CN107287314A - It is a kind of to detect that hereditary hearing impairment gene builds storehouse kit and application - Google Patents
It is a kind of to detect that hereditary hearing impairment gene builds storehouse kit and application Download PDFInfo
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Abstract
Storehouse kit and application are built the present invention relates to a kind of detection hereditary hearing impairment gene, the kit includes the amplimer group for being used to expand hereditary hearing impairment gene；The amplimer group includes the primer according to GJB2, GJB3, SLC26A4 gene and the Design for polymorphism in the mutational site of 12s rRNA genes.The present invention uses multiple PCR technique, and single reaction can detect more than 20 deaf gene mutational sites；Coordinate " double labels " system of use, make on the DNA cloning product of each sample with two sets of independent sequence labels, therefore it is sequenced simultaneously after the amplified production of all samples can be mixed, realizes that high flux is detected, so as to significantly reduce the testing cost of single sample.
Description
Technical field
The present invention relates to technical field of biological, and in particular to one kind builds storehouse kit and its preparation method and application,
More particularly to a kind of detect that hereditary hearing impairment gene builds storehouse kit and application.
Background technology
Deafness is a very common sensory disturbance disease for having a strong impact on human lives, and its cause of disease is complicated, single
Or multiple gene mutation can most probably cause severe deafness, and by such environmental effects, such as medicine, wound, extreme
Environmental exposure etc., sudden can also be caused deaf.Global dysaudia number reaches 3.6 hundred million to the years of WHO 2013 according to estimates, and according to
The Second National Disability Sampling Survey key data publication result that in December, 2006 announces shows that the language hearing of China hinders
Number is hindered to be up in 27,800,000, neonate newly-increased 20,000~30,000 deaf youngsters every year, the incidence of disease is up to 1 ‰~3 ‰.
Hereditary hearing impairment has higher heterogeneity, i.e., different deaf Disease-causing genes can cause mutually isophenic auditory function
Obstacle, and the different mutation of same gene can cause the deafness of different clinical phenotypes, thereby result in the cause of disease of deafness
It is extremely complex.Therefore, the research for causing deaf inherent cause is become probe into deafness disease because and effective treatment means breach,
And effective detection deaf-related gene is taken precautions against deaf early stage and played an important role with treatment.It is oriented at present to cause deaf gene
There are kind more than 150, but molecular epidemiology data display, most of hereditary hearing impairments are drawn by several relatively conventional hot spot mutations
Rise, the mutation of Chinese population common deafness mainly includes GJB2, GJB3, SLC26A4 and mitochondrial 12S rRNA genes are prominent
Become, the mutant proportion in crowd is up to 40%.
In the non-syndrome of autosomal recessive inheritance is deaf, about half is due to that GJB2 gene mutations cause
's.GJB2 reports that most mutational sites are 167delT, 235delC, 35delG and 176_191del16 at present.Lot of documents
It has been shown that, GJB2 genes cause there is very big difference on the deaf site being mutated in not agnate.In America and Europe, particularly in north
In the Caucasian in Europe, southern Europe and the U.S., main mutant form is 35delG, accounts for the 70% of all GJB2 mutation；Another
Mutantional hotspot is 167delT, common in the deaf crowd of Jew, and 40%, the 35delG mutation for accounting for all pathologic mutation are only accounted for
18%；And in asian population, then predominantly 235delC is mutated, in Japan and China, 235delC site mutations are in crowd
Carrying rate be 1.0%-1.3%, account for the 80% of all GJB2 pathologic allele.The non-syndrome ear related to GJB2
Deaf patient's ordinary circumstance is generally that ears are involved simultaneously, deaf in symmetry, and majority shows as congenital deafness, deafness
From slightly to pole severe, most of is severe and pole severe deafness, and the extent of damage is relevant with the type being mutated and site, phase
The individual impaired hearing degree of homogenic type is there is also difference, and clinical manifestation has polymorphism, shows that the expression of gene may be by
To the influence of modifier or environment.
GJB3 gene mutations can cause autosomal dominant or recessive inheritance Non-syndromic deafness.Xia Jiahui academician etc.
The successful clone gene in the world first in 1998, finds that GJB3 is positioned at 1p33- using fluorescence in situ hybridization technique
P35, and two GJB3 mutational sites 547G ＞ A and 538C ＞ T are reported earliest：42 hereditary hearing impairments of their examinations
Family, is found that a missense mutation, the 547th bit base of Cx31 gene coding regions is by G in a deaf family in Zhejiang
It is mutated into A so that connection PROTEIN C x31 No. 183 amino acid becomes lysine by glutamic acid；They are also in Hunan simultaneously
It is found that another is mutated in one family, GJB3 the 538th bit base becomes T by C, causes No. 180 amino acid to be changed into eventually
Only codon.
SLC26A4 DNA encoding the protein Pendrin.Pendrin belongs to solute Protein S LC26, passes through its transport function
Intraor extracellular chlorine/iodide ion can be achieved to exchange.SLC26A4 gene mutations and large vestibular aqueduct syndrome and PendredShi are comprehensive
Simulator sickness is relevant, and clinical manifestation is that congenital nerve deafness shows with different degrees of aphasis, Thyroid Gland Swell, CT or MRI
Show cochlea depauperation, Large Vestibular Aqueduct, mutation rate is only second to GJB2 in the hereditary hearing impairment patient of Chinese population.Arrive
Have found that mutation type and occurrence frequency have very big difference in more than 150 kinds of SLC26A4 mutation type, different crowd so far
It is different, IVS7-2A>G mutation types are most common in Chinese large vestibular aqueduct syndrome patient, there are some researches prove IVS7-2A ＞ G also
For founder effect.
Mitochondrial DNA (mtDNA) is unique DNA being present in cytoplasm, because the mitochondria of filial generation is carried by egg cell
For, therefore mitochondrial inheritance belongs to matrilinear inheritance.Genes in aminoglycoside-induced deafness patient is largely 1555A>G、1095T>C、
1494C>The carrier of T mutation, these mutation add sensitiveness of the individual to aminoglycoside medicaments ototoxicity, often occur
The situation of " pin causes deaf ", is detected by early gene, clinical application suggestion can be provided, simultaneously because the mutation belongs to maternal something lost
Pass, can maternal members all to family play security role.
Mainly there are Sanger PCR sequencing PCRs, ARMS-PCR for the detection method that hereditary hearing impairment gene diagnosis is applied at present
Method, fluorescent PCR method, PCR flow hybridizations method, DNA microarray technology, time-of-flight mass spectrometry (TOFMS) etc..
Sanger PCR sequencing PCRs are that dideoxy nucleotide end terminates PCR sequencing PCR, are the goldstandard methods of detection in Gene Mutation,
Its principle is to extend the primer with reference on sequence template to be measured using a kind of archaeal dna polymerase, until mixing a kind of chain termination
Untill nucleotides.Sequencing is individually reacted by a set of four and constituted each time, and each reaction contains all four deoxidation cores
Thuja acid triphosphoric acid (dNTP), and it is mixed into coupling on four kinds of different dideoxyribonucleoside triphosphates (ddNTP) of limitation, the ddNTP
The fluorophor of 4 kinds of different colours is gone up.Because ddNTP lacks the 3-OH groups required for extension, make the oligonucleotides of extension
Acid is optionally terminated at G, A, T or C.Terminating point sends corresponding fluorescence in reaction depending on corresponding ddNTP.Often
A kind of dNTPs and ddNTPs relative concentration can be adjusted, and reaction is obtained the chain termination production of one group long hundreds to thousands base
Thing.They have common starting point, but terminate on different nucleotides, can be of different sizes by capillary electrophoresis separation
Fragment, and the fluorescence signal of every kind of chain termination product is gathered, so as to read the base sequence of sequence template.But Sanger is direct
Each reaction of PCR sequencing PCR can only detect a site, and flux is low, and detection site is few, cause testing cost high, and data analysis
Manual read is relied on, speed is slow.
It is ARMS-PCR methods (amplification refractory mutation to be mutated Retardation of amplification system
System) it is a kind of improved PCR method, its general principle is, if 3 ' the end bases and template base of primer are not complementary,
It can not be extended with general Taq archaeal dna polymerases, this method carries out base mutation detection using 4 primers, first in mutation to be detected
Design upstream and downstream primer (2 outer primers) on the outside of site, then draw in the upstream and downstream design two close to mutational site to be detected
Thing, 3 ' end bases of a primer are mutant bases, and 3 ' end bases of another primer are wild-type base, if existing prominent
Become, then produce the product of mutant bases, if without mutation, producing the product of wild-type base, passing through agarose gel electrophoresis
Detect the presence of amplified fragments and fragment length determines whether there is point mutation, or remember 2 different fluoresceins in inner primer subscript, pass through
It is that although it is understood that to whether there is point mutation in template to product fluorescence analysis.Each reaction of ARMS-PCR methods can only detect one
Or two sites, detection site is less, causes testing cost higher.
Fluorescent PCR method is to add fluorescence labeling probe on the basis of Standard PCR to realize that base mutation is detected.This method is directed to
The different genotype in same site to be measured designs two different Taqman probes, they is added to simultaneously PCR reaction systems
In, the probe only matched completely with target sequence can just be hydrolyzed and discharge fluorescence signal.Two probe marks difference respectively
Fluorophor, respectively with corresponding fluorescence detection channel detection fluorescence signal in real-time fluorescence quantitative PCR instrument.If only one
Bar probe can discharge fluorescence signal, then the site detected is homozygote, if two probes have fluorescence signal, the position detected
Point is heterozygote.But each reaction of fluorescent PCR method can only detect one or two site, and detection site is less, cause detection
High expensive.
PCR flow hybridizations method is similar with DNA microarray technical principle, is all the detection side designed according to solid-phase hybridization principle
Method, PCR flow hybridization methods are to use the primer with biotin to enter performing PCR target DNA to expand, PCR primer and film with biotin
Fixed capture probe hybridization, adds the Streptavidin of crosslinking alkaline phosphatase, washes away uncombined Streptavidin, plus
Enter substrate colour developing, thus detect whether target DNA makes a variation, and biotin is changed to fluorophor by DNA microarray technology, is passed through and is known
The fluorescence signal of other diverse location and determine whether DNA make a variation.But PCR flow hybridizations method and DNA microarray technology according to
Rely probe specificity, easily error result occur when other nucleotide variations occurs in probe location.
Time-of-flight mass spectrometry (TOFMS) (MALDI-TOF-MS) by designing specific primer, amplifies site to be measured first
Nucleotide fragments, then for site to be measured, wall scroll special primer is designed, the base that this primer 3 ' is held during annealing is just with treating location
The previous base of point is combined.DdNTP and archaeal dna polymerase are added in reaction system, as a certain ddNTP and site base to be measured
Complementary and when combining, chain extension reaction is terminated, and obtains extending the extension products of a base.Due to 4 kinds of bases molecular weight not
Together, the principle being directly proportional using the flight time of sample molecule in the electric field to the charge-mass ratio of molecule, by detecting extension products
Flight time, extension products molecular weight is measured, so that it is determined that the base type in site to be measured.But time-of-flight mass spectrometry (TOFMS) according to
Rely the specificity of extension primer, probe combines mistake or probe location mutation occurs and easily false negative result occurs, while Yi Shougan
Disturb material influence and error result occur.
The content of the invention
It is an object of the invention to provide it is a kind of detect hereditary hearing impairment gene build storehouse kit and application, it is described to build storehouse
Kit detects hereditary hearing impairment gene mutation by multiple PCR method and high-flux sequence method.
To reach the purpose of this invention, the present invention uses following technical scheme：
In a first aspect, the invention provides it is a kind of detect hereditary hearing impairment gene build storehouse kit, it is described to build storehouse reagent
Box includes the amplimer group for being used to expand hereditary hearing impairment gene；
The amplimer group includes the mutational site according to GJB2, GJB3, SLC26A4 gene and 12s rRNA genes
The primer of Design for polymorphism；
Wherein, the GJB2 genes include 35delG, 167delT, 176-191del16,299_300delAT and
235delC genes；The GJB3 genes include 538C>T and 547G>A；The SLC26A4 genes include 281C>T、589G>A、
IVS7-2A>G、1174A>T、1226G>A、1229C>T、1975G>C、2027T>A、2162C>T、2168A>G and IVS15+5G>
A；The 12s rRNA genes include 1494C>T、1555A>G and 1095T>C.
According to the present invention, the amplimer group is as shown in SEQ ID NO.1-24.
Primer shown in the SEQ ID NO.1-24 is as follows：
According to the present invention, 5 ' ends of every primer of the amplimer group add sample sequence label.
According to the present invention, the length of the sample sequence label is 7-10bp, preferably 7bp.
According to the present invention, the sample sequence label is as shown in SEQ ID NO.25-120.
Sequence label shown in the SEQ ID NO.25-120 is as follows：
In the present invention, described label those skilled in the art can be adjusted as needed, as long as can enter to sample
It is all feasible that row, which is distinguished, and the application is preferred to use the sequence label shown in above-mentioned SEQ ID NO.25-120.
Second aspect, the present invention provides a kind of library constructing method of hereditary hearing impairment related gene, comprises the following steps：
Storehouse kit is built using described in first aspect, adds and is built described in the kit of storehouse at 5 ' ends of the primer sets
Sample sequence label, adds in reaction solution and carries out multi-PRC reaction to sample, by multiple samples with different sample label sequences
This PCR primer equal proportion mixes a written library sample, obtains the library of the hereditary hearing impairment related gene.
According to the present invention, the library constructing method also comprises the following steps：(1) PCR primer phosphorylation；(2) end is repaiied
It is multiple；(3) sequence measuring joints are connected；(4) PCR expands library；(5) library is cyclized；(6) prepared by nanosphere；(7) sequencing analysis.
According to the present invention, the system of the multi-PRC reaction is nuclease-free water 5.5 μ L, PCR amplification liquid 12.5 μ L, expanded
Increase the μ L of the primer sets 2 and μ L of DNA sample 5.
According to the present invention, the condition of the multi-PRC reaction is 94 DEG C of 2min；94 DEG C of 20s, 56 DEG C of 30s, 60 DEG C of 20s, 35
Individual circulation；72℃5min；PCR amplification end is stored in 12 DEG C.
In the present invention, the step of the step of structure in the library also includes phosphorylation plus A bases and sequence measuring joints connection
The step of, the conventional steps the step of phosphorylation plus the step of A bases and the step of sequence measuring joints are connected for this area,
Those skilled in the art can be selected according to actual needs.
The third aspect, the present invention provides a kind of method as described in second aspect and builds obtained hereditary hearing impairment dependency basis
The library of cause.
Fourth aspect, the present invention provides a kind of detection method of hereditary hearing impairment related gene for non-diagnostic purpose,
The library of hereditary hearing impairment related gene described in the third aspect is surveyed using joint probe grappling polymerization PCR sequencing PCR (cPAS)
Sequence.
In the present invention, the sequencing uses BGISEQ-500 sequenators.
In the present invention, the amplimer group not only can be only used for the sequenator based on cPAS technologies, other structures
The method for building library sequencing is also feasible.
Also include library mixing in the present invention, before the sequencing and the step of cyclization and prepare DNA nanospheres
Step, the above-mentioned steps are in order to further improve the concentration of fragment to be measured, so as to improve sequencing throughput and the sequencing degree of accuracy.
5th aspect, what the present invention provided the detection hereditary hearing impairment gene described in first aspect builds storehouse kit, such as the
Library described in three aspects or the detection method as described in fourth aspect are used for people's hereditary hearing impairment gene of non-diagnostic purpose
Detection.
Compared with prior art, the device have the advantages that：
(1) present invention uses multiple PCR technique, and single reaction can detect more than 20 deaf gene mutational sites；
(2) present invention uses " double labels " system, makes on the DNA cloning product of each sample with two sets of independent marks
Sequence is signed, therefore will can be simultaneously sequenced after the amplified production mixing of all samples, realizes that high flux is detected, so as to significantly reduce
The testing cost of single sample；
(3) present invention uses advanced cPAS high throughput sequencing technologies, base information in site to be measured is directly read, not by upper
Base pair downstream mutation influence, testing result is accurately and reliably.
Brief description of the drawings
Fig. 1 is the hereditary hearing impairment genetic test schematic diagram of the invention based on cPAS sequencing technologies；
The hereditary hearing impairment genetic test flow chart that Fig. 2 is sequenced for the present invention based on cPAS；
Fig. 3 is that obtained structural dna sequence figure is sequenced in cPAS of the present invention；
Fig. 4 is the DNA fragmentation size and concentration after amplified library of the present invention.
Embodiment
Further to illustrate the technological means and its effect of the invention taken, below in conjunction with being preferable to carry out for the present invention
Example further illustrates technical scheme, but the present invention is not limited in scope of embodiments.
In the examples where no specific technique or condition is specified, according to the technology or condition described by document in the art,
Or carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be can be by regular channel commercially available from
The conventional products of acquisition.
Embodiment 1：Design of primers
1st, design of primers specifically includes following steps：
Select 21, the mutational site of related GJB2, GJB3, SLC26A4 gene of hereditary hearing impairment and 12s rRNA genes
As detection site, it is respectively：35delG, 167delT, 176-191del16,299_300delAT of GJB2 genes with
The 538C of 235delC, GJB3 gene>T and 547G>The 281C of A, SLC26A4 gene>T、589G>A、IVS7-2A>G、1174A>
T、1226G>A、1229C>T、1975G>C、2027T>A、2162C>T、2168A>G and IVS15+5G>A, chondriogen
1494C>T, 1555A>G, 1095T>C.The pcr amplification primer thing in said gene site is designed using primer-design software, amplification is drawn
The length of thing is in 20 bases or so.Using Software for Design evaluate sample sequence label, sequence label includes 7 bases, design
The pcr amplification primer thing formation hair of the high sequence of the primer similarity that should avoid the occurrence of and be sequenced during sequence label and the upper label of addition
The secondary structure such as card structure or dimer.For above-mentioned 4 deaf genes, 21 mutational sites pcr amplification primer thing referring to table 1,
Sample sequence label is referring to table 2.
The pcr amplification primer thing in 1 hereditary hearing impairment gene of table, 21 sites
The sample sequence label of table 2
Embodiment 2 builds library and is sequenced
In the present embodiment, the clinical DNA of above-mentioned site information (Sanger sequencings detection) to be detected known to 24 parts is detected
Sample, numbering is 1~24, wherein 1~No. 21 sample is mutation positive, 22~No. 24 samples are mutation negative sample.Institute
The principle of detection is stated as shown in figure 1, the flow of detection is as shown in Figure 2.
Specifically include following steps：
2nd, pcr amplification reaction
2.1 prepare pcr amplification reaction mixed liquor according to the ratio of table 3 in suitable centrifuge tube, are pressed after mixing on ice
18 μ L are dispensed into 96 hole PCR reaction plates per reacting hole；
The pcr amplification reaction mixture formula of table 3
2.2 draw the PCR reaction primer additions that 2 μ L are stored in 96 hole PCR reaction plates in order using eight road pipettors
Into the corresponding aperture of the PCR reaction plates of step (1), sealed membrane is sticked, 4000rmp in board-like centrifuge is placed in and centrifuges 1 minute；
2.3 add 24 DNA samples to be detected into 96 hole PCR reaction plates of step (2) respectively, and each sample adds 5 μ
L.Sealed membrane is sticked on PCR reaction plates after completing sample-adding, 4000rmp in board-like centrifuge is placed in and centrifuges 1 minute；
2.4 are placed in PCR reaction plates in PCR instrument, the PCR response procedures of table 4；
The PCR response procedures of table 4
2.5.PCR reaction takes out PCR reaction plates after terminating, and is placed in 4000rpm in board-like centrifuge and centrifuges 1 minute.
2.6. each 10 μ L of 96 PCR reaction products of PCR reaction plates of step 2.5 are taken into 1.5mL centrifuge tubes, vibration is mixed
Hand held centrifuge is placed in after even 5 seconds.
3rd, magnetic beads for purifying
3.1. the equilibrium at room temperature magnetic bead of 30 minutes is vibrated and mixed, take 180 μ L into 1.5mL centrifuge tubes, add 100 μ L steps
Rapid 2.6 mixing sample, is mixed, is stored at room temperature 10 minutes, and 10 minutes are placed on magnetic frame to clarifying, supernatant is abandoned.
3.2. the ethanol of 350 μ L 70% is added to centrifuge tube, it is ensured that magnetic bead is completely immersed in 70% ethanol, and horizontal 180 degree is fast
Speed rotates centrifuge tube, stands 1 minute, the magnetic bead on tube wall is migrated to opposing pipe wall, again horizontal 180 degree quick rotation centrifugation
Pipe, stands 1 minute, abandons supernatant.
3.3. repeat step 3.2, open centrifuge tube lid, and room temperature is placed 5 minutes, ethanol is fully volatilized.
3.4. centrifuge tube is removed, 25 μ L DNA lysates is added, is fully mixed with magnetic bead, be stored at room temperature 5 minutes, be placed in magnetic
Power frame is up to clarified, and takes supernatant into new 1.5mL centrifuge tubes.
3.5. sample after purification is quantitatively detected using the fluorescent quantitative detectors of Qubit 3.0, quality measurement concentration
For 42.5ng/ μ L.
4th, prepared by library
4.1. phosphorylation reaction
4.1.1. phosphorylation reaction mixed liquor is prepared in suitable centrifuge tube according to the ratio of table 5, on ice after mixing
Often react and be dispensed into 200 μ L PCR reaction tubes by 6 μ L.
The phosphorylation reaction mixture formula of table 5
| Reagent name | One reaction normal amount |
| Phosphorylation Buffer | 4μL |
| Phosphorylase | 2μL |
| Cumulative volume | 6μL |
4.1.2. according to the mass concentration (M) of the quantitative detection of step 3.5, library system is calculated according to formula (V=200/M)
Standby sample is 4.7 μ L using volume, and taking the samples after purification of 4.7 μ L steps 3.5, to be added to 200 μ L PCR in step 4.1.1 anti-
Ying Guan, the nuclease-free water for adding 29.3 μ L is mixed, and it is 40 μ L end is repaired reaction system final volume.
4.1.3. 37 DEG C in reaction tube placement PCR instrument are incubated 30 minutes, hand held centrifuge are placed in after terminating 5 seconds,
Obtain phosphorylated reacted phosphorylation library DNA.
4.2. joint coupled reaction
4.2.1. coupled reaction mixed liquor is prepared in suitable centrifuge tube according to the ratio of table 6, treated after mixing on ice
With.
The coupled reaction mixture formula of table 6
| Reagent | One reaction normal amount |
| Ligase buffer solution | 27.2μL |
| 100mM adenosine triphyosphates | 0.8μL |
| Ligase | 10μL |
| Cumulative volume | 38μL |
4.2.2. as shown in table 7,38 μ L steps 4.2.1 coupled reaction mixed liquor is taken to step 4.1.3 200 μ L PCR
In reaction tube, then 2 μ L label joints are taken to be added in centrifuge tube.Sequence measuring joints sequence is as follows, and the base of highlighted mark is library
Sequence label：
1 chain：TTGTCTTCCTAAGGAACGACATGGCTACGATCCGACTT
2 chains：
/Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAATAGGTCCGATCAACTCCTTGGCTCACA
The coupled reaction system of table 7
4.2.3. step 4.2.2 centrifuge tube is vibrated and mixed, be placed in hand held centrifuge and centrifuge 5 seconds, be placed in PCR instrument
Upper to close heat lid, 25 DEG C are incubated 20 minutes.
4.2.4. after reaction terminates, PCR reaction tubes is placed on hand held centrifuge and centrifuged 5 seconds, and total overall reaction liquid is turned
Into new 1.5mL centrifuge tubes.
4.3. magnetic beads for purifying
4.3.1. the equilibrium at room temperature magnetic bead of 30 minutes is vibrated and mixed, in the centrifuge tube for taking 72 μ L to step 4.2.4, mixed
It is even, it is stored at room temperature 10 minutes, 10 minutes is placed on magnetic frame to clarifying, supernatant is abandoned；
4.3.2. the ethanol of 180 μ L 70% is added to centrifuge tube, it is ensured that magnetic bead is completely immersed in 70% ethanol, horizontal 180 degree
Quick rotation centrifuge tube, stands 1 minute, the magnetic bead on tube wall is migrated to opposing pipe wall, again horizontal 180 degree quick rotation from
Heart pipe, stands 1 minute, abandons supernatant.
4.3.3. repeat step 4.3.2, opens centrifuge tube lid, and room temperature is placed 5 minutes, ethanol is fully volatilized；
4.3.4. centrifuge tube is taken out, 25 μ L DNA lysates is added, is fully mixed with magnetic bead, be stored at room temperature 5 minutes, be placed in
Magnetic frame is up to clarified, and takes 7 μ L of supernatant liquid into 200 new μ L PCR reaction tubes.
4.4. amplified library
4.4.1. pcr amplification reaction mixed liquor is prepared in suitable centrifuge tube according to the ratio of table 8, on ice after mixing
It is dispensed into 200 μ L PCR reaction tubes of 4.3.4 reaction products, mixes per reacting hole by 18 μ L.
The pcr amplification reaction mixture formula of table 8
4.4.2. PCR reaction tubes are placed in hand held centrifuge and centrifuged 5 seconds.PCR reaction tubes are placed in PCR instrument, transported
The PCR response procedures of row table 9.
The amplified library response procedures of table 9
4.4.3.PCR reaction takes out reaction tube after terminating, and is placed in hand held centrifuge and centrifuges 5 seconds.
4.5. magnetic beads for purifying
4.5.1. the equilibrium at room temperature magnetic bead of 30 minutes is vibrated and mixed, take 100 μ L steps 4.4.3 mix products to arrive
In 1.5mL centrifuge tubes, add 90 μ L magnetic beads and mix.It is stored at room temperature 10 minutes, 10 minutes is placed on magnetic frame to clarifying, is abandoned
Clearly；
4.5.2. the ethanol of 350 μ L 70% is added to centrifuge tube, it is ensured that magnetic bead is completely immersed in 70% ethanol, horizontal 180 degree
Quick rotation centrifuge tube, stands 1 minute, the magnetic bead on tube wall is migrated to opposing pipe wall, again horizontal 180 degree quick rotation from
Heart pipe, stands 1 minute, abandons supernatant.
4.5.3. repeat step 4.5.2, opens centrifuge tube lid, and room temperature is placed 5 minutes, ethanol is fully volatilized.
4.5.4. centrifuge tube is taken out, 25 μ L DNA lysates is added, is fully mixed with magnetic bead, be stored at room temperature 5 minutes, be placed in
Magnetic frame is up to clarified, and takes supernatant into new 1.5mL centrifuge tubes.
4.5.5. with the detecting step 4.5.4 Chinese libraries amplified production DNA fragmentation size of agilent bio-analyser 2100 and
DNA concentration, as a result as shown in figure 4, amplified production DNA fragmentation main peak scope is 240bp~310bp, library concentration is 35.8ng/
μL。
5th, cyclization
5.1, according to step 4.5.5 library concentration, take 4.5 μ L (about 160ng) amplified library product, will with molecular level water
Volume is supplemented to 48 μ L, of short duration centrifugation after fully mixing, and is placed in 95 DEG C of incubation 5min in PCR instrument, incubation is finished takes out PCR at once
Pipe is put on ice for cooling.
5.2 add 11.6 μ L cyclizations buffer solutions, 0.5 μ L ligases in above-mentioned PCR pipe, fully mix, of short duration centrifugation,
37 DEG C of incubation 30min.
6th, prepared by DNA nanospheres
6.1 take in μ L to the 0.2mL PCR pipes of cyclized DNA library 20 of step 5.2.Add 20 μ L DNB and prepare buffer solution,
Vortex oscillator concussion is mixed, and centrifugation, which is placed in after 5 seconds in PCR instrument, reacts.Reaction condition is：95 DEG C of 1min, 65 DEG C of 1min, 40 DEG C
1min, 4 DEG C of holdings.
PCR pipe is taken out after the completion of 6.2 reactions, is centrifuged 5 seconds, 40 μ L DNB polymerase mix I and 4 μ L DNB polymerizations are added
Enzyme mixation II, vortex oscillator concussion is mixed, and centrifugation is placed in PCR instrument at once after 5 seconds starts reaction, reaction condition：30℃
10min, 4 DEG C of holdings.
PCR pipe is taken out after the completion of 6.3 reactions as on ice chest, adding 20 μ LDNB stop buffers at once, with pipettor and
Wealthy mouth suction nozzle slowly blows and beats mixing, is sure not concussion and acutely piping and druming, is placed in 4 DEG C and saves backup.
7th, DNA nanospheres are loaded
Operated with reference to the full-automatic sample loading system product descriptions of BGIDL-50, by the DNA nanospheres of step 6.3
Solution is loaded on sequence testing chip.
8th, machine is sequenced on DNA nanospheres
Reference gene sequenator (BGISEQ-500) product description is operated, and will be loaded with the sequencing of DNA nanospheres
Chip is placed in progress cPAS sequencings in sequenator, obtained structural dna sequence figure is sequenced as shown in Figure 3.
9th, data analysis
After the completion of sequencing, " hereditary hearing impairment genetic analysis software " is used to analyze sequencing data, as a result such as table 10-
Shown in 11：
10 24 sample cPAS sequencing assay results of table
11 24 sample cPAS sequencing assay results of table and Sanger sequencing result comparison sheets
Can be seen that from table 10-11 24 samples 21 sites testing result with Sanger sequencing assay results
Unanimously, 21 site primer accuracys are 100%.
Applicant states that the present invention illustrates the method detailed of the present invention, but not office of the invention by above-described embodiment
It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implemented.Art
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., within the scope of all falling within protection scope of the present invention and being open.
SEQUENCE LISTING
<110>Hua Da Gene science
<120>It is a kind of to detect that hereditary hearing impairment gene builds storehouse kit and application
<130> 2017
<160> 120
<170> PatentIn version 3.3
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cgcccagagt agaagatgg 19
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gttgcagaca aagtcggcct 20
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ggtgtgggga gatgagcagg 20
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gatgaacttc ctcttcttct 20
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ccaacatcgt ggactgctac a 21
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ttgcagcgtg gccactagcc 20
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agtgccctga ctctgctggt 20
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acgtaattta gaaaataatt 20
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gtctgtatgg cagttaagga a 21
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atgtaga 7
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atgaact 7
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cactctg 7
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caaggat 7
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cctacct 7
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tgtgaga 7
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agcatga 7
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agtgcta 7
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tcgcaga 7
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gaaggta 7
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gcgaagt 7
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ggtggac 7
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gttattg 7
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cgatcta 7
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taatcgg 7
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tataaga 7
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ctcgctg 7
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agtctga 7
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ctgtatg 7
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cagtata 7
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tccagag 7
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ctacgta 7
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tgccagt 7
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tacgtga 7
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tgctcac 7
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cagacac 7
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tgctaga 7
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catgata 7
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atatcga 7
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cacgcta 7
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cacagta 7
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cgactac 7
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tgatatg 7
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ctgtgta 7
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ctctata 7
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tgtctac 7
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cagacta 7
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cgtagta 7
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tcgacac 7
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gagcatc 7
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<400> 74
gtacgtc 7
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<400> 75
agagtac 7
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<400> 76
agcgaga 7
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<400> 77
cgtatga 7
<210> 78
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<400> 78
ctcatga 7
<210> 79
<211> 7
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<400> 79
cgagata 7
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<400> 80
agtgatc 7
<210> 81
<211> 7
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<400> 81
agtacac 7
<210> 82
<211> 7
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<400> 82
gctcgta 7
<210> 83
<211> 7
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<400> 83
agctatc 7
<210> 84
<211> 7
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<400> 84
actcata 7
<210> 85
<211> 7
<212> DNA
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<400> 85
ctagcga 7
<210> 86
<211> 7
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<400> 86
catactg 7
<210> 87
<211> 7
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<400> 87
tactatg 7
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<211> 7
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<400> 88
tcagctc 7
<210> 89
<211> 7
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<400> 89
tatgatg 7
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<211> 7
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<213>Artificial synthesized sequence
<400> 90
tcgagtc 7
<210> 91
<211> 7
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<213>Artificial synthesized sequence
<400> 91
cgatatc 7
<210> 92
<211> 7
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<213>Artificial synthesized sequence
<400> 92
cactatc 7
<210> 93
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 93
atcgtga 7
<210> 94
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 94
gtcacac 7
<210> 95
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 95
gatcgtc 7
<210> 96
<211> 7
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<213>Artificial synthesized sequence
<400> 96
atctgta 7
<210> 97
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 97
cgtgatg 7
<210> 98
<211> 7
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<213>Artificial synthesized sequence
<400> 98
agacaga 7
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<211> 7
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<213>Artificial synthesized sequence
<400> 99
tagatga 7
<210> 100
<211> 7
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<213>Artificial synthesized sequence
<400> 100
atactga 7
<210> 101
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<213>Artificial synthesized sequence
<400> 101
cgtcgtc 7
<210> 102
<211> 7
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<213>Artificial synthesized sequence
<400> 102
atgtctc 7
<210> 103
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 103
tgcagta 7
<210> 104
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 104
tacactg 7
<210> 105
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 105
acagatc 7
<210> 106
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 106
gatatac 7
<210> 107
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 107
tagagta 7
<210> 108
<211> 7
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<213>Artificial synthesized sequence
<400> 108
ctctcac 7
<210> 109
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 109
tcacgtc 7
<210> 110
<211> 7
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<213>Artificial synthesized sequence
<400> 110
cagctac 7
<210> 111
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 111
tatctga 7
<210> 112
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 112
gtctaga 7
<210> 113
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 113
ctagatg 7
<210> 114
<211> 7
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<213>Artificial synthesized sequence
<400> 114
ctcacta 7
<210> 115
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 115
gctgtga 7
<210> 116
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 116
gacgctg 7
<210> 117
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 117
gtgcgta 7
<210> 118
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 118
ctgatac 7
<210> 119
<211> 7
<212> DNA
<213>Artificial synthesized sequence
<400> 119
tgagatc 7
<210> 120
<211> 7
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<213>Artificial synthesized sequence
<400> 120
gtgatga 7
Claims (10)
1. a kind of detect that hereditary hearing impairment gene builds storehouse kit, it is characterised in that the storehouse kit built includes being used to expand
Increase the amplimer group of hereditary hearing impairment gene；
The amplimer group is polymorphic including the mutational site according to GJB2, GJB3, SLC26A4 gene and 12s rRNA genes
Property design primer；
Wherein, the GJB2 genes include 35delG, 167delT, 176-191del16,299_300delAT and 235delC base
Cause；The GJB3 genes include 538C>T and 547G>A；The SLC26A4 genes include 281C>T、589G>A、IVS7-2A>G、
1174A>T、1226G>A、1229C>T、1975G>C、2027T>A、2162C>T、2168A>G and IVS15+5G>A；The 12s
RRNA genes include 1494C>T、1555A>G and 1095T>C.
2. according to claim 1 build storehouse kit, it is characterised in that the amplimer group such as SEQ ID NO.1-24
It is shown.
3. according to claim 1 or 2 build storehouse kit, it is characterised in that every primer of the amplimer group
5 ' ends add sample sequence label.
4. build storehouse kit according to any one of claim 1-3, it is characterised in that the length of the sample sequence label
Spend for 7-10bp, preferably 7bp.
5. build storehouse kit according to any one of claim 1-4, it is characterised in that the sample sequence label is such as
Shown in SEQ ID NO.25-120.
6. a kind of library constructing method of hereditary hearing impairment related gene, it is characterised in that comprise the following steps：
Storehouse kit is built using any one of claim 1-5, storehouse kit is built in 5 ' end additions of the primer sets
Described in sample sequence label, add reaction solution in sample carry out multi-PRC reaction, different sample label sequences will be carried
The PCR primer equal proportions of multiple samples be mixed into a library sample, obtain the library of the hereditary hearing impairment related gene；
Preferably, the library constructing method also comprises the following steps：(1) PCR primer phosphorylation；(2) end is repaired；(3) even
Connect sequence measuring joints；(4) PCR expands library；(5) library is cyclized；(6) prepared by nanosphere；(7) sequencing analysis.
7. library constructing method according to claim 6, it is characterised in that the system of the multi-PRC reaction is seedless
Sour enzyme water 5.5 μ L, PCR amplification liquid 12.5 μ L, the μ L of amplimer group 2 and the μ L of DNA sample 5；
Preferably, the condition of the multi-PRC reaction is 94 DEG C of 2min；94 DEG C of 20s, 56 DEG C of 30s, 60 DEG C of 20s, 35 circulations；
72℃5min；PCR primer is stored in 12 DEG C.
8. a kind of method as claimed in claims 6 or 7 builds the library of obtained hereditary hearing impairment related gene.
9. the detection method of a kind of hereditary hearing impairment related gene for non-diagnostic purpose, it is characterised in that by claim 8
The library of the hereditary hearing impairment related gene is sequenced using joint probe grappling polymerization PCR sequencing PCR.
10. detection hereditary hearing impairment gene any one of claim 1-5 builds storehouse kit, such as claim 8 institute
The library stated or detection method as claimed in claim 9 are used for the detection of people's hereditary hearing impairment gene of non-diagnostic purpose.
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| CN107974497A (en) * | 2017-12-11 | 2018-05-01 | 国家卫生计生委科学技术研究所 | Utilize the deaf Disease-causing gene detection kit of ionization time of flight |
| CN108624673A (en) * | 2018-02-07 | 2018-10-09 | 新开源锦和河北生物科技有限公司 | A kind of diagnostic kit and diagnostic method of pregnant woman's preeclampsia |
| CN108949951A (en) * | 2018-05-18 | 2018-12-07 | 中国人民解放军陆军军医大学第附属医院 | A kind of while Non-invasive detection GJB2 and SLC26A4 gene mutation method and kit |
| CN109554463A (en) * | 2018-12-29 | 2019-04-02 | 中国人民解放军第四军医大学 | A kind of phonosensitive nerve deafness Disease-causing gene GJB2 abrupt climatic change kit |
| CN109694908A (en) * | 2017-10-24 | 2019-04-30 | 深圳乐土生物科技有限公司 | Detect the quick library constructing method and detection method in 20 mutational sites of deaf gene |
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