CN101812523B - SNP (Single Nucleotide 0olymorphism) detection specific primer, liquid-phase chip and detection method of RYR1 (Ryanodine Receptors 1) gene - Google Patents
SNP (Single Nucleotide 0olymorphism) detection specific primer, liquid-phase chip and detection method of RYR1 (Ryanodine Receptors 1) gene Download PDFInfo
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Abstract
The invention discloses an SNP (Single Nucleotide Polymorphism) detection specific primer, a liquid-phase chip and a detection method of an RYR1 (Ryanodine Receptors 1) gene. The liquid-phase chip comprises wild type and mutant type ASPE (Allele Specific Primer Extension) primer pairs respectively designed aiming at the mutational sites of each type, microballoons respectively coated with a specific anti-tag sequence and primers used for respectively amplifying target RYR1 gene sequences with G341R, R614C, T2206M and/or G2434R sites. The SNP detection liquid-phase chip of the RYR1 gene has very good signal-to-noise ratio, and a designed probe and the anti-tag sequences have no cross reactions basically. The designed ASPE primers have very good specificity and can accurately distinguish various types of mutational sites. The detection method has simple steps, and four SNP sites can finish being detected in one step, thus the operation is convenient; and moreover, various uncertain factors existing in the processes of multiple operations are avoided, thus the detection precision is greatly improved.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, concrete RYR1 gene SNP detection specific primer, liquid-phase chip and the detection method of relating to.
Background technology
Ryanodine acceptor 1 (Ryanodine receptors 1, RYR1) as a kind of calcium channel albumen, the myocyte eventually not the pond calcium ion discharge and inspire the myocyte and play critical effect in shrinking.It is empty myopathy (the central core disease of the axis of centres that the RYR1 sudden change has been confirmed to be; CCD), empty sick (the multiminicore disease of how small axle; MmD), malignant hyperthermia (malignant hyperthermia; MH) and axle sky/rhabdos sick (Core/rod disease, major cause CRD).
After zhang report RYR1 in 1993 was the Disease-causing gene of CCD, up to the present, existing 44 kinds of RYR1 sudden change report was relevant with CCD, comprises 39 missense mutation and 5 deletion mutantions.Research shows, most CCD is by due to the RYR1 transgenation.RYR1 equally also is the Disease-causing gene of MmD, but the numerical example report is only arranged so far, and is extremely rare.CRD refers in the empty sick muscle pathology of axle; Rhabdos appears in the myofiber more than 10% simultaneously; Recently different RYR1 transgenations is determined in the sick family of axle sky/rhabdos, and this shows that rhabdos possibly be the Secondary cases pathological change of some CCD.And MH is the unusual hypermetabolism state of Skelettmuskel that is triggered by volatility inhalation anesthetic and sux-cert, is a kind of heredofamilial subclinical myonosus that has.The RYR1 gene pleiomorphism is the molecular biology basis that most of MH takes place, so the detection of RYR1 gene pathogenic mutation type necessitates.
The sudden change multidigit of RYR1 leak path in N end and middle section (as: Tyr523Ser, Arg2163His, Arg2435Leu); And the excited sudden change multidigit of losing the lotus root couplet of shrinking is held (as: Gly4890Arg, Arg4892Trp, Ile4897The in C; Gly4898Glu; Gly4898Arg, Ala4905Val, Arg4913Gly).Yet the Ile4795Cys of C end, generation but be leak path.
At present, the modal catastrophe point of RYR1 is R614C, G2434R and G341R, and they account for 10% of malignant hyperthermia case.Wherein, R614C and G341R are common in the Continent of Europe, and G2434R is common in NA.Japan report sport R1667C, P1773S, Q3756E and P1763S mostly.It is T2206M that only there is 1 routine its catastrophe point of malignant hyperthermia transgenation report the China's Mainland.
Almost do not detect the launch of RYR1 gene pleiomorphism at present both at home and abroad, major part still is in the experimental study stage, as yet not commercialization; Existing detection technique mainly is based upon on the basis of PCR, like direct sequencing, and quantitative fluorescent PCR; PCR-single-strand conformation polymorphism analysis (SSCP) detects, and these technology exist sensitivity low, the easy pollution of sample, the shortcoming that false positive rate is high; Simultaneously, can not satisfy clinical needs owing to detect the limitation of flux.And polymerase chain reaction one restriction fragment length polymorphism (PCR-RFLP) analytical technology once can only be carried out a kind of detection of sudden change, time and effort consuming.
Summary of the invention
One of the object of the invention provides the RYR1 gene SNP detection liquid-phase chip.This liquid-phase chip can be used for detecting the variation of normal genotype and four kinds of common allele type G341R, R614C, T2206M and G2434R of RYR1 gene.
The technical scheme that realizes above-mentioned purpose is following:
A kind of RYR1 gene SNP detection liquid-phase chip mainly includes:
(A). designed ASPE primers is right respectively to the SNP site of RYR1 gene: every kind of ASPE primer is made up of to the specific primer sequence in goal gene SNP site the tag sequence and the 3 ' end of 5 ' end, and said specific primer sequence is respectively: to the SEQ ID NO.9 in G341R site and SEQ ID NO.10, to the SEQ ID NO.11 and the SEQ ID NO.12 in R614C site, be directed against the SEQ ID NO.13 and the SEQ ID NO.14 in T2206M site; And/or to the SEQ ID NO.15 and the SEQ ID NO.16 in G2434R site; Said tag sequence is selected from the sequence among the SEQ ID NO.1-8;
(B). be coated with the microballoon of special anti-tag sequence respectively, above-mentioned every kind of microballoon has the different colours coding; Said anti-tag sequence is selected from the sequence among SEQ ID NO.17～SEQ ID NO.24, and said anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). be used for increasing respectively have G341R, the amplimer of the RYR1 gene target sequence in R614C, T2206M and/or G2434R SNP site.
Preferably, said amplimer is: to the SEQ ID NO.25 in G341R site and SEQ ID NO.26, to the SEQ ID NO.27 in R614C site and SEQ ID NO.28, to the SEQ ID NO.29 in T2206M site and SEQ ID NO.30 and/or to the SEQ ID NO.31 and the SEQ ID NO.32 in G2434R site.
Preferably, said ASPE primer is to being: to the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.9 in G341R site and the sequence of being made up of SEQ ID NO.2 and SEQ ID NO.10, reach the sequence of being made up of SEQ ID NO.8 and SEQ ID NO.16 to the sequence of being made up of SEQ ID NO.7 and SEQ ID NO.15 that the sequence of being made up of SEQ ID NO.3 and SEQ ID NO.11 in R614C site reaches the sequence be made up of SEQ ID NO.4 and SEQ ID NO.12, the sequence of being made up of SEQ ID NO.5 and SEQ ID NO.13 that is directed against the T2206M site reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.14 and/or is directed against the G2434R site.
Preferably, also be provided with the spacerarm sequence in the middle of said anti-tag sequence is connected with microballoon.
Another object of the present invention provides the method for using above-mentioned liquid-phase chip that the RYR1 gene SNP is detected.
Concrete technical scheme is following:
A kind of method of using above-mentioned liquid-phase chip to the detection of RYR1 gene SNP mainly may further comprise the steps:
(1) pcr amplification testing sample DNA;
(2) the PCR reaction product is carried out enzyme with the ExoSAP-IT test kit and is cut processing;
(3) carry out primer extension reaction with said ASPE primer, in reaction process, mix biotin labeled dCTP, thereby make a plurality of biotin labeling on the reacted product band;
(4) microballoon of the anti-tag sequence that being coated with of corresponding A SPE primer is special and the product behind the above-mentioned extension carry out hybridization;
(5) product behind the hybridization and Streptavidin-phycoerythrin react;
(6) detect through fluorescence detector.
Another object of the present invention provides and is used for the Auele Specific Primer that the RYR1 gene SNP detects.
Concrete technical scheme is following:
Be used for the Auele Specific Primer that the RYR1 gene SNP detects, include: to the SEQ ID NO.9 in G341R site and SEQ IDNO.10, to the SEQ ID NO.11 in R614C site and SEQ ID NO.12, to the SEQ ID NO.13 in T2206M site and SEQ ID NO.14 and/or to the SEQ ID NO.15 and the SEQ ID NO.16 in G2434R site.
Major advantage of the present invention is:
1. the identical rate of detection method provided by the present invention and PCR sequencing PCR is up to 100%.Prepared RYR1 gene SNP detection liquid-phase chip has extraordinary signal-NR, and does not have cross reaction basically between institute's designed probe and the anti-tag sequence.
2. designed ASPE Auele Specific Primer of the present invention has extraordinary specificity, can accurately distinguish the genotype of various types, and can under the reaction conditions of homogeneous, carry out hybridization, and does not have non-specific binding basically between the various primer, probe.
3. detection method step of the present invention is simple; One step multiplex PCR can be accomplished four amplifications with the target sequence in SNP site; Many uncertain factors of existing in the complex operations processes such as repeated multiple times PCR have been avoided; Thereby can improve the detection accuracy rate greatly, embodied qualitative, quantitative analysis characteristic of accurate while, special coincidence detection application need.
4. not only to have overcome traditional solid phase chip susceptibility not high in the present invention, and the defective of the repeatability difference of detected result is improved existing liquid-phase chip technology simultaneously, makes prepared microballoon can be applicable to the different detection project, has very strong expansion.The fluorescent signal value that detects improves greatly, thereby makes the sensitivity that detects be further enhanced, and SNR strengthens, and detected result more accurately and reliably.Simultaneously, the combination of multiple ASPE Auele Specific Primer makes liquid-phase chip and detection method form one and detects the intact system of effect.
Embodiment
Embodiment 1RYR1 gene SNP detection liquid-phase chip mainly includes:
One, ASPE primer
Distinguish the primer sequence of designs specificity to four kinds of common SNP site: G341R, R614C, T2206M, the G2434R of RYR1.The ASPE primer is made up of " Tag+ specific primer sequence ".The ASPE primer sequence is as shown in the table:
Table 1ASPE primer sequence (Tag+ specific primer sequence)
Every the ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence to anti-tag sequence on the corresponding microballoon, and 3 ' end is mutant or wild-type specific primer sequence (shown in above-mentioned table 1).All ASPE primers are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with the stock solution of 100pmol/mL respectively with 10mmol/LTris Buffer.
Two, the microballoon that encapsulates of anti-tag sequence
According to institute's designed ASPE specific primer sequence; Select the tag sequence; Reduce between the anti-tag sequence of each microballoon to greatest extent and secondary structure that tag and ASPE specific primer sequence possibly form, corresponding anti-tag sequence is as shown in table 2 on eight kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 2 microballoon numbering and the microballoon
8 kinds of microballoons selecting are available from U.S. Luminex company, with the anti-tag sequence encapsulate with microballoon on.Be connected with the spacerarm sequence of 5-10 T between anti-tag sequence and the microballoon, promptly before each anti-tag sequence, add the spacerarm sequence of the preceding paragraph 5-10 T, the anti-tag sequence is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Synthetic anti-tag sequence is used sterilization ddH
2O is made into the stock solution of 100nmol/ml.Said spacerarm is to be used for anti-tag and microsphere surface is spaced apart or anti-tag placed the sequence of hydrophilic environments.Through the spacerarm sequence of suitable length is set between anti-tag sequence and microballoon, can reduce sterically hinderedly, improve the efficient of hybridization and the specificity of hybridization.Common spacerarm sequence comprises poly dT, i.e. poly (dT), and oligomerization four polyoxyethylene glycol and (CH2) n spacerarm (n >=3) are like (CH2) 12, (CH2) 18 etc.In addition, if exist poly (dA) to disturb, can also use poly (TTG) as spacerarm.Spacerarm of the present invention is preferably 5-10 T.
The process that microballoon encapsulates is following:
Get 5 * 10 respectively
6The carboxylated microballoon of individual above-mentioned numbering (available from Luminex company) is suspended in the MES solution of 50ul 0.1mol/L (pH4.5), adds 10ul synthetic anti-tag molecule (100nmol/ml).EDC (N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide) (available from the Pierce Chemical company) working fluid of preparation 10ng/ml.Add the EDC working fluid of 2.5ul in the microballoon suspension, constant temperature was hatched 30 minutes, added the EDC working fluid of 2.5ul again, and constant temperature was hatched 30 minutes again.After reaction finished, the Tween-20 washing with 0.02% was once washed once with 0.1% SDS liquid again.The microballoon that is coated with the anti-tag sequence after the washing is resuspended in the Tris-EDTA solution of 100ul, and [10mmol/L Tris (pH8.0), among the 1mmol/LEDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of target sequence with SNP site
4 kinds of common SNP site G341R of target detect RYR1, R614C, T2206M, G2434R.Utilize Primer5.0 design four pairs of primers (seeing table 3), amplify four target sequences respectively with SNP site.
Table 3 amplifies the primer of the target sequence with SNP site
All primers are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.
Embodiment 2 utilization embodiment 1 said RYR1 gene SNP detection liquid-phase chip is to the detection of sample
The prescription of said various solution is following:
MES damping fluid (pH5.0) prescription (250ml) of 50mM:
| Reagent | The source | Final concentration | The consumption of every 250ml |
| MES(2[N-Morpholino] ethanesulfonic?acid) | Sigma?M-2933 | 0.05M | ?2.44g |
| 5MNaOH | Fisher?SS256-500 | --- | 5 |
2 * Tm hybridization buffer
| Reagent | The source | Final concentration | The consumption of every 250ml |
| 1MTris-HCl，pH8.0 | SigmaT3038 | 0.2M | ?50ml |
| 5MNaCl | Sigma?S5150 | 0.4M | ?20ml |
| Triton?X-100 | Sigma?T8787 | 0.16％ | ?0.4ml |
Be stored in 4 ℃ after the filtration.
The ExoSAP-IT test kit is available from U.S. USB company.
Biotin labeled dCTP is available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
One, the DNA extraction of sample:
Extract the test kit explanation in a small amount with reference to AxyPrep whole blood genome, obtain DNA to be detected.
Two, the pcr amplification of testing sample
Utilize four pairs of primers of Primer5.0 design, one step of multiplex PCR amplifies the target sequence that RYR1 has the SNP site, and the product size is respectively 260bp, 374bp, 268bp, 400bp.Primer sequence (SEQ NO.25-32) is seen shown in the above-mentioned table 3.
At first prepare the multiple PCR primer working fluid: the primer stock solution 100ul that respectively gets SEQ NO.25～32 respectively mixes and is the multiple PCR primer working fluid in the 1.5ml Eppendorf tube.The multi-PRC reaction system is following:
10 * damping fluid (contains Mg
2+) 5ul
DNTP (each 2.5mmol/L) 4ul
Taq enzyme (5U/ul) 0.5ul
Multiple PCR primer working fluid (each 16.7pmol/mL) 6ul
Template DNA (10ng/ul) 1ul
ddH
2O 33.5ul
Be total to 50ul
The pcr amplification program is: 95 ℃ of 3min; 94 ℃ of 20s, 56 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 10min; 4 ℃ of preservations are subsequent use.
Three, the enzyme of PCR product is cut processing
With reference to the explanation of ExoSAP-IT test kit, detailed step is following:
1. get the reacted product of 7.5ul PCR, add 3ul ExoSAP-IT enzyme;
2.37 ℃ hatch 15min.Hatch 15min for 80 ℃, make unnecessary enzyme-deactivating.The product that enzyme is cut after the processing directly is used for follow-up ASPE primer extension reaction.
Four, site-specific primer extension reaction (ASPE)
Utilize the locus specificity primer of above-mentioned design to carry out primer extension reaction, in reaction process, mix biotin labeled dCTP, thereby make a plurality of biotin labeling on the reacted product band.
At first prepare blended ASPE primer working fluid: respectively get the corresponding ASPE primer of G341R-w and G341R-m, R614C-w and R614C-m, T2206M-w and T2206M-m, G2434R-w and G2434R-m stock solution 10ul respectively in the 1.5ml Eppendorf tube; Add 10mmol/L Tris Buffer and mend, mix and be ASPE mix primer working fluid to 200ul.The system of ASPE reaction is following:
10 * damping fluid 2ul
MgCl
2(50mmol/L) 0.5ul
Biotin-dCTP(400umol/L) 0.25ul
DATP, dGTP, dTTP mixed solution (each 100umol/L) 1ul
Tsp enzyme (5U/ul) 0.25ul
Blended ASPE primer working fluid (each 500nmol/L) 1ul
Enzyme is cut the pcr amplification product 5ul of processing
ddH
2O 10.ul
Be total to 20ul
The PCR program is: 96 ℃ of 2min; 94 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 4 ℃ of preservations are subsequent use.
Five, hybridization
1. according to designed ASPE primers, (microballoon concentration is 2.5 * 10 to select corresponding eight kinds of optimum microballoons
5Individual/ml);
2. the microballoon of getting every kind of numbering of 1ul respectively is in the Eppendorf tube of 1.5ml;
3. microballoon is in >=centrifugal the 1-2min of 10000g;
4. supernatant discarded, microballoon is resuspended in 2 * Tm hybridization buffer of 100ul, the vortex mixing;
5. get the above-mentioned microballoon suspension of 25ul in the corresponding hole of 96 hole filter plates, control wells adds the ddH of 25ul
2O;
6. the ASPE reaction solution of getting 5-25ul is used ddH in corresponding hole
2O complements to 50ul;
7. encase 96 orifice plates with lucifuge with masking foil, 95 ℃ of 60s, 37 ℃ of 15min are hatched hybridization;
8. the microballoon after the hybridization is in >=centrifugal the 2-5min of 3000g;
9. remove supernatant, microballoon is resuspended in 1 * Tm hybridization buffer of 75ul;
10. microballoon is in >=centrifugal the 2-5min of 3000g;
11 are resuspended in microballoon in 1 * Tm hybridization buffer of 75ul, and adding 15ul concentration is Streptavidin-phycoerythrin (SA-PE) of 10ug/ml;
12.37 ℃ hatch 15min, on the Luminex instrument, detect.
Six, the result detects and data analysis
The reaction after product is through Luminex serial analysis instrument detecting.With the carrier of polystyrene microsphere, as detection platform, nucleic acid molecule is carried out high-throughout many indexs parallel detection with fluorescence detector as reaction.In the manufacturing processed of microballoon, mix the ruddiness and the infrared light staining agent of different ratios, thereby form the microballoon of 100 kinds of different colours codings of as many as.Different microballoon covalent attachment to the nucleic acid molecule of different things to be detected as probe molecule, reporter molecules is with biotin labeling, and uses high-sensitive fluorescent dyeing.These microballoons and determinand, reporter molecules, fluorescent marker just form complete microballoon detection architecture and are used for reading of Luminex system.Excitated red respectively laser of Luminex reading system and green laser are used for the detection of microsphere system, and detected result is shown in table 4 and table 5.
Fluorescent value (MFI) and data processing there are following requirement:
1. each site need have an allelotrope MFI at least greater than 300 and greater than 10 * PCR negative control MFI;
2.NET MFI=sample MFI-PCR negative control MFI (NET MFI less than 0 with 0 the expression);
3. satisfy the data of above two conditions, calculate sudden change ratio by following formula:
Sudden change ratio=mutant NET MFI ÷ (mutant NET MFI+ wild-type NET MFI)
4. rule of thumb the sudden change ratio of each detection site is confirmed threshold value (cut-off value), to divide wild-type homozygote, heterozygote and mutant homozygote.
Use present method to detect the RYR1 gene pleiomorphism of great amount of samples, experimental data meets above-mentioned requirements, therefore can calculate their sudden change ratio.Being provided with as follows of threshold value (cut-off value): the sudden change ratio range is regarded as the wild-type homozygote at 0%-20%; 30%-70% is regarded as heterozygote; 80%-100% is regarded as the anomaly homozygote.Compare with the liquid-phase chip result with the PCR sequencing PCR detection, calculate the identical rate of classifying method detected result provided by the present invention.Present method detects 20 increments RYR1 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.It is thus clear that RYR1 gene SNP detection liquid-phase chip provided by the present invention can detect the SNP type of RYR1 gene exactly, and the result is reliable and stable.
Table 4 pattern detection result (MFI)
| Sequence number NO. | G341R-w | G341R-m | R614C-w | R614C-m | T2206M-w | T2206M-m | G2434R-w | G2434R-m |
| Negative control | 13 | 24 | 14 | 24 | 23 | 16 | 12 | 18 |
| 1 | 4296 | 239 | 3532 | 240 | 2605 | 232 | 1530 | 1600 |
| 2 | 2875 | 239 | 4313 | 237 | 3701 | 286 | 2894 | 225 |
| 3 | 2425 | 239 | 2935 | 244 | 3417 | 230 | 2807 | 289 |
| 4 | 3999 | 234 | 2015 | 243 | 3586 | 253 | 4280 | 266 |
| 5 | 3614 | 138 | 1459 | 1286 | 3833 | 236 | 4404 | 179 |
| 6 | 2943 | 248 | 2305 | 248 | 3329 | 257 | 2481 | 265 |
| 7 | 2807 | 242 | 3481 | 235 | 4288 | 188 | 2824 | 246 |
| 8 | 3853 | 148 | 2845 | 245 | 3054 | 287 | 4279 | 235 |
| 9 | 3408 | 238 | 2055 | 246 | 3657 | 235 | 3678 | 232 |
| 10 | 3991 | 238 | 2451 | 247 | 3805 | 239 | 2973 | 255 |
| 11 | 3292 | 144 | 3059 | 243 | 3951 | 258 | 3200 | 239 |
| 12 | 145 | 2914 | 2951 | 244 | 4393 | 240 | 3955 | 235 |
| 13 | 2993 | 144 | 2694 | 244 | 4008 | 189 | 2643 | 238 |
| 14 | 3265 | 243 | 3780 | 326 | 2164 | 309 | 3825 | 237 |
| 15 | 2091 | 229 | 3773 | 146 | 2186 | 302 | 3552 | 230 |
| 16 | 4233 | 239 | 3710 | 147 | 4114 | 216 | 4365 | 240 |
| 17 | 4310 | 225 | 2594 | 226 | 3411 | 228 | 3101 | 258 |
| 18 | 2305 | 238 | 3614 | 227 | 240 | 2055 | 2635 | 251 |
| 19 | 2674 | 248 | 3186 | 226 | 3416 | 223 | 4037 | 237 |
| 20 | 2891 | 236 | 2178 | 248 | 3769 | 235 | 4090 | 282 |
Table 5 sample RYR1 Polymorphism Analysis result
The liquid-phase chip of the ASPE primer that embodiment 3 is different detects the detection of transgenation to the RYR1 gene SNP
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
Detection liquid-phase chip with RYR1 gene G341R site mutation is an example; To the wild-type of G341R and the specific primer sequence of mutant design ASPE primer 3 ' end; The Tag sequence of ASPE primer 5 ' end then is selected from 6 among the SEQ ID NO.1-SEQID NO.8; Accordingly, encapsulate anti-tag sequence selection SEQID NO.17-SEQ ID NO.24 on microballoon and corresponding tag sequence complementary pairing.Specifically design shown in following table (table 6).It is said like embodiment 1 and embodiment 2 that synthetic, the anti-tag sequence of ASPE primer encapsulates microballoon, amplimer, detection method.
The design of table 6 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned designing and preparing, by embodiment 2 said testing processes and method sample 21-40 is detected, detected result is following:
Table 7 pattern detection result (MFI) and Polymorphism Analysis
More than be to the specifying of possible embodiments of the present invention, but this embodiment is not in order to limiting claim of the present invention, does not allly break away from equivalence of the present invention and implement or change, all should be contained in the claim of the present invention.
Sequence table
< 110>Guangzhou Yishan Biotechnology Co., Ltd.
< 120>RYR1 gene SNP detection specific primer, liquid-phase chip and detection method
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<212>DNA
< 213>artificial sequence
<400>28
tcctggcacc?gcaagtct 18
<210>29
<211>22
<212>DNA
< 213>artificial sequence
<400>29
gtcttctacc?aacacccgaa?cc 22
<210>30
<211>18
<212>DNA
< 213>artificial sequence
<400>30
cacgcagcag?ctcaccca 18
<210>31
<211>20
<212>DNA
< 213>artificial sequence
<400>31
tggaggagaa?cgccaatgtg 20
<210>32
<211>19
<212>DNA
< 213>artificial sequence
<400>32
ccgagcaggt?cgatcaagg 19
Claims (6)
1. a RYR1 gene SNP detection liquid-phase chip is characterized in that, mainly includes:
(A). designed ASPE primers is right respectively to the SNP site of RYR1 gene: every kind of ASPE primer is made up of to the specific primer sequence in goal gene SNP site the tag sequence and the 3 ' end of 5 ' end, and said specific primer sequence is respectively: to the SEQ ID NO.9 in G341R site and SEQ ID NO.10, to the SEQ ID NO.11 and the SEQ ID NO.12 in R614C site, be directed against the SEQ ID NO.13 and the SEQ ID NO.14 in T2206M site; And/or to the SEQ ID NO.15 and the SEQ ID NO.16 in G2434R site; Said tag sequence is selected from the sequence among SEQ ID NO.1～SEQ ID NO.8;
(B). be coated with the microballoon of special anti-tag sequence respectively, above-mentioned every kind of microballoon has the different colours coding; Said anti-tag sequence is selected from the sequence among SEQ ID NO.17～SEQ ID NO.24, and said anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). be used for increasing respectively have G341R, the amplimer of the RYR1 gene target sequence in R614C, T2206M and/or G2434R SNP site.
2. RYR1 gene SNP detection liquid-phase chip according to claim 1; It is characterized in that said amplimer is: to the SEQ ID NO.25 in G341R site and SEQ ID NO.26, to the SEQ ID NO.27 in R614C site and SEQ ID NO.28, to the SEQ ID NO.29 in T2206M site and SEQ ID NO.30 and/or to the SEQ ID NO.31 and the SEQ ID NO.32 in G2434R site.
3. RYR1 gene SNP detection liquid-phase chip according to claim 1 and 2; It is characterized in that said ASPE primer is to being: to the sequence of forming by SEQ ID NO.1 and SEQ ID NO.9 in G341R site and the sequence of forming by SEQ ID NO.2 and SEQ IDNO.10, reach the sequence of forming by SEQ ID NO.8 and SEQ ID NO.16 to the sequence of forming by SEQ ID NO.7 and SEQ ID NO.15 that the sequence of being made up of SEQ ID NO.3 and SEQ ID NO.11 in R614C site reaches the sequence be made up of SEQID NO.4 and SEQ ID NO.12, the sequence of being made up of SEQ ID NO.5 and SEQ ID NO.13 that is directed against the T2206M site reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.14 and/or is directed against the G2434R site.
4. RYR1 gene SNP detection liquid-phase chip according to claim 1 is characterized in that, also is provided with the spacerarm sequence in the middle of said anti-tag sequence is connected with microballoon.
5. RYR1 gene SNP detection liquid-phase chip according to claim 4 is characterized in that, said spacerarm sequence is 5-10 T.
6. be used for the Auele Specific Primer that the RYR1 gene SNP detects, it is characterized in that: include: to the SEQ ID NO9 in G341R site and SEQ ID NO.10, to the SEQ ID NO.11 in R614C site and SEQ ID NO.12, to the SEQ ID NO.13 in T2206M site and SEQ ID NO.14 and/or to the SEQ ID NO.15 and the SEQ IDNO.16 in G2434R site.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010148409XA CN101812523B (en) | 2010-04-09 | 2010-04-09 | SNP (Single Nucleotide 0olymorphism) detection specific primer, liquid-phase chip and detection method of RYR1 (Ryanodine Receptors 1) gene |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201010148409XA CN101812523B (en) | 2010-04-09 | 2010-04-09 | SNP (Single Nucleotide 0olymorphism) detection specific primer, liquid-phase chip and detection method of RYR1 (Ryanodine Receptors 1) gene |
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| CN101812523A CN101812523A (en) | 2010-08-25 |
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| CN201010148409XA Expired - Fee Related CN101812523B (en) | 2010-04-09 | 2010-04-09 | SNP (Single Nucleotide 0olymorphism) detection specific primer, liquid-phase chip and detection method of RYR1 (Ryanodine Receptors 1) gene |
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| CN101984072B (en) * | 2010-09-21 | 2013-04-24 | 广州益善生物技术有限公司 | PDGFRA gene mutation detection liquid-phase chip |
| CN103187280A (en) * | 2011-12-29 | 2013-07-03 | 中芯国际集成电路制造(上海)有限公司 | Manufacturing method of fin type field effect transistor |
| CN106222282A (en) * | 2016-08-10 | 2016-12-14 | 北京市畜牧总站 | A kind of test kit and the purposes of the mutation research for blue Buddhist nun's alkali receptor thereof |
| CN109082466A (en) * | 2018-08-21 | 2018-12-25 | 宁波海尔施基因科技有限公司 | A kind of multiple gene detection kit and its application method detecting CACNA1S gene pleiomorphism |
| CN109022555B (en) * | 2018-08-21 | 2021-05-25 | 宁波海尔施基因科技有限公司 | Multiplex gene detection kit for detecting RYR1 gene polymorphism and use method thereof |
| CN109207579B (en) * | 2018-09-06 | 2022-03-22 | 宁波海尔施基因科技股份有限公司 | Multiple detection kit for detecting malignant high fever susceptibility gene and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1516596A (en) * | 2001-05-17 | 2004-07-28 | ���Ĵ���ѧ | Method of modulating activity of calcium channels in cardiac cells and reagents therefor |
| CN101146785A (en) * | 2005-03-23 | 2008-03-19 | 纽约市哥伦比亚大学理事会 | Novel anti-arrythmic and heart failure drugs that target the leak in the ryanodine receptor (RYR2) |
| CN101500576A (en) * | 2005-08-25 | 2009-08-05 | 纽约市哥伦比亚大学理事会 | Agents for preventing and treating disorders involving modulation of the RYR receptors |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1516596A (en) * | 2001-05-17 | 2004-07-28 | ���Ĵ���ѧ | Method of modulating activity of calcium channels in cardiac cells and reagents therefor |
| CN101146785A (en) * | 2005-03-23 | 2008-03-19 | 纽约市哥伦比亚大学理事会 | Novel anti-arrythmic and heart failure drugs that target the leak in the ryanodine receptor (RYR2) |
| CN101500576A (en) * | 2005-08-25 | 2009-08-05 | 纽约市哥伦比亚大学理事会 | Agents for preventing and treating disorders involving modulation of the RYR receptors |
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