagnosis of the Wadenberg syndrome I patient.

Description

Detection kit for mutation of Wadenberg syndrome type I pathogenic gene PAX3

Technical Field

The invention relates to the field of gene detection, in particular to a PAX3 gene single mutation site c.773T > C (p.L258P) typing detection kit applied to clinical diagnosis of Wardenberg syndrome type I.

Background

Waardenberg Syndrome (WS) is a clinically common disease causing syndromic deafness, and the genetic mode is autosomal dominant inheritance, and at least 2 percent of congenital deafness is caused by Waardenberg syndrome. The pathogenesis of the disease is a syndrome caused by abnormal function of neural crest cells caused by loss of melanocytes originated from the neural crest. Warderberg syndrome is mainly manifested by sensorineural deafness and abnormal pigment, including abnormal color of iris, white forehead hair, premature gray hair, hypopigmentation of skin or freckle deposition; other manifestations include angular ectopy, high and wide nasal root, hirsutism, flush of eyebrow or middle part of eyebrow. The vardenberg syndrome can be divided into 4 subtypes according to clinical manifestations: type I (WS1) is manifested by congenital sensorineural deafness, pigment abnormality and angular ectopy; type II (WS2) has no angular heterotropia, and other clinical manifestations are the same as WS 1; type iii (WS3) combined upper limb deformity on WS1 basis; type IV (WS4) merged the ganglion-free megacolon over WS 2. The Wardnberg syndrome has various clinical manifestations, and the clinical manifestations of the Wardberg syndrome can also be different among different individuals in the same family due to different penetrance rates or other reasons, and few individuals have all the manifestations.

The Wardnberg syndrome is a group of heterogeneous diseases caused by mutation of transcription factor related genes, the gene PAX3(PairBox 3, pairing box gene) is located in chromosome 2q 35-2 q37, the cDNA sequence has a full length of 1440bp, and the encoded PAX3 protein is one of members of the pairing box transcription factor PAX family. The PAX3 protein encoded by the PAX3 gene consists of 479 amino acids, mainly contains a pairing box structural domain, a homologous structural domain, a highly conserved zinc peptide sequence and a transcription activation domain rich in serine-threonine-proline, and has a molecular weight of about 53 kD. PAX3 was first expressed in the dorsal neural tube before neural crest migration and then gradually and widely expressed in melanocytes of the central nervous system, joints, skeletal muscles, and neural crest-derived tissues such as the peripheral nervous system, myocardial interstitium, smooth muscle, thymus, adrenal medulla, skin hair, and inner ear following neural tube development. PAX3 is involved in the regulation of embryonic growth and development by regulating the development of neural crest, and plays an important role in the formation process of skeletal muscle and melanin. In the development of melanocytes, PAX3 can activate or inhibit the expression of microphthalmia-associated transcription factor (MITF), dopachrome tautomerase (DCT) and tyrosinase-related protein 1 (TRP 1) mainly through direct or synergistic action with other transcription factors, and can promote and inhibit the synthesis of melanin by regulating the growth, development and differentiation of melanocytes. Animal experiment research shows that melanocyte dysplasia can cause melanocyte-derived intermediate cell deficiency in blood vessel veins, further cause the degeneration and degeneration of Corti apparatus, and finally cause sensorineural deafness.

Mutation of the PAX3 gene led to WS by affecting neural crest development, particularly neural crest-derived melanocyte development. Various mutations of PAX3 gene can be detected in 90% of Wardnberg syndrome I, and complete deletion of PAX3 gene is found in partial cases. Because the mutation varieties of the PAX3 gene are various, the function of the protein is affected differently after each site is mutated, and some sites are polymorphic after mutation, the difficulty of determining the pathogenicity of new mutation sites is increased. At present, no report about C.773T > C (p.L258P) mutation of PAX3 gene is found.

Disclosure of Invention

The invention aims to provide a detection kit for mutation of Warderberg syndrome type I pathogenic gene PAX 3.

In order to achieve the purpose, the invention adopts the following technical scheme:

a kit for detecting C.773T > C (p.L258P) mutation of PAX3 gene comprises PCR reaction reagents for amplifying DNA fragments, wherein the PCR reaction reagents comprise PCR primers, and a target fragment amplified by the PCR primers comprises a base corresponding to 773 th (reference sequence NM-181457.4) of a CDS region of human PAX3 gene.

Preferably, the kit further comprises a reagent for extracting template DNA required for PCR amplification from an individual to be tested.

Preferably, the kit further comprises reagents for sequencing the PCR amplified target fragment.

Preferably, the PCR primer is selected from primer pair P1, and the sequence of primer pair P1 is:

PAX3-F-1:5’-ACCCCAATCAGATGAAGGC-3’；

PAX3-R-1:5’-GGTGGACTTCTGTGTGTCGT-3’。

preferably, the PCR primer is selected from primer pair P2, and the sequence of primer pair P2 is:

PAX3-F-2:5’-GGGGCAGAAAGATAGAGTAGGA-3’；

PAX3-R-2:5’-AATAGTGGCTGGGACAGAGTAA-3’。

preferably, the PCR primer is selected from primer pair P3, and the sequence of primer pair P3 is:

PAX3-F-3:5’-AGGCTCTGATATTGACTCTG-3’；

PAX3-R-3:5’-TATGCCACCTTGGGTAGTT-3’。

the method for detecting C.773T > C (p.L258P) mutation of the PAX3 gene by using the kit comprises the following steps:

1) collecting blood, body fluid or tissue of an individual to be detected, and then extracting DNA;

2) taking the DNA extracted in the step 1) as a template, and carrying out PCR reaction by using the PCR primer to obtain a PCR reaction product; separating the target fragment amplified by the PCR reaction from the PCR reaction product, and typing and identifying the base contained in the target fragment and corresponding to 773 th (reference sequence NM-181457.4) of the CDS region of the human PAX3 gene.

Preferably, the genotyping method adopts a method of directly sequencing the target fragment, and the genotype or the allelic type of the tested individual corresponding to 773 th (reference sequence NM-181457.4) of the CDS region of the human PAX3 Gene is determined by comparing the sequencing result with the reference sequence (reference sequence Gene ID: 5077).

Preferably, the genotype determined by the alignment includes wild homozygous T/T, mutant heterozygous T/C or mutant homozygous C/C.

The kit is applied to analysis of Warderberg syndrome type I etiology. The kit is used for detecting whether a c.773T > C mutation exists at the 773 rd site (reference sequence NM-181457.4) of a CDS region of a human PAX3 gene in a sample (a PAX3 gene fragment of a patient) to be detected, so as to judge the hereditary cause of the occurrence of the Wadenberg syndrome I of the patient. Among them, c.773T > C mutation of PAX3 gene makes the corresponding base at 773 (reference sequence NM-181457.4) of CDS region of PAX3 gene generate mishaped mutation, the mutation makes 258 leucine changed into proline (p.L258P), the amino acid position is highly conserved among species, the mutation affects function.

The invention has the beneficial effects that:

the kit provided by the invention can be used for rapidly detecting the specific mutation site of the PAX3 gene, and can judge the occurrence reason of Wardnberg syndrome I of a patient by detecting whether the DNA sample from the patient has C.773T > C mutation of the PAX3 gene, thereby providing a basis for clinical diagnosis.

The kit provided by the invention is used for diagnosing Warderberg syndrome I: 1) the invention provides a convenient and reliable method for screening susceptibility genes in Waardenberg syndrome type I patients; 2) through prenatal diagnosis and screening, whether the fetus is a c.773T > C mutation carrier is determined, the birth rate of the deaf infant can be reduced, and the burden of the society and families is reduced.

Drawings

FIG. 1 shows the amino acid conservation analysis of the coding region of PAX3 gene: the mutation is located at 773 th T base of CDS region of PAX3 gene, and enclosed by a box is amino acid before mutation.

FIG. 2 is a flow chart of PCR reaction: reaction conditions (reaction temperature and time) are shown, where ↓ represents a 0.5 ℃ decrease per cycle.

FIG. 3A is a diagram showing the sequencing result of the PAX3 gene of the tested individual: the heterozygote sequence was mutated, with the position of the mutation site indicated by the arrow (reverse sequencing).

FIG. 3B is a diagram showing the sequencing result of the PAX3 gene of the tested individual: wild type sequence, arrow indicates mutation site position (reverse sequencing).

FIG. 4 is an electropherogram of the amplification product: the left lane is Marker and the right lane is amplified target fragment (395 bp).

Detailed Description

The invention is described in detail below with reference to the drawings and examples, which are provided to illustrate the invention and not to limit the scope of the invention.

The invention screens 100 non-syndrome Wardberg syndrome type I patients and 100 controls with normal hearing and no family history by using a candidate gene screening method, and a c.773T > C mutation is found in PAX3 gene of one Wardberg syndrome type I patient. The audiological test result of the patient is extremely severe hearing loss, CT and MRI results show no abnormality, bilateral irises are in light blue, the nasal root is flat, and the inner canthus is widened. The hearing of the parents is normal, and the hearing of the brother is normal. The patient's genotype was a c.773T > C heterozygous mutation, and both the patient's parental and brother genotypes were wild-type. Patients with c.773T > C belonging to spontaneous mutation. The PAX3 gene mutation was cosegregated with the vardenberg syndrome type I phenotype. The Wadenberg syndrome type I associated with PAX3 gene mutation is transmitted in an autosomal dominant manner. There are more than 100 mutations related to Wardenberg syndrome type I reported at present, and no c.773T > C mutation is reported.

The mutation (c.773t > C) described above resulted in a missense mutation of the base corresponding to position 773 of the CDS region of the PAX3 gene (NM — 181457.4), and the mutation changed leucine at position 258 to proline (p.l258p), a site highly conserved among species (fig. 1).

The detection of the above mutation (c.773T > C) can be carried out by various methods for detecting a mutation, for example, PCR (polymerase chain reaction) -sequencing method, hybridization method using a labeled PAX3 gene DNA probe, method using restriction fragment length polymorphism or sequence-specific primer, etc. Wherein, the method for detecting the sample by adopting a PCR amplification-direct sequencing method comprises the following steps:

1) collecting a sample of an individual to be tested, such as blood, and extracting genomic DNA;

2) carrying out PCR reaction by using the DNA as a template and a PCR primer designed near 773 th base of a CDS region of the PAX3 gene to obtain a PCR amplification product;

3) carrying out target fragment sequencing analysis on the obtained PCR amplification product, comparing a sequence obtained by sequencing with a PAX3 Gene reference sequence (a standard sequence Gene ID:5077 of a wild-type PAX3 Gene), and determining whether the C.773T > C mutation exists in the PAX3 Gene of an individual to be detected;

4) and judging whether the individual to be detected is Wardenberg syndrome I type caused by PAX3 gene mutation c.773T > C according to the result.

The PCR primer used in the above step 2) can be designed based on the known primer nucleotide sequence: usually 15 to 30 bases, and GC content of about 45 to 50%, and specifically binds to the terminal at an appropriate temperature. Primers can be designed using existing computer programs.

If the PCR reaction product obtained in step 2) above is detected using a hybridization probe, the hybridization probe used may be a probe that hybridizes with the normal PAX3 nucleotide sequence, or with the mutated nucleotide sequence, or with the complementary sequence thereof. These probes may be labeled with a radioisotope, a chromogenic substance or a fluorescent substance, and particularly, allele-specific probes may be used.

According to different detection methods, the kit for detecting C.773T > C mutation of PAX3 gene comprises PCR reaction reagents and reagents for detecting PCR amplification products, wherein the reagents are specifically selected from sequencing detection reagents, restriction length polymorphism detection reagents, sequence-specific primer detection reagents and probe hybridization detection reagents.

The kit container contains reagent components for detecting C.773T > C mutation of PAX3 gene, and provides manufacturing, using and selling information of medicine or biological products approved by government drug regulatory agency. The PCR reaction reagent may contain, for example, amplification primers, dNTPs, DNA polymerase used for PCR reaction, a buffer therefor, and the like.

Example 1

Various Wadenberg syndrome type I patients are collected through a deafness outpatient service and a resource collection network, and a resource library is established. On the premise of patient's voluntary, after signing the informed consent, blood samples are taken, and an outpatient medical record database is established to record the patient's condition, the incidence of disease in the family and the contact way in detail. Then, genomic DNA was extracted by protease degradation, quantified and stored at-20 ℃ with each DNA sample corresponding in detail to the clinical data of the registered patients. Then, primers (amplification target region: exon 3 of PAX3 Gene, reference sequence Gene ID: 5077; NM-181457.4) were designed using online primer design software Primer5.0, and PCR amplification was performed on a BIORAD My Cycle thermal cycler using genomic DNA as a template. The sequencing primer is the same as the PCR amplification primer, and is used for forward and reverse sequencing by using an ABI 3730DNA sequencer. The sequence obtained by sequencing was compared with the sequence in Genbank (Gene ID:5077) to determine the PAX3 c.773T > C mutation. The method comprises the following specific steps:

first, extracting blood sample to be tested and PCR amplification of coding region of PAX3 gene

1. Preparation of DNA in blood sample of subject to be tested

1.1 study object

The PAX3 gene was screened for 100 patients with non-syndromic Wardberg syndrome type I and 100 normal hearing controls without family history as follows.

The sporadic non-syndrome deafness subjects are collected from deafness patients who are subjected to deafness gene screening in otolaryngology head and neck surgery outpatient service of Xijing hospital (xi' an city, Shaanxi province). The normal hearing control is a normal hearing subject without family history of deafness, and the medical history and family history of all participants are investigated in detail and subjected to physical examination, wherein the otological examination comprises otoscopy and audiological evaluation. 5-10 mL of blood samples are collected by each person after signing an informed consent, and the collection time is 10 months in 2009.

1.2 genomic DNA extraction

1.2.1 preparation before experiment and important precautions

(1) Protease K was dissolved by adding a predetermined amount of protease K Storage Buffer, and stored at-20 ℃. The prepared Proteinase K is not stored for a long time at room temperature, and repeated freeze thawing is avoided, so that the activity of the Proteinase K is not influenced.

(2) All centrifugation operations were done at room temperature.

(3) Storage of blood sample: the blood sample added with anticoagulant can be stored at 2-8 ℃ for 10 days at most, for some experiments such as Southern hybridization and the like, complete and full-length genome DNA is required, please store the blood sample at 2-8 ℃ for 3 days at most, and the degradation degree of the genome DNA is light.

1.2.2 working steps

1) The blood sample was centrifuged at low speed until it was stratified, and the supernatant serum was removed with a pipette, taking care not to aspirate or damage the middle buffy coat layer.

2) Transferring all blood cells into a 5mL centrifuge tube, adding erythrocyte lysate to the total volume of 4mL, and turning upside down and mixing uniformly for 20 times until the precipitate is fully dispersed.

3)6500g, centrifuged for 10min and the supernatant discarded.

4) 3mL of Buffer FG1 was added and vortexed for 15s to thoroughly disperse the precipitate.

5) And centrifuging at 6500g for 10min, discarding supernatant, reversely buckling the centrifugal tube on clean absorbent paper, and sucking water.

6) Preparing a mixed solution of the DNA extracting solution and the proteinase K according to the mixing ratio of the DNA extracting solution and the proteinase K being 100:1, mixing, fully and uniformly mixing by whirling for 15s, and preparing as required.

7) Adding 1mL of the prepared mixed solution of the DNA extracting solution and the proteinase K into the sample, immediately and fully whirling and oscillating for 1min until the solution has no lumps.

8) The sample was incubated in a 65 ℃ water bath for 15min, during which time the mixture was reversed and mixed 3 times until the color of the sample changed from red to light green, indicating complete digestion of the protein.

9) 2mL of isopropanol was added to the sample and mixed by inversion 10 times until a white flocculent precipitate was visible.

10) A clean sterile 1.5mL centrifuge tube was labeled and 500. mu.L of pre-cooled 75% ethanol was added.

11) Picking the white flocculent precipitate in the step 9) by using a clean and sterile 1mL pipette tip, transferring the white flocculent precipitate into 75% ethanol prepared in the step 10), reversing and mixing the white flocculent precipitate for 10 times, and slowly pouring off the supernatant, wherein the white flocculent precipitate is not poured off.

11) Add 500. mu.L of pre-cooled 75% ethanol again, mix well 10 times by inversion, pour off the supernatant slowly and suck to dryness.

12) The tube cap was opened and dried at room temperature for 15min until all liquid was completely evaporated.

13) 380. mu.L of a DNA solution was added, and the mixture was incubated in a water/metal bath at 65 ℃ for 2 hours while shaking to dissolve the DNA sufficiently.

14) And quantifying and detecting the purity by a spectrophotometer.

15) The DNA was stored at-20 ℃.

2. PCR amplification of the coding region of the PAX3 Gene

2.1 primer sequences

Primer5 Primer design software (reference sequences Gene ID: 5077; NM-181457.4) was used for this test after sequence synthesis, with a design completion time of 2019, 8 months:

the upstream primer PAX3-F-1: 5'-ACCCCAATCAGATGAAGGC-3',

the downstream primer PAX3-R-1: 5'-GGTGGACTTCTGTGTGTCGT-3'.

The size of the fragment obtained by PCR amplification using this primer was 395 bp.

2.2 establishment of PCR reaction System (Table 1)

TABLE 1 PCR reaction System for PAX3 Gene

In this case, the PCR Mix of Tiangen was used for PCR amplification.

Reaction conditions are as follows: the PCR reaction was carried out on a BIORAD My Cycle thermal cycler, and the reaction sequence (including temperature and time) is shown in FIG. 2:

1) pre-denaturation at 94 ℃ for 4 min;

2) denaturation at 94 ℃ for 30 seconds, annealing at 61 ℃ (initial annealing temperature, 0.5 ℃ reduction per cycle) for 30 seconds, extension at 72 ℃ for 40 seconds, 12 cycles;

3) then denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension at 72 ℃ for 40 seconds, and 30 cycles;

4) after the reaction, the reaction was further extended at 72 ℃ for 5 minutes and stored at 4 ℃.

Electrophoresis process of PCR products:

1) gel (1% agarose): 0.4g of agarose was weighed and suspended in 40mL of XTAE (500mL Erlenmeyer flask).

2) Sol: heating with high fire in a microwave oven until boiling, continuously boiling for several minutes, taking out and mixing.

3) Cooling the glue: after the gel was completely dissolved, the gel was removed from the microwave oven, cooled to about 60 deg.C, added 1 drop of EB (about 10. mu.L, 10mg/mL), and shaken well.

4) Spreading glue: the two ends of the plate are sealed with adhesive tapes, 250mL of glue solution is poured into the plate, and a comb ruler is inserted.

5) Gluing: the plate was placed in an electrophoresis tank containing an electrophoresis solution (0.5 × TAE, liquid level 1 to 2mm from the surface of the gel), and the comb ruler was pulled out.

6) Sample adding: and (4) adding the sample according to a specified format by using a pipettor, and finally adding the MarkerDL 2000.

7) Glue spreading: covering the electrophoresis tank cover, checking the positive and negative levels, starting the electrophoresis apparatus, and adjusting the electrophoresis voltage.

8) Quantification: when the bromophenol blue leaves the sample adding hole by 1.5-2 cm, the electrophoresis apparatus is closed, the gel is carefully taken out, and the gel is placed into a camera for photographing. After electrophoresis, 6 bands can be seen, the lengths of the fragments of the MarkerDL2000(TaKaRa) are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp respectively, and the total concentration of DL2000 is 300ng/5 mu L. Since 5. mu.LDL 2000 was used for electrophoresis, the content of each band was 50 ng. When the PCR product was electrophoresed, 5. mu.L (PCR product) was electrophoresed. The size and content of the PCR product were judged by comparing the gray value of the PCR product after electrophoresis with the gray value of DL2000 (see FIG. 4).

Purification and quantification of PCR amplification products from the coding region of the (II) PAX3 Gene

Purification of PCR amplification products (96-well plate method):

1) after the electrophoresis of the PCR amplification product is finished, a target strip is cut off by a scalpel under a long-wave 365nm ultraviolet transilluminator, the mass of the cut gel block is less than 3g, and the gel block is placed in the plate hole number corresponding to the plate hole number.

2) Centrifuging at 4000rpm for 1min, adding 500ul sol solution, covering with sealing film, and water bathing at 65 deg.C for 15 min.

3) Checking whether the glue block in each hole is completely dissolved, if not, carrying out water bath at 65 ℃ for 3min again, uncovering the sealing film, adding 10 mu L of uniformly mixed magnetic beads in each hole by using a continuous liquid adding device, covering a silica gel pad, carrying out vortex oscillation for 30s, and transferring to a horizontal oscillator at 600-800 rpm for oscillation for 5 min.

4) And (3) clamping the 96-well plate into a magnetic frame, carrying out magnetic attraction for 30s, slightly reversing the magnetic frame and the sample for 3 times, and standing for magnetic attraction for 1min again.

5) Discarding the waste liquid, slightly knocking on absorbent paper, transferring 500 mu L of lotion into each hole by using a 50-1200 mu L8-channel electric pipettor, covering a silica gel pad for vortex oscillation for 30s, clamping a 96-hole plate into a magnetic frame, magnetically attracting for 30s, slightly reversing the magnetic frame and the sample for 3 times, and standing for magnetic attraction for 1min again.

6) Discarding the waste liquid, slightly knocking on absorbent paper, moving 500 mu L70% ethanol to each hole by using a 50-1200 mu L8-channel electric pipettor, covering a silica gel pad on each hole, carrying out vortex oscillation for 30s, clamping a 96-hole plate into a magnetic frame, carrying out magnetic attraction for 30s, slightly reversing the magnetic frame and the sample for 3 times, and standing for magnetic attraction for 1min again.

7) Abandoning the waste liquid, slightly knocking on absorbent paper, inverting and centrifuging to 600rpm, and horizontally shaking for 5 min.

8) Centrifuging to 1000rpm, clamping the 96-well plate into a magnetic frame, and magnetically attracting for 1 min.

9)2 mu L of sample +6 mu L of 1.4X bromophenol blue are mixed and dripped into 0.8% identification gel, the identification gel is transversely dripped according to the vertical sequence of A01-H01, 2 holes are left in the middle, 1 mu L of DL2000 and 2 mu L of DL2000 are respectively added, and 300V electrophoresis is carried out for 11 min.

10) And (3) putting the identification gel into a gel imager to collect images, wherein the images must ensure that the marker strips are clear.

11) And (3) contrasting gel images before and after purification, marking the concentration of the sequencing template obtained after the amplification product of each hole is purified on a PCR record table according to a PCR quantitative standard, diluting to the specified concentration, and carrying out electrophoretic re-identification on the recovered electrophoretic sample without bands according to 4 mu L sample +5 mu L1.4X bromophenol blue.

12) Centrifuging the diluted template for 2min to 4000rpm, marking the state of the Lims system template, checking the state of the template again before confirming submission, and storing the template in a refrigerator at 4 ℃ after confirming no error.

(III) direct sequencing of PCR amplification products of the coding region of the purified PAX3 Gene

1. The purity and amount requirements of the PCR product DNA template are shown in Table 2.

DNA purity: OD260/OD280 is 1.6-2.0.

DNA concentration: PCR product 10 ng/. mu.L.

TABLE 2 DNA dosage

PCR product Length (bp)	Template addition for sequencing reactions (ng)
		100～200	1～3
200～500	3～10
		500～1000	5～20
1000～2000	10～40
		>2000	40～100

2. Sequencing reactions

1) The reagents required for the sequencing reaction should be freshly prepared, and the reagents that need to be autoclaved must be sterilized before use. The equipment required for the sequencing reaction (e.g., 96-well plates, tip heads, etc.) should also be clean and sterile.

2) In order to ensure the freshness of the sequencing sample and the reaction reagent, the sample should be loaded on ice.

3) The current reaction system is 5. mu.L, and the amounts of various reagents added are shown in Table 3.

TABLE 3 sequencing reaction System for PCR amplification product of PAX3 Gene

Among them, BDT is a fluorescent dye produced by applied biosystems of America (ABI) for sequencing reactions. 5 XGCbuffer is the buffer for sequencing reactions produced by applied biosystems, Inc. (ABI) of USA.

4) The samples were placed on a PCR machine (thermal cycler) and the course of the reaction is shown in Table 4.

TABLE 4 sequencing reaction procedure for PCR amplification product of PAX3 gene

5) The reacted sample needs to be taken down from a PCR instrument (thermal cycler) in time, the sample to be purified in a short time is placed in a refrigerator at 4 ℃, and the sample which can be purified only after more than one day is placed in a refrigerator at-20 ℃ for freezing.

3. Purification and sequencing of sequencing reactions

1) Adding 20 μ L of 80% ethanol into each well, and centrifuging at 4000rpm for 30 min; putting the sample plate on a folded paper towel, and reversely throwing the sample plate in a centrifugal machine, wherein the speed rate cannot exceed 1000rpm when the sample plate is reversely thrown;

2) adding 30 μ L70% ethanol into each well, centrifuging at 4000rpm for 10min, and back-throwing;

3) repeating step 2) two more times;

4) placing the sample plate in a clean drawer, and drying for 30min in a dark place;

5) adding 5 mu L of formamide, sealing the membrane, centrifuging and placing in a refrigerator at the temperature of minus 20 ℃;

6) denaturalizing at 95 deg.C for 5min before loading, placing on ice for 2min, centrifuging, and loading on ABI 3730 sequencer.

The sequencing results are shown in FIGS. 3A and 3B. Of 100 patients, 1 patient with Wardenberg syndrome type I was found to have c.773T > C heterozygous mutation in the PAX3 gene test. No c.773T > C mutant was found in the screening of 100 normal hearing subjects.

(IV) kit for detecting deafness related gene PAX3 mutation site (c.773T > C) and application thereof

1. Composition of the kit

(1) Amplification primers:

upstream primer PAX3-F-1: 5'-ACCCCAATCAGATGAAGGC-3'

Downstream primer PAX3-R-1: 5'-GGTGGACTTCTGTGTGTCGT-3'

(2) PCR Mix 2 for PCR amplification

(4)dNTP 2.5mM

(5) Big-Dye mix (manufactured by applied biosystems of America (ABI))

2. Application method

1) PCR amplification

Software Primer5.0 was used to design PCR primers for the coding region of the PAX3 gene, and the reaction conditions are shown in FIG. 2.

2) PCR product purification

And (3) performing electrophoresis on the PCR product, purifying the gel and performing electrophoresis quantification.

3) Sequencing reaction and validation

And (3) carrying out sequencing reaction by taking the PCR primer as a sequencing primer and carrying out sequencing reaction on a BIORAD My Cycle thermal cycler. After the reaction is finished, the extension product is loaded on an ABI 3730DNA sequencer. The resulting sequencing map was analyzed and compared to the normal sequence (Gene ID:5077) to determine the presence or absence of a mutation.

Example 2

The amplification primers (design completion time 2019, 8 months) were as follows, otherwise as in example 1:

the upstream primer PAX3-F-2: 5'-GGGGCAGAAAGATAGAGTAGGA-3',

the downstream primer PAX3-R-2: 5'-AATAGTGGCTGGGACAGAGTAA-3'.

Example 3

The amplification primers (design completion time 2019, 8 months) were as follows, otherwise as in example 1:

the upstream primer PAX3-F-2: 5'-AGGCTCTGATATTGACTCTG-3',

the downstream primer PAX3-R-2: 5'-TATGCCACCTTGGGTAGTT-3'.

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Claims (10)

1. A kit for detecting C.773T > C mutation of PAX3 gene, which is characterized in that: the kit comprises PCR reaction reagents for amplifying DNA fragments, wherein the PCR reaction reagents comprise PCR primers, and a target fragment amplified by the PCR primers comprises a base corresponding to 773 th position of a CDS region of a PAX3 gene.

2. The kit for detecting the c.773T > C mutation of the PAX3 gene according to claim 1, wherein: the kit also comprises a reagent for extracting template DNA required by PCR amplification from an individual to be detected.

3. The kit for detecting the c.773T > C mutation of the PAX3 gene according to claim 1, wherein: the kit also includes reagents for sequencing the PCR amplified target fragment.

4. The kit for detecting the c.773T > C mutation of the PAX3 gene according to claim 1, wherein: the PCR primer is selected from a primer pair P1, and the sequence of the primer pair P1 is as follows:

PAX3-F-1:5’-ACCCCAATCAGATGAAGGC-3’；

PAX3-R-1:5’-GGTGGACTTCTGTGTGTCGT-3’。

5. the kit for detecting the c.773T > C mutation of the PAX3 gene according to claim 1, wherein: the PCR primer is selected from a primer pair P2, and the sequence of the primer pair P2 is as follows:

PAX3-F-2:5’-GGGGCAGAAAGATAGAGTAGGA-3’；

PAX3-R-2:5’-AATAGTGGCTGGGACAGAGTAA-3’。

6. the kit for detecting the c.773T > C mutation of the PAX3 gene according to claim 1, wherein: the PCR primer is selected from a primer pair P3, and the sequence of the primer pair P3 is as follows:

PAX3-F-3:5’-AGGCTCTGATATTGACTCTG-3’；

PAX3-R-3:5’-TATGCCACCTTGGGTAGTT-3’。

7. a method for detecting C.773T > C mutation of PAX3 gene is characterized in that: the method comprises the following steps:

1) collecting blood, body fluid or tissue of an individual to be detected, and then extracting DNA;

2) taking the DNA extracted in the step 1) as a template, and carrying out PCR reaction by using a PCR primer to obtain a PCR reaction product; separating the target fragment amplified by the PCR reaction from the PCR reaction product, and carrying out typing identification on the base contained in the target fragment and corresponding to 773 th position of the CDS region of the PAX3 gene.

8. The method for detecting the c.773T > C mutation of the PAX3 gene according to claim 6, wherein the C is selected from the group consisting of: the genotyping identification adopts a method of directly sequencing the target fragment, and determines the genotype or allele type of the 773 th site of the CDS region of the PAX3 gene of the individual to be detected by comparing the sequencing result with a reference sequence.

9. The method for detecting the c.773T > C mutation of the PAX3 gene according to claim 6, wherein the C is selected from the group consisting of: the genotype determined by the alignment includes wild homozygous T/T, mutant heterozygous C/T or mutant homozygous C/C.

10. Use of the kit of claim 1 for analysis of the etiology of Waardenberg syndrome type I.

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