CN107083435A - Detect the SNaPshot kits of 10 site deaf gene polymorphisms - Google Patents

Detect the SNaPshot kits of 10 site deaf gene polymorphisms Download PDF

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CN107083435A
CN107083435A CN201710418325.5A CN201710418325A CN107083435A CN 107083435 A CN107083435 A CN 107083435A CN 201710418325 A CN201710418325 A CN 201710418325A CN 107083435 A CN107083435 A CN 107083435A
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pcr
snapshot
primer
sequencing primer
sequencing
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赵薇薇
胡昌明
徐艳艳
陈白雪
贺书香
刘晶星
于世辉
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Guangzhou Kingmed Diagnostics Central Co Ltd
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    • C12Q2600/16—Primer sets for multiplex assays

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Abstract

The invention provides a kind of SNaPshot kits for detecting a variety of deaf gene polymorphisms, the deaf gene pleomorphism site of detection includes GJB2 gene locis rs80338943, rs111033204,508_511dupAACG；SLC26A4 genes rs111033318, rs121908362, rs121908363, rs200455203, rs111033380, rs111033313；Mitochondrial DNA rs267606617.

Description

Detect the SNaPshot kits of 10 site deaf gene polymorphisms
Technical field
The present invention relates to diagnosis of molecular biology field and hereditary disease field.Specifically, the invention provides one kind detection The SNaPshot kits of a variety of deaf gene polymorphisms.
Background technology
Congenital deafness is one of most common inborn defect, is also most common mankind sensory system's disease, the incidence of disease For 0.1%-0.3%.60% deafness is related to inherent cause.In these inherent causes, mainly including GJB2, SLC26A4, GJB3 gene and mitochondrial DNA are m.1555A>G and m.1494C>T sites.In the mutation of neonates in China deaf gene In examination, GJB2 gene mutations have higher carrying rate, and about 2.6%, SLC26A4 gene mutation carrying rates are about 1.9%, Neonate's mitochondrial DNA is m.1555A>The equal cytoplasmic mutations of G account for 0.1%, GJB3 gene mutations and found only in small number of patients.
The Cx26 of GJB2 gene codes is expressed in cochlea, positioned at vessels of internal ear line, basilar memebrane and spiral limbus, is lost with congenital Severe deafness is related in transmissibility, after GJB2 gene mutations, and the circulation that K+ enters endolymph fluid is affected, and causes often aobvious or often hidden Phonosensitive nerve deafness (DFNB1 or DFNA3).
The Cx31 of GJB3 gene codes is a member of inserted by connexin, is expressed in cochlea and auditory nerve, and mutation can be led Often aobvious or normal hidden non-comprehensive deafness, peripheral nerve disease are caused with hearing disability.
SLC26A4 genes mainly cause Chinese's Dilated Vestibular Aqueduct Syndrome, can be detected with reference to temporal bone CT.This disease goes out Hearing may be normal when raw, or has a slight hearing loss to middle severe, because bed of falling, children play or sports in it is slight Collision or flu can cause obvious Hearing, and whether its clinical manifestation is with age of onset, deaf degree and with dizzy It is dizzy to have close relationship.
Mitochondrial DNA Mutation is matrilinear inheritance, mitochondrial DNA m.1494C ＞ T, m.1555A ＞ G and aminoglycosides medicine Thing causes deaf relevant with non-syndromic cleft lip and palate.
In the non-syndromic cleft lip and palate patient of deifferent regions.China, the mutant proportion of each gene difference, Primary mutations Gene is GJB2 genes, and SLC26A4 genes and mitochondrial DNA are m.1555A>G and m.1494C>T sites, GJB3 gene mutations Found only in small number of patients, GJB2 gene Primary mutations mode is c.235delC, to account for the 63.6- of GJB2 gene mutations 76.8%, next to that c.299_300delAT and c.176_191del16 accounting for 8.7%-13.1%, c.508_511dupAACG Account for 4.3%-6.3%.C.2168A the Primary mutations of SLC26A4 genes is>G and c.919-2A>G, accounts for SLC26A4 genes The 77.1%-84.67% of mutation, c.1975G>C and c.2162C>T etc. accounts for 1%-2.5% respectively.Mitochondrial DNA deafness base Because m.1555A Primary mutations is>G, m.1494C>T.
Due to there is the degree of accuracy other sequencing detection methods more, specificity, the problem of precision is not enough, current deaf base Because polymorphic detection is typically still carried out using sanger methods, it is only capable of detecting a site in once testing, and when spending Between it is longer.The deaf gene polymorphic detection that this difficulty is caused, particularly can cover extensive deaf gene pleomorphism site Examination is difficult to popularize, even in Beijing, the city such as Shanghai, the free deaf gene screening of neonate also only cover two GJB2 and SLC26A4 sites are, it is necessary to which the further examination of hundreds of members also only many 4 sites at one's own expense, such examination can undoubtedly be omitted largely Potential hereditary hearing impairment patient, adverse effect is brought to its health care and treatment from now on.In addition, the limitation of above-mentioned detection means Also so that the work of hereditary disease related scientific research is difficult to extensive development.
Snapshot carries out Snapshot reactions by designing the primer of different length for different loci, and product is through electrophoresis point From, fluoroscopic examination, Gene mapper analyses, can disposably detect in multiple SNP sites, and every secondary response only needs very small amount DNA samples.More complicated than general PCR method, Snapshot detection architectures, particularly multiple Snapshot detection architectures are set Need the factor considered numerous in meter：Primer length, Tm values, tail structure selection, and template and the ratio of primer all may Cause non-specific amplification and subsequent base is misread.The multiple Snapshot detection architectures of reliable design are needed to design of primers Careful consideration is carried out, largely verifies and adjusts accordingly.
The example that the detection technique is applied into deaf SNP site detection at present only has the A of CN 103911452 and CN 102618624 A, the former detection object includes GJB2 genes 35,109,176-199,235,299-300 sites, SLC26A4 Gene 11 74,1226,1229,1975,2027,2162,2168, IVS7-2 sites, mitochondrial DNA 1494,1555,3243, The mutation in 7444 sites.The detection object of the latter include GJB2 genes 235,299-300 sites, SLC26A4 genes 2168, IVS7-2 sites, the site of mitochondrial DNA 1555,3243,7445.Both of which has been authorized.But still have part deaf related SNP site, such as c.508_511dupAACG, SLC26A4-919-2, GJB3 GJB3-547,538 sites etc. are not covered with, It is therein c.508_511dupAACG, the higher mutational site of occurrence rate in SLC26A4-919-2 or population of China.Design energy Include more extensive deaf related locus, the Snapshot kits in the site that particularly existing kit is not directed to, for losing The deaf prevention of transmissibility and scientific research are significant.
The content of the invention
The present invention disposably detects multiple deaf gene pleomorphism sites using Snapshot technologies, and speed is fast and saves Time and expense.Vast scale can be detected, the Chinese heredity non-syndromic cleft lip and palate crowd that prior art is omitted helps It is that the diagnosis and treatment in later stage are carried in finding to carry the infant (including Delayed onset auditorily handicapped infant) that deaf gene is mutated early For scientific basis, contribute to the generation for taking intervening measure to prevent disfluency in time, be effectively reduced the deaf and dumb incidence of disease.
On the one hand, the invention provides a kind of SNaPshot kits for detecting a variety of deaf gene polymorphisms, detection Deaf gene pleomorphism site includes GJB2 gene locis rs80338943, rs111033204,508_511dupAACG； SLC26A4 genes rs111033318, rs121908362, rs121908363, rs200455203, rs111033380, rs111033313；12s rRNA genes (mitochondrial DNA) rs267606617.
Further, the amplimer and sequencing primer for detecting these pleomorphism sites are included in kit.
Further, amplimer is respectively：
Further, sequencing primer is respectively：
Further, amplimer is when being expanded, and amplifing reagent ratio is as follows：
Wherein, the ratio of amplimer is 1：1；
Further, amplified production is when carrying out micro sequence, and sequencing reagent ratio is as follows：
Reagent Volume (μ L)
SNaPshot Ready Mix 1.25
ddH2O 4.25
The sequencing buffer solution of 5 times of dilutions 1.5
Primer mixture 2
total 9.0
Further, amplified production is when carrying out micro sequence, and sequencing primer concentration ratio is as follows：
Further, the kit application method includes step：
DNA is extracted；
Amplified production is purified；
Extension products are purified；
Sequencing analysis.
On the other hand, multiple deaf gene polymorphisms are disposably detected using Snapshot technologies the invention provides one kind The non-diagnostic research methods in site, specifically include step：
DNA is extracted：
DNA offers are carried out with the collecting sample of human blood, it is relevant with specific reference to company《Poba gene group DNA extraction standards are grasped Make flow》, extract the DNA finished and calculate its concentration and be diluted to 5-10 μ g/mL；
Reagent prepares：
A) the Master Mix of two times of configuration, among these including dNTP, MgCl2, it is the super fidelity dna polymerase of thermal starting, slow Fliud flushing, after room temperature is melted, of short duration centrifugation；Get out appropriate number of PCR reaction tubes；
B) primer mixture is prepared：The ratio of each primer is=1:1, concussion is mixed；It is standby after of short duration centrifugation；
C) reaction system is prepared
D) concussion is mixed, after of short duration centrifugation, 23 μ L of packing to the PCR reaction tubes marked.It is transferred to sample and prepares area；
Sample-adding
If a) sample and quality-control product are stored in -20 DEG C, using preposition thaw at RT, and of short duration centrifugation；
B) 10ngDNA templates are added into the PCR reaction tubes dispensed, and are diluted；
C) cover after lid, concussion is mixed, of short duration centrifugation is transferred to PCR amplification regions；
PCR is expanded：
PCR pipe is put to be put in PCR instrument.Response procedures set as follows：
95℃5min；95℃30s；55℃30s；72℃1min；45 circulations；72℃5min；25 DEG C of insulations；
Pcr amplification product is purified：
2ul ExoSAP-IT is taken to be dispensed into 200ul EP pipes, often pipe adds PCR primer 5ul, mixes micro- from by following journey Sequence enters performing PCR：7ul 37℃15min；80℃15min；4℃forever
SNaPshot PCR are expanded
A) 8 connecting legs are taken out in reaction after terminating, and prepare primer mixture, and ratio is as follows：
B) following system configurations are pressed：
C) Snapshot extensions are carried out respectively to the amplified productions of 2 pipes after purification, 1ul purified products are added, mix it is micro- from, Enter performing PCR by following procedure：96℃10s；96℃10s；50℃5s；60℃30s；25 circulations；4 DEG C of insulations；
SNE product purifications：
A) toward addition FastAP 1ul, buffer2ul, 7ul water, 10ulSNaPshot PCR in SNaPshot PCR primers Product, performing PCR is entered by following procedure：
b)37℃10min；75℃5min；1 circulation；4 DEG C of insulations；
Electrophoresis：
95 DEG C of denaturation 3min of product 1ul+8.8ul HiDi+0.2ul 120Liz that SAP has been handled, cooled on ice, upper machine Interpretation of result is with explaining：
Analysis carries out initial analysis using GENEMAPPERID V4.1 softwares, determines SNP site；
Analyzing the result finished needs to preserve, and GENEMAPPERID V4.1 software documents forms and SNaPshot peak figures are each Preserve a；
GeneMapper4.1 software analysis.
On the other hand, the SNaPshot reagents of a variety of deaf gene polymorphisms of detection are being prepared the invention provides primer sets In application, amplimer is respectively：
Sequencing primer is respectively：
Further, sequencing primer concentration ratio is as follows：
Brief description of the drawings
Fig. 1 is the Snapshot detection examples of moiety site.
Fig. 2 is moiety site SNaPshot testing results example (feminine gender).
Embodiment
The detection process of embodiment 1
1. the specific implementation process of detection
1.1DNA extract
DNA offers are carried out with the collecting sample of human blood, it is relevant with specific reference to company《Poba gene group DNA extraction standards are grasped Make flow》, extract the DNA finished and calculate its concentration and be diluted to 5-10 μ g/mL.
1.2 reagents prepare
E) the Master Mix of two times of configuration, among these including dNTP, MgCl2, it is the super fidelity dna polymerase of thermal starting, slow Fliud flushing, after room temperature is melted, of short duration centrifugation；Get out appropriate number of PCR reaction tubes.
F) primer mixture is prepared：The ratio of each primer is=1:1, concussion is mixed；It is standby after of short duration centrifugation.
G) reaction system is prepared
D) concussion is mixed, after of short duration centrifugation, 23 μ L of packing to the PCR reaction tubes marked.It is transferred to sample and prepares area.
1.3 sample-adding
If d) sample and quality-control product are stored in -20 DEG C, using preposition thaw at RT, and of short duration centrifugation.
E) 10ngDNA templates are added into the PCR reaction tubes dispensed, and are diluted.
F) cover after lid, concussion is mixed, of short duration centrifugation is transferred to PCR amplification regions.
1.4PCR amplification
PCR pipe is put to be put in PCR instrument.Response procedures set as follows：
95℃5min；95℃30s；55℃30s；72℃1min；45 circulations；72℃5min；25 DEG C of insulations；
1.5PCR amplified productions are purified
2ul ExoSAP-IT is taken to be dispensed into 200ul EP pipes, often pipe adds PCR primer 5ul, mixes micro- from by following journey Sequence enters performing PCR：7ul 37℃15min；80℃15min；4℃forever
1.6SNaPshot PCR are expanded
A) 8 connecting legs are taken out in reaction after terminating, and prepare primer mixture, and ratio is as follows：
B) following system configurations are pressed：
Reagent Volume (μ L)
SNaPshot Ready Mix 1.25
ddH2O 4.25
The sequencing buffer solution of 5 times of dilutions 1.5
Primer mixture 2
total 9.0
C) Snapshot extensions are carried out respectively to the amplified productions of 2 pipes after purification, 1ul purified products are added, mix it is micro- from, Enter performing PCR by following procedure：96℃10s；96℃10s；50℃5s；60℃30s；25 circulations；4 DEG C of insulations；
1.7SNE product purification
C) toward addition FastAP 1ul, buffer2ul, 7ul water, 10ulSNaPshot PCR in SNaPshot PCR primers Product, performing PCR is entered by following procedure：
d)37℃10min；75℃5min；1 circulation；4 DEG C of insulations；
1.8 electrophoresis
95 DEG C of denaturation 3min of product 1ul+8.8ul HiDi+0.2ul 120Liz that SAP has been handled, cooled on ice, on Machine
2. interpretation of result is with explaining
2.1 interpretation of result methods
Analysis carries out initial analysis using GENEMAPPERID V4.1 softwares, determines SNP site.
Analyzing the result finished needs to preserve, and GENEMAPPERID V4.1 software documents forms and SNaPshot peak figures are each Preserve a.
2.2 interpretation of result
GeneMapper4.1 software analysis, as a result such as accompanying drawing 1.
3.QC rules
Two Quality Controls are set up in this detection altogether, and Quality Control criterion is with reference to detection SOP：
1.POS, is positive control, is typically chosen the sample that 1 or multiple site is heterozygous mutant.
2.NTC, is no template control.
4. primer specificity
1) amplified fragments of all primers designed by the present invention cover corresponding detection site, without other homologous bases Cause, details refer to subordinate list 2；
2) present invention is expanded to detection sample respectively using amplimer, and is surveyed using Sanger PCR sequencing PCRs Sequence, sequencing result shows that each primer amplified fragments coincide (seeing attached list 3, only display portion result) with gene reference sequence.
3) present invention carries out micro sequence using SNaPshot sequencing primers to corresponding amplified production, and sequencing result is shown, The base of relative position and the sequencing reaction incorporation at each product peak is consistent with expection, and (sees attached list 4 without other Interference Peaks, only show Show partial results).
5. the degree of accuracy
This accuracy in detection is defined as this detection method testing result with other laboratories or the uniformity of known results.
The DNA sample that the application is included, is detected that testing result is shown in Table 3 using SNaPshot PCR sequencing PCRs.All detections As a result expection is met, it is 100% that calculating, which obtains this accuracy in detection,.
Remarks：
1. the known results of other samples refer to the result determined using Sanger PCR sequencing PCRs.
2.NMD=NO MUTATION DETECTED.
The degree of accuracy experimental data table of table 3
6. detection specificity and detection sensitivity
The detection specificity of the application detection is defined as negative match-rate, and detection sensitivity is defined as positive coincidence rate.
64 samples of primer pair that this detection is used are detected, all DNA samples with other laboratory results or by Qualification organization is estimated, and detailed results are shown in Table 3.Testing result is negative (site of deaf gene 22 is not detected by mutation) Sample is completely the same, and the detection specificity of this detection is 100%.Testing result is positive (any one position in the site of deaf gene 22 Point detect mutation or detect mutation, think this sample for mutation the positive) sample it is completely the same, mutational site and class Type is also completely the same, and the detection sensitivity of this detection is 100%.
Table 4 detects specificity and detection sensitivity
7. precision
The precision of this detection is defined as that sample is carried out to repeat the ability that detection obtains same result.
7.1 withinrun precision
To 1 positive sample, (wherein DFN-POS-1 known results are GJB2 for this detection:C.235delC 3 multiple holes) have been carried out Detection, as a result shows, the testing result between the different holes of same sample is consistent.The withinrun precision of this detection is 100%.
The withinrun precision of table 5
Sample number Hole 1 Hole 2 Hole 3
DFN-POS-1 GJB2:c.235delC GJB2:c.235delC GJB2:c.235delC
7.2 betweenrun precisions (Precision-Between Runs)
To 1 positive sample, (wherein DFN-POS-1 known results are GJB2 for this detection:C.235delC) carried out four times Detection.The testing result of same sample different batches is consistent.This detection withinrun precision is 100%.
The betweenrun precision of table 6
Note：I：Instrument is numbered；T：Technician
Above-mentioned data demonstrate the application method.Accuracy, specificity can meet detection and require, can be clinically Substitute Sanger PCR sequencing PCRs.
The present processes are grouped by scientific design and collocation primer, are realized the complete detection in a reaction tube and are showed There is technology to omit c.508_511dupAACG, SLC26A4-919-2 deafness related locus and other several sites occurred frequently, tool There are good market value and important medical significance.
Subordinate list:Primer amplified fragments
SEQUENCE LISTING
<110>Golden domain
<120>Detect the SNaPshot kits of 10 site deaf gene polymorphisms
<160> 22
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence
<400> 1
gagaagtctc cctgttctgt cc 22
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<400> 2
acaaagcagt ccacagtgtt g 21
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
tctcgtatcc agcagcaatg 20
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence
<400> 4
gggttccagg aaattacttt gt 22
<210> 5
<211> 30
<212> DNA
<213>Artificial sequence
<400> 5
aattgtggta agtagaatat gtagttagaa 30
<210> 6
<211> 24
<212> DNA
<213>Artificial sequence
<400> 6
aaggagtatc agtgaaatga agct 24
<210> 7
<211> 22
<212> DNA
<213>Artificial sequence
<400> 7
ttctatggca atgtcgatgg tt 22
<210> 8
<211> 23
<212> DNA
<213>Artificial sequence
<400> 8
ctacacaaag ggaagagggt cta 23
<210> 9
<211> 21
<212> DNA
<213>Artificial sequence
<400> 9
gaactctgag cttccagtca a 21
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence
<400> 10
gcccatgtat ttgccctgtt 20
<210> 11
<211> 19
<212> DNA
<213>Artificial sequence
<400> 11
tggagcaatg cgggttctt 19
<210> 12
<211> 25
<212> DNA
<213>Artificial sequence
<400> 12
cttgagattt cacttggttc tgtag 25
<210> 13
<211> 43
<212> DNA
<213>Artificial sequence
<400> 13
tttttttttt ttttttttta tctcccacat ccggctatgg gcc 43
<210> 14
<211> 51
<212> DNA
<213>Artificial sequence
<400> 14
tttttttttt tttttttttt ttttttgcca tgcacgtggc ctaccggaga c 51
<210> 15
<211> 59
<212> DNA
<213>Artificial sequence
<400> 15
tttttttttt tttttttttt tttttttttt ctccatgcag cggctggtga agtgcaacg 59
<210> 16
<211> 26
<212> DNA
<213>Artificial sequence
<400> 16
ttgccagtgc cctgactctg ctggtt 26
<210> 17
<211> 44
<212> DNA
<213>Artificial sequence
<400> 17
tttttttttt tttttttttt tttatggcag tagcaattat cgtc 44
<210> 18
<211> 21
<212> DNA
<213>Artificial sequence
<400> 18
aatgtatcaa gtccacagta a 21
<210> 19
<211> 34
<212> DNA
<213>Artificial sequence
<400> 19
tttttttttt ttttaccaga accttaccac ccgc 34
<210> 20
<211> 59
<212> DNA
<213>Artificial sequence
<400> 20
tttttttttt tttttttttt tttttttttt tttttttgat atagctccac agtcaagca 59
<210> 21
<211> 41
<212> DNA
<213>Artificial sequence
<400> 21
tttttttttt tacttggttc tgtagataga gtatagcatc a 41
<210> 22
<211> 47
<212> DNA
<213>Artificial sequence
<400> 22
tttttttttt tttttttttt tttttagaaa ggacacattc tttttga 47

Claims (10)

1. a kind of SNaPshot kits for detecting a variety of deaf gene polymorphisms, the deaf gene pleomorphism site of detection includes GJB2 gene locis rs80338943, rs111033204,508_511dupAACG；SLC26A4 genes rs111033318, rs121908362、rs121908363、rs200455203、rs111033380、rs111033313；Mitochondria DNArs267606617。
2. the kit described in claim 1, wherein including the amplimer and sequencing primer that detect these pleomorphism sites.
3. the kit described in claim 2, wherein amplimer are respectively：
Sequencing primer is respectively：
4. the kit described in claim 3, amplimer is when being expanded, and amplifing reagent ratio is as follows：
Wherein, the ratio of amplimer is 1：1.
5. the kit described in claim 4, amplified production is when carrying out micro sequence, and sequencing reagent ratio is as follows：
Reagent Volume (μ L) SNaPshot Ready Mix 1.25 ddH2O 4.25 The sequencing buffer solution of 5 times of dilutions 1.5 Primer mixture 2 total 9.0
6. the kit described in claim any one of 3-5, amplified production is when carrying out micro sequence, and sequencing primer concentration ratio is such as Under：
Primer Final concentration (pmol) Sequencing primer SNE1-DFN-235delC 0.15 Sequencing primer SNE1-DFN-delAT 0.2 Sequencing primer SNE1-DFN-dupAACG 0.6 Sequencing primer SNE4-SLC26A4-589 0.4 Sequencing primer SNE4-SLC26A4-919-2 2 Sequencing primer SNE3-SLC26A4-1707+5 0.5 Sequencing primer SNE3-SLC26A4-2027 0.2 Sequencing primer SNE3-SLC26A4-1975 0.4 Sequencing primer SNE3-SLC26A4-2168 0.2 Sequencing primer SNE3-SLC26A4-2162 0.6
7. the kit described in claim any one of 1-6, the kit application method includes step：
DNA is extracted；
Amplified production is purified；
Extension products are purified；
Sequencing analysis.
8. a kind of utilization Snapshot technologies disposably detect the non-diagnostic research methods of multiple deaf gene pleomorphism sites, tool Body includes step：
DNA is extracted：
DNA offers are carried out with the collecting sample of human blood, it is relevant with specific reference to company《Poba gene group DNA extraction standard operation streams Journey》, extract the DNA finished and calculate its concentration and be diluted to 5-10 μ g/mL；
Reagent prepares：
A) the Master Mix of two times of configuration, among these including dNTP, MgCl2, the super fidelity dna polymerase of thermal starting, buffer solution, After room temperature is melted, of short duration centrifugation；Get out appropriate number of PCR reaction tubes；
B) the amplimer mixture listed by claim 3 is prepared：The ratio of each primer is=1:1, concussion is mixed；It is of short duration from It is standby after the heart；
C) reaction system is prepared
D) concussion is mixed, after of short duration centrifugation, 23 μ L of packing to the PCR reaction tubes marked.It is transferred to sample and prepares area；
Sample-adding
If a) sample and quality-control product are stored in -20 DEG C, using preposition thaw at RT, and of short duration centrifugation；
B) 10ngDNA templates are added into the PCR reaction tubes dispensed, and are diluted；
C) cover after lid, concussion is mixed, of short duration centrifugation is transferred to PCR amplification regions；
PCR is expanded：
PCR pipe is put to be put in PCR instrument.Response procedures set as follows：
95℃5min；95℃30s；55℃30s；72℃1min；45 circulations；72℃5min；25 DEG C of insulations；Pcr amplification product Purifying：
Take 2ul ExoSAP-IT to be dispensed into 200ul EP pipes, often pipe adds PCR primer 5ul, mix micro- from entering by following procedure Performing PCR：7ul 37℃15min；80℃15min；4℃forever
SNaPshot PCR are expanded
A) 8 connecting legs are taken out in reaction after terminating, and prepare the sequencing primer mixture listed by claim 3, and ratio is as follows：
B) following system configurations are pressed：
Reagent Volume (μ L) SNaPshot Ready Mix 1.25 ddH2O 4.25 The sequencing buffer solution of 5 times of dilutions 1.5 Primer mixture 2 total 9.0
C) Snapshot extensions are carried out respectively to the amplified productions of 2 pipes after purification, 1ul purified products are added, mix it is micro- from,
Enter performing PCR by following procedure：96℃10s；96℃10s；50℃5s；60℃30s；25 circulations；4 DEG C of insulations；
SNE product purifications：
A) toward addition FastAP 1ul, buffer2ul, 7ul water, 10ulSNaPshot PCR productions in SNaPshot PCR primers Thing, performing PCR is entered by following procedure：
b)37℃10min；75℃5min；1 circulation；4 DEG C of insulations；
Electrophoresis：
95 DEG C of denaturation 3min of product 1ul+8.8ul HiDi+0.2ul 120Liz that SAP has been handled, cooled on ice, upper machine result Analysis is with explaining：
Analysis carries out initial analysis using GENEMAPPERID V4.1 softwares, determines SNP site；
Analyzing the result finished is needed to preserve, and GENEMAPPERID V4.1 software documents forms and SNaPshot peak figures are respectively preserved It is a；
GeneMapper4.1 software analysis.
9. the answering in the SNaPshot reagents for preparing a variety of deaf gene polymorphisms of detection of the primer sets listed by claim 3 With.
10. the application described in claim 9, wherein sequencing primer concentration ratio is as follows：
CN201710418325.5A 2017-06-06 2017-06-06 Detect the SNaPshot kits of 10 site deaf gene polymorphisms Pending CN107083435A (en)

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CN109852685A (en) * 2019-02-18 2019-06-07 南通大学 HPR1 gene mutation body and its application in the deaf diagnostic reagent of preparation
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Application publication date: 20170822