CN106755496A - Multiplex PCR specific primer, kit and method based on high throughput sequencing technologies detection hereditary hearing impairment gene - Google Patents
Multiplex PCR specific primer, kit and method based on high throughput sequencing technologies detection hereditary hearing impairment gene Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C12Q2600/16—Primer sets for multiplex assays
Abstract
The invention belongs to field of gene detection, more particularly to a kind of multiplex PCR specific primer based on high throughput sequencing technologies detection hereditary hearing impairment gene, kit and method.The primer includes 52 pairs of specific primers, its nucleotide sequence such as SEQ ID NO：1 to SEQ ID NO：Shown in 104；The kit also includes the specific primer, universal primer, enzymatic mixture, quality-control product, nuclease free water.The detection method of hereditary hearing impairment gene is captured and expanded with a pcr amplification reaction the invention also discloses with the primer or kit.The present invention program effectively improves detection efficiency, accuracy rate, while reduces cost, simplified operating procedure.
Description
Technical field
It is more particularly to a kind of to detect hereditary hearing impairment base based on high throughput sequencing technologies the invention belongs to field of gene detection
The multiplex PCR specific primer of cause, kit and detection method.
Background technology
Deafness belongs to clinical common inborn defect, and in China, hearing and speech impairments patient occupies all kinds of deformity more than 27,000,000
First of.In neonate colony, the incidence of congenital hearing disorder is up to 1-3/1000, it is estimated that China increases 3-6 newly every year
Ten thousand deafness patients, and what these deaf deformity about 60% were caused by inherent cause.
Hereditary hearing impairment genetic test is by detecting person under inspection's deafness Disease-causing gene situation, instructing deafness patient or wind high
The intervention and treatment of dangerous crowd, while by the analysis to deaf gene testing result, determine mode of inheritance, to patient home into
The risk of member, gene carries risk, filial generation risk etc. and makes Scientific evaluation, instructs science marriage and childbirth, pre- from source
Anti- deaf inborn defect.
Detection deaf inheritance ospc gene common methods have Sanger methods, gene chips, fluorescence quantitative PCR method, many at present
Color fluorescence solubility curve analytic approach, high-flux sequence method etc..The sequencing of Sanger PCR sequencing PCRs every time can only be to a certain of sample
Section region is sequenced, and detection efficiency is low, high cost, and sensitivity is low (20%).Gene chips, fluorescence quantitative PCR method inspection
Deaf inheritance ospc gene is surveyed, the method can only be directed to one or the site design detection probe of several known mutations forms detection core
Piece, therefore detection unknown mutation site is not used to, the result of detection is also just not comprehensive enough, accurate.Multicolor fluorescence solubility curve
Analytic approach, fluorescent dye used is general double-stranded DNA intercalator, and primer dimer and non-specific amplification product cannot be known
Not, false positive is easily caused.High-flux sequence method is mainly the supporting library construction of sequenator provided by sequencing instrument provider
Kit, have passed through the strict Quality Control assessment of supply company, with preferably building storehouse effect, but have the disadvantage kit delivery date it is long,
Kit package specification fixes, test flexibility not enough etc..Some reagent manufacturers are according to the experimental technique for sharing report in document
Carry out building storehouse, but the experiment effect for being completed is uneven, and sequencing quality is not high, influences clinical reference value.
At present, on the market detection deaf gene main flow be GJB2 (gene mutation clinical manifestation is deaf congenital severe),
(gene mutation clinical manifestation is congenital for GJB3 (gene mutation clinical manifestation be the day after tomorrow high frequency phonosensitive nerve deafness), SLC26A4
Property or delayed it is deaf), (gene mutation clinical manifestation is drug-induced deafness to MT-RNR1, carries the individuality of gene mutation to amino
Glycoside medicaments insensitive, is exactly " pin cause deaf " often said) 4 common inherited deaf genes, and with site examination more than detection
Based on, the gene mutation information of detection is few, is not enough to reflect the mutation overall condition of hereditary hearing impairment.
As can be seen here, prior art could be improved.
The content of the invention
In consideration of it, being necessary to provide a kind of based on high throughput sequencing technologies detection hereditary hearing impairment gene regarding to the issue above
Multiplex PCR specific primer, kit and detection method.Primer covering GJB2, GJB3, SLC26A4, MT-RNR1,
It is all common with rare mutational site in six kinds of full extrons of gene of FOXI1, KCNJ10；Can simultaneously detect that multiple samples are more
Individual hereditary hearing impairment tumor susceptibility gene, effectively improves detection efficiency, accuracy rate, while reduces cost, simplified operating procedure.
To reach above-mentioned purpose, the present invention uses following technical scheme：
A kind of multiplex PCR specific primer that hereditary hearing impairment Disease-causing gene is detected based on high throughput sequencing technologies, including
52 pairs of specific primers, its nucleotide sequence such as SEQ ID NO：1 to SEQ ID NO：Shown in 104.
Further, the specific primer is the primer by modifying.
Further, the method for modifying of the specific primer is thio-modification, Brdurd is modified, 5 ' reverse dT are repaiied
One or more in decorations.
Further, the Tm values of the specific primer are 58-62 DEG C.
A kind of kit that hereditary hearing impairment Disease-causing gene is detected based on high throughput sequencing technologies, including in above-mentioned primer
At least two pairs.
Further, the kit also includes universal primer, enzymatic mixture, quality-control product, nuclease free water；The enzyme is mixed
Compound includes：High-fidelity DNA polymerase, PCR Buffer, dNTP mixtures etc..
Further, the quality-control product behaviour normal gene group DNA, from commercially available.
Further, the end of specific primer 3 ' and 5 ' ends mark Tag1 and Tag2 respectively.
Further, the end Tag1 sequences of specific primer 3 ' and target area complete complementary or partial complementarity；Specifically
Property the end of primer 5 ' Tag2 and universal primer complete complementary or partial complementarity.
Further, the universal primer is used to expand the amplified production of special primer amplification target area again
Increase, the target area amplified production to different testing samples is marked.
Further, the universal primer 3 ' holds Tag3 and special primer Tag2 complete complementaries or partial complementarity, and length is
50-70bp。
Further, the specific primer or kit are applied to the detection of hereditary hearing impairment Disease-causing gene.
The method that hereditary hearing impairment Disease-causing gene is detected based on high throughput sequencing technologies, including：
S1：Nucleic acid extraction is carried out to sample using paramagnetic particle method, the measure of concentration and purity is then carried out, determined qualified laggard
The quantitative dilution of row, it is standby；
S2：The target area of the nucleic acid with the reaction system containing the specific primer or the kit to being extracted in S1
Domain carries out a PCR amplification, collects amplified production standby；
S3：Nucleic acid purification is carried out to amplified production in S2 using paramagnetic particle method, the measure of concentration and purity is carried out, sample mixing is fixed
Amount, upper machine sequencing.
Further, the reaction system containing the specific primer is：
The enzymatic mixture includes：High-fidelity DNA polymerase, PCR Buffer, dNTP mixtures etc..
Further, the response procedures of the PCR amplifications are：
Further, the hereditary hearing impairment Disease-causing gene includes FOXI1 (primer sequence SEQ ID NO:1-SEQ ID
NO:10), KCNJ10 (primer sequence SEQ ID NO:11-SEQ ID NO:34), SLC26A4 (primer sequence SEQ ID NO:
35-SEQ ID NO:78), GJB2 (primer sequence SEQ ID NO:79-SEQ ID NO:90), GJB3 (primer sequence SEQ ID
NO:91-SEQ ID NO:100), MT-RNR1 (primer sequence SEQ ID NO:101-SEQ ID NO:104) six genes.
Further, the target area of the nucleic acid described in step (2), including but not limited to GJB2, GJB3, SLC26A4,
Internal sequence, the outside regulating and controlling sequence of gene on six genes of MT-RNR1, FOXI1, KCNJ10；The gene internal sequence,
Including but not limited to gene includes subregion, exon region, contains the region of introne and extron simultaneously.
Further, the detection sample of the detection method includes in vitro whole blood, mouth desquamated cells and tissue sample
Deng；The tissue sample is including paraffin-embedded tissue, flesh tissue and frozen section etc..
Ion Torrent/ of the above-mentioned high-flux sequence platform including but not limited to Illumina HiSeq/MiSeq, Life
The microarray datasets such as Proton platforms, the BGISEQ-500/50 of Hua Da gene.
Beneficial effect of the present invention：
The multiplex PCR specific primer that hereditary hearing impairment gene is detected based on high throughput sequencing technologies that the present invention is provided,
Detection gene scope is detected except common GJB2, GJB3, SLC26A4, MT-RNR1 gene whole extron, also including other
Deaf Disease-causing gene related gene FOXI1 (jaw transcription factor family), KCNJ10 (inward rectification type potassium channel family) gene
The detection of whole extrons, covers above-mentioned six kinds of genes all common with rare mutational site；Can detect simultaneously multiple samples,
Multiple hereditary hearing impairment tumor susceptibility genes, effectively improve detection efficiency, accuracy rate, while reduces cost, simplified operating procedure etc..
The present invention provide primer be directed to hereditary hearing impairment Disease-causing gene GJB2, GJB3, SLC26A4, MT-RNR1,
The primer of the full extron design of FOXI1, KCNJ10, wide coverage, detection site are more.Each pair primer amplified target
Area size is 450-550bp, and all primer Tm Unitings are 58-62 DEG C.
Modification, such as the modification of thio-modification, Brdurd, 5 ' are introduced when the multiple PCR primer that the present invention is provided is designed
The method of modifying such as reverse dT modifications, the primer after modification is difficult to be degraded by nuclease, it is ensured that ensure while PCR specific amplifications
Its amplification efficiency；And with relatively uniform Tm values, good specificity, stability and homogeneous are shown in multiplex amplification
Property, while not existing non-specific amplification, the method saves the work of many wheel primer screenings.Specifically, the primer after thio-modification
The derivative that the oxygen atom of sequence oligonucleotides chain upper band double bond is replaced by sulphur atom, can resist the degraded of nuclease, enhancing
Primer stability；Each deoxythymidine is deoxidized uracil and replaces in primer oligonucleotide sequence after Brdurd modification
In generation, double-strand solution temperature can be made to increase by 1.7 DEG C, so as to increase the stability of double-strand；Primer widow's core after 5 ' reverse dT modifications
Reverse dT is contained in the end of nucleotide sequence 5 ', so as to form crosslinking, the crosslinking of formation can suppress during archaeal dna polymerase extends
Exonuclease degradation.
The Disease-causing gene higher for the hereditary hearing impairment incidence of disease based on high-flux sequence platform of the invention it is multiple
PCR primer, kit and detection method, can be captured and the multiple sample multiple heredity ears of amplification with a pcr amplification reaction
Deaf Disease-causing gene, the genetic mutation such as including point mutation, short-movie section insertion and deletion, effectively improves detection efficiency, accuracy rate, while
Reduces cost, the flexibly operating procedure that simplifies, offer kit package specification etc..
The present invention breaks through the limitation of traditional sensing techniques using high flux gene sequencing technology, improves detection sensitivity；Together
When can also detect the variation situation including known mutations and unknown mutation site, obtain comprehensive and accurate testing result.By right
Hereditary hearing impairment Disease-causing gene and its mutational site are sequenced, and can obtain specific gene sequence information, and then assess deaf
Genetic risk, the intervention and treatment of deafness patient or High risk group are instructed, while by deaf gene testing result
Analysis, determines mode of inheritance, to the risk of patient home member, carries risk, filial generation risk etc. and makes science
Assessment, instructs science marriage and childbirth, and deaf inborn defect is prevented from source.
The present invention eliminates the centrifugation pillar purifying that tradition is built in storehouse by the way of magnetic beads for purifying nucleic acid, simple to operate；
Secondly, the present invention only needs to a wheel PCR for the primer of high-flux sequence Platform Designing, by choosing thermograde, time ladder
Degree, period optimize PCR response procedures by a large amount of orthogonal experiments, and the fusion of amplification twice that sample is built into storehouse is anti-in a PCR
Answering system is carried out, and the inventive method directly captures target area with primer and expanded in target gene chain, it is not necessary to
Target gene is carried out to block the treatment such as modification, detection time and cost is saved.
Brief description of the drawings
Fig. 1 is the method flow diagram for detecting hereditary hearing impairment Disease-causing gene.
Fig. 2 builds the schematic diagram of storehouse PCR amplifications for target gene.
Fig. 3 primer specificity the result figures；Wherein, M：Marker；N：Negative control；P：Positive control；1-3：No. 1-3
Whole blood sample is verified；4-6：4-6 mouth desquamated cells sample is verified；7-9：7-9 paraffin organizations sample is verified.
Fig. 4 primer sensitivity the result figures；Wherein, M：Marker；N：Negative control；P：Positive control；1-3：No. 1 complete
Three kinds of template concentrations sensitivity checkings of blood sample；4-6：The three kinds of template concentrations sensitivity checkings of No. 4 mouth desquamated cells samples；7-
9：The three kinds of template concentrations sensitivity checkings of No. 7 paraffin organization samples.
Fig. 5 primers repeatability the result figure；Wherein, M：Marker；N：Negative control；P：Positive control；1-3：Experiment
Member A detects No. 2 whole blood samples, No. 5 mouth desquamated cells samples, No. 8 paraffin organization sample the results；4-6：Laboratory technician B is examined
Survey No. 2 whole blood samples, No. 5 mouth desquamated cells samples, No. 8 paraffin organization sample the results.
Specific embodiment
In order to problem solved by the invention, the technical scheme for being used and the effect for being reached is better described, now tie
Close specific embodiment and related data is expanded on further.It should be noted that present invention is including but not limited to following implementation
Example and combinations thereof implementation method.
Embodiment 1 is based on the multiplex PCR specific primer of high throughput sequencing technologies detection hereditary hearing impairment Disease-causing gene, examination
Agent box and detection method
1st, the design of primer
It is described that University of California Santa Cruz are selected to hereditary hearing impairment Disease-causing gene sequence
(UCSC) database, the full extron design of primers of tumor susceptibility gene is carried out from Primer3 primer-design softwares, and scope of design is included
All common and uncommon mutantional hotspot in the gene.
The primer being designed to the full extron of hereditary hearing impairment Disease-causing gene totally 52 pairs, each pair specific primer expands
Gaining mark area size is 450-550bp, and wide coverage, detection site are more；Special primer introduces special sex modification when designing,
Primer after modification is difficult to be degraded by nuclease, and with relatively uniform Tm values, Uniting is 60 DEG C or so (± 2 DEG C)；
The primer shows good specificity, stability and homogeneity in multiplex amplification, while not existing non-specific amplification.Tool
Body, the primer specific method of modifying is the one kind or several in thio-modification, Brdurd modification, 5 ' reverse dT modifications
Kind.The derivative that the oxygen atom of the primer sequence oligonucleotide chain upper band double bond after the thio-modification is replaced by sulphur atom, energy
The degraded of nuclease is enough resisted, strengthens primer stability；It is every in primer oligonucleotide sequence after the Brdurd modification
Individual deoxythymidine is deoxidized uracil replacement, can increase by 1.7 DEG C of double-strand solution temperature, so as to increase the stabilization of double-strand
Property；Reverse dT is contained in the end of primer oligonucleotide sequence 5 ' after described 5 ' reverse dT modifications, so that crosslinking is formed, the friendship of formation
Connection can suppress the degradation of the exonuclease during archaeal dna polymerase extends.
Sequencing primer of the invention is by special Mdification primer, it can be ensured that ensure that it expands while PCR specific amplifications
Increasing Efficiency.
2nd, hereditary hearing impairment Disease-causing gene kit is detected
The kit of detection hereditary hearing impairment Disease-causing gene method of the invention, including：
Hereditary hearing impairment Disease-causing gene specific primer：For expanding multiple target areas in testing sample, scope is expanded
At least full exon region of coverage goal gene；
Universal primer：For in library construction process, the amplified production to special primer amplification target area to be carried out again
Secondary amplification, the sequencing library to different testing samples is marked, and then distinguishes different samples；
PCR reaction solutions：Including enzymatic mixture, nuclease free water；The enzymatic mixture includes that high-fidelity DNA polymerase (is used
It is 1.25U/ reactions to measure), PCR Buffer (consumption be 1 ×), dNTP mixtures (consumption is 200 μM/kind/reaction)；
Quality-control product：People normal gene group DNA, from normal person's whole blood sample；Quality-control product of the present invention is from commercially available.
3rd, the method that hereditary hearing impairment Disease-causing gene is detected based on high throughput sequencing technologies
Comprise the following steps：
S1：Using nanoscale, superparamagnetic carboxyl magnetic bead under different salinity and hydrophobic environment with the reversible suction of nucleic acid molecules
Attached principle, Specific adsorption nucleic acid molecules, by subsequently washing and elution step, obtain the sample of nucleic acid of high-purity, use
Qubit or NanoDrop carries out the measure of concentration and purity, according to the sample of nucleic acid concentration results for determining, uses 10mM Tris
Sample of nucleic acid is diluted to 50-250ng/ μ L, preferred 50-100ng/ μ L build the initial concentration in storehouse as amplification.
Using 1.5% agarose gel electrophoresis 40min, the quality to nucleic acid after dilution is estimated, solidifying according to agarose
The homogeneity and integrality of gel electrophoresis result judge to extract the quality of nucleic acid, and nucleic acid integrality is good or generation slight degradation does not influence
Build storehouse effect.Enter group after the detection of the nucleic acid concentration that is extracted, purity testing and agarose gel electrophoresis is qualified and enter rower respectively
Note.The qualified standard of the sample is nucleic acid concentration ＞ 50ng/ μ L, OD260/OD280=1.7-2.0,1% Ago-Gel electricity
Swimming detection sample strip is complete, homogeneous, without substantially hangover.
S2：The Disease-causing gene target area in testing sample in S1 is carried out with specific primer of the invention or kit
PCR amplification, collects amplified production, obtains building storehouse product；The amplified production fragment length is special primer amplified fragments
With universal primer sum；
Pcr amplification reaction system is：The μ L of special primer mixture 2；The μ L of universal primer 2；Template/quality-control product 100ng；Enzyme is mixed
The μ L of compound 12.5；Volume is complemented to 25 μ L by nuclease free water.
Pcr amplification reaction program is：
When hereditary hearing impairment Disease-causing gene to be measured has multiple, the different hereditary hearing impairment Disease-causing genes of same testing sample
Amplification can carry out simultaneously or part while carrying out.In specific experimentation, can as needed from any of the above-described kind of side
Case is carried out.If, it is necessary to ensure that the amount of the amplified production for building sequencing library is consistent when being expanded respectively.When to be measured
When sample has multiple, the amplification of the hereditary hearing impairment Disease-causing gene of different samples must be carried out respectively.
S3：The storehouse product of building of each sample is purified respectively using magnetic bead is purified.Remaining primer in removal PCR primer
And dNTP etc., reclaim amplified production；Carry out concentration, purity testing and 1% agarose gel electrophoresis detection sample quality, the sample
This qualified standard detects sample for nucleic acid concentration ＞ 2ng/ μ L, OD260/OD280=1.7-2.0,1% agarose gel electrophoresis
Band is complete, homogeneous, without substantially hangover.All qualified nucleic acid samples (being no more than 96) concentration are diluted to 2nmol/L respectively,
And isometric sample mixing, machine sequencing in unification.
Check experiment：
Positive control reaction system includes：The μ L of special primer mixture 2；The μ L of universal primer 2；The μ L of enzymatic mixture 12.5；Matter
Control product 100ng；Volume is complemented to 25 μ L by nuclease free water.Whether just positive control be used to check the amplification ability of reaction system
Whether normal and sample is abnormal.
Negative control reaction system includes：The μ L of special primer mixture 2；The μ L of universal primer 2；The μ L of enzymatic mixture 12.5；Nothing
Volume is complemented to 25 μ L by nuclease water.Negative control is used to check that reaction system pollutes with the presence or absence of nucleic acid substances.
The response procedures of positive control and negative control with step S2 amplified reaction program.
The primer specificity of embodiment 2 is verified
Using S1 methods in embodiment 1 from whole blood sample (numbering：1-3), mouth desquamated cells sample (numbering：4-6), stone
Wax tissue sample (numbering：Nucleic acid is extracted in 7-9), after carrying out concentration, purity testing, taking qualified samples will using 10mM Tris
Every Sample Dilution detects every sample quality (same embodiment of criterion of acceptability to 100ng/ μ L using 1% agarose gel electrophoresis
S1 steps in 1), it is qualified after enter group and be marked respectively.Above-mentioned 9 qualified samples are carried out using S2 methods in embodiment 1
Amplification, applied sample amount is each 1 μ L, carries out 1% agarose gel electrophoresis detection (the same embodiment of criterion of acceptability after amplification after product purification
S3 steps in 1).9 samples are expanded using hereditary hearing impairment Disease-causing gene special primer and detection method is detected.Control examination
Same embodiment one is tested, detection method is identical with sample testing method.Testing result is shown in Fig. 3.
Testing result represents that negative control, without any non-specific band, illustrates that reaction system is without dirt in addition to residual primers
Dye；Positive control band becomes clear, and size is correct, illustrates that the amplification ability of reaction system is normal.9 sample amplified productions of detection
600bp is concentrated mainly on, without other non-specific bands, is consistent with desired design, and primer dimer content is few, and primer
Dimer size is obvious with amplification target fragment difference.It can thus be seen that pcr amplification primer thing of the invention and detection method energy
The limitation that conventional method produces a large amount of primer dimers is broken through, primer specificity is enhanced.
The primer detection sensitivity of embodiment 3 is verified
Using the qualified whole blood sample of quality inspection in embodiment 1 (numbering：1), mouth desquamated cells sample (numbering：4), paraffin
Tissue sample (numbering：7) genomic samples for extracting carry out primer detection sensitivity checking.Every sample initial concentration is
100ng/ μ L, dilute according to 5 times, 10 times, 20 times of concentration gradients, and every sample concentration is respectively 20ng/ μ L, 10ng/ μ after dilution
L and 5ng/ μ L, and sample ID and concentration markers are carried out respectively.Using S2 methods in embodiment 1 to above-mentioned 9 qualified diluted samples
Originally expanded, applied sample amount is each 1 μ L, 9 samples are expanded and detection method using hereditary hearing impairment Disease-causing gene special primer
Detected.Check experiment is with embodiment one, and detection method is identical with sample testing method.Testing result is shown in Fig. 4.
Testing result represents that negative control, without any non-specific band, illustrates that reaction system is without dirt in addition to residual primers
Dye；Positive control band becomes clear, and size is correct, illustrates that the amplification ability of reaction system is normal.9 sample amplified productions of detection
600bp is concentrated mainly on, without other non-specific bands, is consistent with desired design, while primer dimer content is few, and drawn
Thing dimer size is obvious with amplification target fragment difference.Meanwhile, even if template content as little as 5ng, still being capable of Successful amplification
Go out target stripe, it can thus be seen that pcr amplification primer thing of the invention and detection method can break through the limitation of conventional method, carry
Detection sensitivity high.
The repeatability checking of the primer detection of embodiment 4
Using the qualified whole blood sample of quality inspection in embodiment 1 (numbering：2), mouth desquamated cells sample (numbering：5), paraffin
Tissue sample (numbering：8) genomic samples for extracting carry out primer detection repeatability checking.Every sample is by 2 different behaviour
Work person's S2 methods in 2 different lab platforms are according to embodiment 1 are expanded to above-mentioned 3 samples, and applied sample amount is 1 μ
L, 3 samples are using hereditary hearing impairment Disease-causing gene special primer by different operating person in different laboratory amplifications and detection side
Method is detected.Check experiment is with embodiment one, and detection method is identical with sample testing method.Testing result is shown in Fig. 5.
Testing result represents that negative control, without any non-specific band, illustrates that reaction system is without dirt in addition to residual primers
Dye；Positive control band becomes clear, and size is correct, illustrates that the amplification ability of reaction system is normal.Totally 6 expansions of 3 samples of detection
Volume increase thing is concentrated mainly on 600bp, without other non-specific bands, is consistent with desired design, while primer dimer content pole
It is few, and primer dimer size is obvious with amplification target fragment difference.Meanwhile, different operators and different experiment porch examine
Survey result indifference.It can thus be seen that pcr amplification primer thing of the invention and detection method are reproducible.
Embodiment described above only expresses several embodiments of the invention, and its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Shield scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
<110>Guangzhou Qi Hui bio tech ltd
<120>Multiplex PCR specific primer, kit and side based on high throughput sequencing technologies detection hereditary hearing impairment gene
Method
<160> 104
<170> PatentIn version 3.3
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<213>Artificial sequence
<400> 9
tccaggtcta tgtgatcctc ag 22
<210> 10
<211> 19
<212> DNA
<213>Artificial sequence
<400> 10
ctggggtggg aggatttag 19
<210> 11
<211> 20
<212> DNA
<213>Artificial sequence
<400> 11
ggtgagggtt aggagtcagc 20
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence
<400> 12
gcaataagaa gcacaatggc 20
<210> 13
<211> 20
<212> DNA
<213>Artificial sequence
<400> 13
agccttcctc ttctcccttg 20
<210> 14
<211> 20
<212> DNA
<213>Artificial sequence
<400> 14
aaagtcaccc tcaccactgc 20
<210> 15
<211> 20
<212> DNA
<213>Artificial sequence
<400> 15
agacacagcc tctgacagcc 20
<210> 16
<211> 20
<212> DNA
<213>Artificial sequence
<400> 16
tattccttac cagggcattg 20
<210> 17
<211> 21
<212> DNA
<213>Artificial sequence
<400> 17
aatgtctgat gacctgttcc c 21
<210> 18
<211> 22
<212> DNA
<213>Artificial sequence
<400> 18
gaccacaaag tgaccatcag ag 22
<210> 19
<211> 19
<212> DNA
<213>Artificial sequence
<400> 19
tggtgacttg aggctggag 19
<210> 20
<211> 20
<212> DNA
<213>Artificial sequence
<400> 20
atattggctt gggtccttcc 20
<210> 21
<211> 27
<212> DNA
<213>Artificial sequence
<400> 21
tgactaacac aagttgttat ctaaacc 27
<210> 22
<211> 20
<212> DNA
<213>Artificial sequence
<400> 22
agggcactgg gaggtgtaac 20
<210> 23
<211> 20
<212> DNA
<213>Artificial sequence
<400> 23
tgttaaggtt tgggcaggag 20
<210> 24
<211> 20
<212> DNA
<213>Artificial sequence
<400> 24
aacaattggc tgagaccacc 20
<210> 25
<211> 20
<212> DNA
<213>Artificial sequence
<400> 25
tggtaccaag gaagcctcac 20
<210> 26
<211> 20
<212> DNA
<213>Artificial sequence
<400> 26
cttggcaatg gataggaagg 20
<210> 27
<211> 24
<212> DNA
<213>Artificial sequence
<400> 27
ccttatttct ttcagtgtgt aggg 24
<210> 28
<211> 19
<212> DNA
<213>Artificial sequence
<400> 28
ggcccatttc tcaccagac 19
<210> 29
<211> 21
<212> DNA
<213>Artificial sequence
<400> 29
cccctacagg gaacaactac c 21
<210> 30
<211> 20
<212> DNA
<213>Artificial sequence
<400> 30
aaatctgggt gtcctacccc 20
<210> 31
<211> 20
<212> DNA
<213>Artificial sequence
<400> 31
acaagaagtg gggaggcttg 20
<210> 32
<211> 20
<212> DNA
<213>Artificial sequence
<400> 32
ctgagtttga tttcaggggc 20
<210> 33
<211> 20
<212> DNA
<213>Artificial sequence
<400> 33
tacactgtgt gtccccatcc 20
<210> 34
<211> 22
<212> DNA
<213>Artificial sequence
<400> 34
gattttatta ggctgacagg gg 22
<210> 35
<211> 20
<212> DNA
<213>Artificial sequence
<400> 35
aggtgatccc gttctttctg 20
<210> 36
<211> 18
<212> DNA
<213>Artificial sequence
<400> 36
ggcctctggg agaagcag 18
<210> 37
<211> 23
<212> DNA
<213>Artificial sequence
<400> 37
gcagaatcca gttcataact ttg 23
<210> 38
<211> 20
<212> DNA
<213>Artificial sequence
<400> 38
cagacctatg gtagctgggg 20
<210> 39
<211> 24
<212> DNA
<213>Artificial sequence
<400> 39
cattgtaagt tgaggacttt ctgc 24
<210> 40
<211> 25
<212> DNA
<213>Artificial sequence
<400> 40
aatgcactta atatagccaa aacac 25
<210> 41
<211> 21
<212> DNA
<213>Artificial sequence
<400> 41
tgcagacaca ttgaacattt g 21
<210> 42
<211> 23
<212> DNA
<213>Artificial sequence
<400> 42
ttccaggaaa ttactttgtt ttg 23
<210> 43
<211> 22
<212> DNA
<213>Artificial sequence
<400> 43
ttttgtgcta taggcaggct ac 22
<210> 44
<211>
<212> DNA
<213>Artificial sequence
<400> 44
ctcctgggct caagcaatc 19
<210> 45
<211> 18
<212> DNA
<213>Artificial sequence
<400> 45
gtgtgtgtgc tcgtgtgc 18
<210> 46
<211> 24
<212> DNA
<213>Artificial sequence
<400> 46
gaaggagtat cagtgaaatg aagc 24
<210> 47
<211> 20
<212> DNA
<213>Artificial sequence
<400> 47
tagcatttga tgagatgggg 20
<210> 48
<211> 20
<212> DNA
<213>Artificial sequence
<400> 48
ctggtgaaag aatccaaccc 20
<210> 49
<211> 22
<212> DNA
<213>Artificial sequence
<400> 49
cctgaaatac tcagcgaagg tc 22
<210> 50
<211> 20
<212> DNA
<213>Artificial sequence
<400> 50
gccttcctct gttgccattc 20
<210> 51
<211> 20
<212> DNA
<213>Artificial sequence
<400> 51
tccagtgagc tggaagacac 20
<210> 52
<211> 23
<212> DNA
<213>Artificial sequence
<400> 52
ggggaatata gtgatatggc agg 23
<210> 53
<211> 20
<212> DNA
<213>Artificial sequence
<400> 53
tgggcaatag agtgtgaccc 20
<210> 54
<211> 21
<212> DNA
<213>Artificial sequence
<400> 54
aacgaaagaa agtggcttca c 21
<210> 55
<211> 20
<212> DNA
<213>Artificial sequence
<400> 55
aacaccagaa tgatgggctc 20
<210> 56
<211> 20
<212> DNA
<213>Artificial sequence
<400> 56
ccctttggct accttgtacg 20
<210> 57
<211> 22
<212> DNA
<213>Artificial sequence
<400> 57
gcaactgtga cttgactcct tg 22
<210> 58
<211> 20
<212> DNA
<213>Artificial sequence
<400> 58
gggtctaggg cctattcctg 20
<210> 59
<211> 24
<212> DNA
<213>Artificial sequence
<400> 59
aatagccttt ccagataaca gttg 24
<210> 60
<211> 25
<212> DNA
<213>Artificial sequence
<400> 60
tttagaccct ctaactgctc tcatc 25
<210> 61
<211> 25
<212> DNA
<213>Artificial sequence
<400> 61
ttggataatt tgatatgaat ggttg 25
<210> 62
<211> 20
<212> DNA
<213>Artificial sequence
<400> 62
tgcaatactg gacaacccac 20
<210> 63
<211> 22
<212> DNA
<213>Artificial sequence
<400> 63
tgaatgctac tgaattatgg gc 22
<210> 64
<211> 20
<212> DNA
<213>Artificial sequence
<400> 64
tcccagatag gagaaagggc 20
<210> 65
<211> 20
<212> DNA
<213>Artificial sequence
<400> 65
agtgagcaat gatgccactg 20
<210> 66
<211> 24
<212> DNA
<213>Artificial sequence
<400> 66
tgaggctcca tgaagttata tagg 24
<210> 67
<211> 20
<212> DNA
<213>Artificial sequence
<400> 67
tttcagtgga gcatcaggtg 20
<210> 68
<211> 20
<212> DNA
<213>Artificial sequence
<400> 68
ggggaattat gttccctgac 20
<210> 69
<211> 26
<212> DNA
<213>Artificial sequence
<400> 69
gatgagtagc agtaagcaat caatac 26
<210> 70
<211> 21
<212> DNA
<213>Artificial sequence
<400> 70
ttacatggtg atcagtgcag c 21
<210> 71
<211> 20
<212> DNA
<213>Artificial sequence
<400> 71
cctctgtggt caagcgagtc 20
<210> 72
<211> 20
<212> DNA
<213>Artificial sequence
<400> 72
acctatacat gcctccatgc 20
<210> 73
<211> 22
<212> DNA
<213>Artificial sequence
<400> 73
cctaaaagct acagaaccag gc 22
<210> 74
<211> 20
<212> DNA
<213>Artificial sequence
<400> 74
aagtccccaa gatcaaagcc 20
<210> 75
<211> 20
<212> DNA
<213>Artificial sequence
<400> 75
tgcatggagg catgtatagg 20
<210> 76
<211> 18
<212> DNA
<213>Artificial sequence
<400> 76
cgcctggcct acattcac 18
<210> 77
<211> 24
<212> DNA
<213>Artificial sequence
<400> 77
tgaaatacaa ccagaaacaa tgtg 24
<210> 78
<211> 21
<212> DNA
<213>Artificial sequence
<400> 78
cgtaactttg gcatttcctt c 21
<210> 79
<211> 18
<212> DNA
<213>Artificial sequence
<400> 79
ccgggaagct ctgaggac 18
<210> 80
<211> 18
<212> DNA
<213>Artificial sequence
<400> 80
gcaaccgctc tgggtctc 18
<210> 81
<211> 20
<212> DNA
<213>Artificial sequence
<400> 81
gcatgcttgc ttacccagac 20
<210> 82
<211> 20
<212> DNA
<213>Artificial sequence
<400> 82
tctgggtttt gatctcctcg 20
<210> 83
<211> 19
<212> DNA
<213>Artificial sequence
<400> 83
ggcctaccgg agacatgag 19
<210> 84
<211> 22
<212> DNA
<213>Artificial sequence
<400> 84
tccctctcat gctgtctatt tc 22
<210> 85
<211> 25
<212> DNA
<213>Artificial sequence
<400> 85
tttgctaatt agatattgtt ctggg 25
<210> 86
<211> 18
<212> DNA
<213>Artificial sequence
<400> 86
aaccccagga ggacaccc 18
<210> 87
<211> 21
<212> DNA
<213>Artificial sequence
<400> 87
ggctgttagg ggttattggt g 21
<210> 88
<211> 21
<212> DNA
<213>Artificial sequence
<400> 88
gacatgaggc catttgctat c 21
<210> 89
<211> 23
<212> DNA
<213>Artificial sequence
<400> 89
tggttatgaa tactttgcag cac 23
<210> 90
<211> 21
<212> DNA
<213>Artificial sequence
<400> 90
tgaaggggta agcaaacaaa c 21
<210> 91
<211> 19
<212> DNA
<213>Artificial sequence
<400> 91
aaactagcgt gggcgagtc 19
<210> 92
<211> 20
<212> DNA
<213>Artificial sequence
<400> 92
gaggattgag aggtacgggg 20
<210> 93
<211> 24
<212> DNA
<213>Artificial sequence
<400> 93
aacaagtcag aactcagaac actg 24
<210> 94
<211> 19
<212> DNA
<213>Artificial sequence
<400> 94
gttgtcgtac agcttggcg 19
<210> 95
<211> 18
<212> DNA
<213>Artificial sequence
<400> 95
cttcgtcaca tgcccctc 18
<210> 96
<211> 19
<212> DNA
<213>Artificial sequence
<400> 96
gaggcttgtc cttgtgcag 19
<210> 97
<211> 20
<212> DNA
<213>Artificial sequence
<400> 97
ccgtctgcat cgtactcacc 20
<210> 98
<211> 20
<212> DNA
<213>Artificial sequence
<400> 98
gtgggttacc tatacccggc 20
<210> 99
<211> 20
<212> DNA
<213>Artificial sequence
<400> 99
tgaccccact ctgagttcac 20
<210> 100
<211> 20
<212> DNA
<213>Artificial sequence
<400> 100
tcctctccac tgcaggaatc 20
<210> 101
<211> 26
<212> DNA
<213>Artificial sequence
<400> 101
gcagtgatta acctttagca ataaac 26
<210> 102
<211> 22
<212> DNA
<213>Artificial sequence
<400> 102
cctttgagtt ttaagctgtg gc 22
<210> 103
<211> 22
<212> DNA
<213>Artificial sequence
<400> 103
ccaaactggg attagatacc cc 22
<210> 104
<211> 20
<212> DNA
<213>Artificial sequence
<400> 104
tgctaaatcc accttcgacc 20
Claims (10)
1. it is a kind of based on high throughput sequencing technologies detect hereditary hearing impairment Disease-causing gene multiplex PCR specific primer, its feature
It is that the multiplex PCR specific primer includes 52 pairs of specific primers, its nucleotide sequence such as SEQ ID NO：1 to SEQ
ID NO：Shown in 104.
2. specific primer according to claim 1, it is characterised in that the specific primer is drawing by modification
Thing.
3. specific primer according to claim 2, it is characterised in that the method for modifying of the specific primer is thio
Modification, Brdurd modification, 5 ' reverse dT modify in one or more.
4. specific primer according to claim 1, it is characterised in that the Tm values of the specific primer are 58-62 DEG C.
5. it is a kind of based on high throughput sequencing technologies detect hereditary hearing impairment Disease-causing gene kit, it is characterised in that including upper
State at least two pairs in the specific primer described in claim any one of 1-4.
6. kit according to claim 5, it is characterised in that the kit also include universal primer, enzymatic mixture,
Quality-control product, nuclease free water.
7. specific primer described in any one of claim 1-4 or the kit described in claim any one of 5-6 are applied to lose
Transmissibility deafness Disease-causing gene detection.
8. the method that hereditary hearing impairment Disease-causing gene is detected based on high throughput sequencing technologies, is comprised the following steps：
S1：Nucleic acid extraction is carried out to sample using paramagnetic particle method, the measure of concentration and purity is then carried out, is determined after measure is qualified
Amount dilution, it is standby；
S2：With specific primer described in any one of 1-4 containing claim or kit described in claim any one of 5-6 is in S1
The target area of the nucleic acid for being extracted carries out a PCR amplification, collects amplified production standby；
S3：Nucleic acid purification is carried out to amplified production in S2 using paramagnetic particle method, the measure of concentration and purity is carried out, sample mixing is quantitative, on
Machine is sequenced.
9. method according to claim 8, it is characterised in that the reaction system containing the specific primer is：
10. method according to claim 8, it is characterised in that the response procedures of the PCR amplifications are：
98 DEG C, activate 20min；Circulation 1 time；
72 DEG C, extend 10min；Circulation 1 time.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710058615.3A CN106755496A (en) | 2017-01-23 | 2017-01-23 | Multiplex PCR specific primer, kit and method based on high throughput sequencing technologies detection hereditary hearing impairment gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710058615.3A CN106755496A (en) | 2017-01-23 | 2017-01-23 | Multiplex PCR specific primer, kit and method based on high throughput sequencing technologies detection hereditary hearing impairment gene |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106755496A true CN106755496A (en) | 2017-05-31 |
Family
ID=58942942
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710058615.3A Pending CN106755496A (en) | 2017-01-23 | 2017-01-23 | Multiplex PCR specific primer, kit and method based on high throughput sequencing technologies detection hereditary hearing impairment gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106755496A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591480A (en) * | 2017-01-23 | 2017-04-26 | 中南大学湘雅医院 | Multiplex PCR specific primers, kit and method for detecting causative genes of large vestibular aqueduct syndrome based on high throughput sequencing technique |
| CN109355374A (en) * | 2018-11-30 | 2019-02-19 | 中国人民解放军第四军医大学 | Large Vestibular Aqueduct/Pendred syndrome Disease-causing gene SLC26A4 mutation detection kit |
| CN111979313A (en) * | 2020-09-21 | 2020-11-24 | 郑州桑林生物科技有限公司 | Specific primer and kit for detecting pathogenic variation of deafness gene based on multiplex PCR and high-throughput sequencing technology and application |
| CN114686580A (en) * | 2020-12-28 | 2022-07-01 | 广东菲鹏生物有限公司 | Compositions, kits, methods, and systems for nucleic acid sample amplification |
| CN114686579A (en) * | 2020-12-28 | 2022-07-01 | 广东菲鹏生物有限公司 | Compositions, kits, methods, and systems for nucleic acid sample amplification |
| CN114686561A (en) * | 2020-12-28 | 2022-07-01 | 广东菲鹏生物有限公司 | Compositions, kits, methods, and systems for nucleic acid sample amplification |
| CN114686562A (en) * | 2020-12-28 | 2022-07-01 | 广东菲鹏生物有限公司 | Compositions, kits, methods, and systems for nucleic acid sample amplification |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591480A (en) * | 2017-01-23 | 2017-04-26 | 中南大学湘雅医院 | Multiplex PCR specific primers, kit and method for detecting causative genes of large vestibular aqueduct syndrome based on high throughput sequencing technique |
| CN109355374A (en) * | 2018-11-30 | 2019-02-19 | 中国人民解放军第四军医大学 | Large Vestibular Aqueduct/Pendred syndrome Disease-causing gene SLC26A4 mutation detection kit |
| CN111979313A (en) * | 2020-09-21 | 2020-11-24 | 郑州桑林生物科技有限公司 | Specific primer and kit for detecting pathogenic variation of deafness gene based on multiplex PCR and high-throughput sequencing technology and application |
| CN111979313B (en) * | 2020-09-21 | 2023-05-26 | 郑州桑林生物科技有限公司 | Specific primer, kit and application for detecting pathogenic variation of deafness gene based on multiplex PCR and high-throughput sequencing technology |
| CN114686580A (en) * | 2020-12-28 | 2022-07-01 | 广东菲鹏生物有限公司 | Compositions, kits, methods, and systems for nucleic acid sample amplification |
| CN114686579A (en) * | 2020-12-28 | 2022-07-01 | 广东菲鹏生物有限公司 | Compositions, kits, methods, and systems for nucleic acid sample amplification |
| CN114686561A (en) * | 2020-12-28 | 2022-07-01 | 广东菲鹏生物有限公司 | Compositions, kits, methods, and systems for nucleic acid sample amplification |
| CN114686562A (en) * | 2020-12-28 | 2022-07-01 | 广东菲鹏生物有限公司 | Compositions, kits, methods, and systems for nucleic acid sample amplification |
| CN114686562B (en) * | 2020-12-28 | 2024-04-30 | 广东菲鹏生物有限公司 | Compositions, kits, methods and systems for nucleic acid sample amplification |
| CN114686580B (en) * | 2020-12-28 | 2024-04-30 | 广东菲鹏生物有限公司 | Compositions, kits, methods and systems for nucleic acid sample amplification |
| CN114686579B (en) * | 2020-12-28 | 2024-04-30 | 广东菲鹏生物有限公司 | Compositions, kits, methods and systems for nucleic acid sample amplification |
| CN114686561B (en) * | 2020-12-28 | 2024-04-30 | 广东菲鹏生物有限公司 | Compositions, kits, methods and systems for nucleic acid sample amplification |
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