CN109355371A - It is a kind of for detecting the kit of MMACHC gene mutation - Google Patents
It is a kind of for detecting the kit of MMACHC gene mutation Download PDFInfo
- Publication number
- CN109355371A CN109355371A CN201811370750.2A CN201811370750A CN109355371A CN 109355371 A CN109355371 A CN 109355371A CN 201811370750 A CN201811370750 A CN 201811370750A CN 109355371 A CN109355371 A CN 109355371A
- Authority
- CN
- China
- Prior art keywords
- kit
- mmachc
- mutation
- gene
- insa
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 101150071413 MMACHC gene Proteins 0.000 title claims abstract description 52
- 206010064571 Gene mutation Diseases 0.000 title claims abstract description 12
- 101150039072 INSA gene Proteins 0.000 claims abstract description 31
- 230000035772 mutation Effects 0.000 claims abstract description 22
- 108091008146 restriction endonucleases Proteins 0.000 claims abstract description 21
- 230000003321 amplification Effects 0.000 claims abstract description 13
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 13
- 238000001514 detection method Methods 0.000 claims abstract description 12
- 230000029087 digestion Effects 0.000 claims description 27
- 238000012408 PCR amplification Methods 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 9
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 8
- 238000013467 fragmentation Methods 0.000 claims description 6
- 238000006062 fragmentation reaction Methods 0.000 claims description 6
- 238000001502 gel electrophoresis Methods 0.000 claims description 6
- 102100023381 Cyanocobalamin reductase / alkylcobalamin dealkylase Human genes 0.000 claims description 5
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 5
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 5
- 101710164985 Cyanocobalamin reductase / alkylcobalamin dealkylase Proteins 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 238000012216 screening Methods 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 10
- 230000000869 mutational effect Effects 0.000 description 8
- 238000012163 sequencing technique Methods 0.000 description 6
- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 201000003694 methylmalonic acidemia Diseases 0.000 description 3
- 101150058299 Cblc gene Proteins 0.000 description 2
- 102100023470 Cobalamin trafficking protein CblD Human genes 0.000 description 2
- 101000941071 Homo sapiens Lysosomal cobalamin transport escort protein LMBD1 Proteins 0.000 description 2
- 102100031335 Lysosomal cobalamin transport escort protein LMBD1 Human genes 0.000 description 2
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000005515 coenzyme Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- OAJLVMGLJZXSGX-CXGXMSGESA-L (2r,3r,4s,5r)-2-(6-aminopurin-9-yl)-5-methanidyloxolane-3,4-diol;cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] 1-[3-[(4z,9z,14z)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8 Chemical compound [Co+3].O[C@@H]1[C@H](O)[C@@H]([CH2-])O[C@H]1N1C2=NC=NC(N)=C2N=C1.O([C@H]1[C@H]([C@H](O[C@@H]1CO)N1C2=CC(C)=C(C)C=C2N=C1)O)P([O-])(=O)OC(C)CNC(=O)CCC1(C)C(CC(N)=O)C2[N-]\C1=C(C)/C(C(C\1(C)C)CCC(N)=O)=N/C/1=C\C(C(C/1(CC(N)=O)C)CCC(N)=O)=N\C\1=C(C)/C1=NC2(C)C(C)(CC(N)=O)C1CCC(N)=O OAJLVMGLJZXSGX-CXGXMSGESA-L 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 102100023376 Corrinoid adenosyltransferase Human genes 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 101001114650 Homo sapiens Corrinoid adenosyltransferase Proteins 0.000 description 1
- 101001013648 Homo sapiens Methionine synthase Proteins 0.000 description 1
- 101001116314 Homo sapiens Methionine synthase reductase Proteins 0.000 description 1
- 101001114654 Homo sapiens Methylmalonic aciduria type A protein, mitochondrial Proteins 0.000 description 1
- 102100031551 Methionine synthase Human genes 0.000 description 1
- 102100024614 Methionine synthase reductase Human genes 0.000 description 1
- 102100023377 Methylmalonic aciduria type A protein, mitochondrial Human genes 0.000 description 1
- 102000019010 Methylmalonyl-CoA Mutase Human genes 0.000 description 1
- 108010051862 Methylmalonyl-CoA mutase Proteins 0.000 description 1
- 108020004485 Nonsense Codon Proteins 0.000 description 1
- 208000033716 Organic aciduria Diseases 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- -1 cblD Proteins 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- JEWJRMKHSMTXPP-BYFNXCQMSA-M methylcobalamin Chemical compound C[Co+]N([C@]1([H])[C@H](CC(N)=O)[C@]\2(CCC(=O)NC[C@H](C)OP(O)(=O)OC3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)C)C/2=C(C)\C([C@H](C/2(C)C)CCC(N)=O)=N\C\2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O JEWJRMKHSMTXPP-BYFNXCQMSA-M 0.000 description 1
- 235000007672 methylcobalamin Nutrition 0.000 description 1
- 239000011585 methylcobalamin Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 208000037890 multiple organ injury Diseases 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000037434 nonsense mutation Effects 0.000 description 1
- 201000008152 organic acidemia Diseases 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of for detecting the kit of MMACHC gene mutation, includes: MMACHC gene-specific amplification primer and BmtI restriction endonuclease in the kit；The sequence of the MMACHC gene-specific amplification primer is as shown in SEQ ID NO.1-SEQ ID NO.2.Using kit of the invention, to MMACHC gene, c.492_493 insA mutation is detected, compared with other detection methods, its is at low cost, easy to operate, result interpretation is intuitive, accuracy rate is high, to equipment and operator without particular/special requirement, suitable for general hospital and the screening that c.492_493 insA is mutated of molecular biosciences labs MMACHC gene.
Description
Technical field
The present invention relates to detection in Gene Mutation technical fields, and in particular to a kind of for detecting the examination of MMACHC gene mutation
Agent box.
Background technique
Methylmalonic acidemia (methylmalonic academia, MMA) is most normal in congenital Organic aciduria
The disease seen, mainly due to methylmalonyl-CoA isomerase (mutaseapoenzyme, mut) self-defect or its coenzyme
Caused by cobalamin (Cobalamin, Cbl, VitB12) metabolic deficiency.Conjugating enzyme defect includes complete defect mut0 type, and part lacks
Fall into mut- type.Cobalamin metabolism defect includes 8 kinds of hypotypes of cblA, cblB, cblC, cblD, cblE, cblF, cblG and cblH,
Wherein cblC, cblD and cblF can lead to coenzyme deoxyadenosyl cobalamin and methyl cobalamin dyssynthesis, cause internal methyl
The metabolins such as malonic acid, homocysteine are accumulated extremely, therefore referred to as methylmalonic acidemia is with plasma homocysteine (letter
Claim combination type MMA).Clinical manifestation is the multiple organ injuries such as nerve, liver, kidney, marrow.
CblC type is the most common type in cobalamin metabolism obstacle, and encoding gene MMACHC is positioned at chromosome
1p34.1 contains 5 exons, and long 10736bp encodes 282 amino acid, has reported more than 40 mutation, mostly missense mutation and
Nonsense mutation.
Mainly there are PCR sequencing PCR and ARMS-PCR method for the detection method of gene mutation at present, but both methods is all deposited
Certain defect wherein, wherein PCR sequencing PCR sensitivity lower about 20%, and complicated for operation, detection time is longer, false negative rate compared with
It is high.ARMS-PCR method can reach 1% sensitivity, but testing cost is excessively high, limit its clinical promotion and application.
Summary of the invention
The present inventor has found a new mutation site c.492_493 insA from MMACHC gene first, and
MMACHC gene new mutation site is determined that it is, the generally patient with the mutation moderate occurs to severe cobalamin metabolism barrier
Hinder.Based on this discovery, inventor further devises the kit of specific primer and the application primer.Utilize the kit
The MMACHC gene c.492_493 mutational site insA can fast and accurately be detected.Thus the invention proposes following technical sides
Case:
The first aspect of the present invention, provide it is a kind of for detecting the kit of MMACHC gene mutation, in the kit
It include: MMACHC gene-specific amplification primer and BmtI restriction endonuclease；
The sequence of the MMACHC gene-specific amplification primer is as shown in SEQ ID NO.1-SEQ ID NO.2.
Further, in the kit further include: PCR reaction solution, digested liquid, positive reference product and negative reference product.
Preferably, containing in the PCR reaction solution of every 10 μ L: archaeal dna polymerase 0.25-1U, dNTPs0.5-1.5mM,
MgCl21-4mM。
It is furthermore preferred that containing in the PCR reaction solution of every 10 μ L: archaeal dna polymerase 0.5U, dNTPs 1.0mM, MgCl2 3mM。
Preferably, the digested liquid is l0 × Buffer.
Preferably, the positive reference product are that c.492_493 digestion can occur for insA mutation containing MMACHC gene
DNA fragmentation.
Preferably, the negative reference product are that c.492_493 digestion cannot occur for insA mutation without MMACHC gene
DNA fragmentation.
The second aspect of the present invention provides the method using kit detection MMACHC gene mutation, including following step
It is rapid:
(1) sample to be tested DNA is extracted, as template；
(2) PCR amplification is carried out using MMACHC gene-specific amplification primer pair template, obtains pcr amplification product；
(3) pcr amplification product is subjected to digestion with BmtI restriction endonuclease, obtains digestion products；
(4) digestion products are subjected to gel electrophoresis, are determined according to gel electrophoresis result；If there is 159bp and 113bp
Two band then determine that sample to be tested contains the c.492_493 insA mutation of MMACHC gene；If there is mono- band of 271bp,
Then without containing MMACHC gene, c.492_493 insA is mutated sample to be tested.
Preferably, in step (2), the condition of PCR amplification are as follows: 95 DEG C of initial denaturation 10min；Subsequent 94 DEG C of 15s, 62 DEG C
20s, 70 DEG C of 25s, totally 30 recycle.
When using kit detection MMACHC gene of the invention, c.492_493 insA is mutated, binding specificity PCR skill
Art and enzyme incision technology, every part of sample carry out PCR amplification with specific primer respectively, then carry out the digestion at mutational site, lead to
The c.492_493 insA mutation of MMACHC gene can be detected in a short time by crossing a PCR.MMACHC gene of the invention
Specificity amplification primer be it is specially designed, 3 ' ends of the specificity amplification primer are mutating alkali yl, and the Tm value of primer is low
4-10 DEG C of renaturation temperature is reacted in PCR, improves the specificity of abrupt climatic change.In addition, the choosing for restriction enzyme type
Select it is also very crucial, different types of restriction enzyme have different recognition sites, in selectional restriction restriction endonuclease need
Comprehensively consider the factors such as the sequence of PCR product and mutational site sequence after expanding, guarantees normal different between mutated gene
The site that base is formed specificity limitation restriction endonuclease during digestion is different, to improve the specificity of digestion；It is also contemplated that
Wild type and saltant type are easy to distinguish after digestion.The selected restriction enzyme of the present invention is BmtI restriction endonuclease, is known
Other site are as follows:
For the MMACHC gene of c.492_493 insA mutation, a restriction enzyme can be generated at mutational site
The restriction enzyme site of enzyme BmtI can generate two band of 159bp and 113bp when carrying out digestion using kit of the invention；And it is right
In the MMACHC gene without c.492_493 insA mutation, the restriction enzyme site of restriction enzyme BmtI is not present, it can not
It is digested, therefore what is generated is mono- band of 271bp.This three band is different in size, and difference is obvious, therefore is easy to pass through gel
Electrophoresis is determined.
Further include having positive reference product and negative reference product in kit of the invention, can be used as point of entire PCR reaction
Sub- internal control index improves the stability of testing result to avoid the appearance of false negative result.
Beneficial effects of the present invention:
Using kit of the invention, to MMACHC gene, c.492_493 insA mutation is detected, and is detected with others
Method is compared, at low cost, easy to operate, result interpretation is intuitive, accuracy rate is high, to equipment and operator without particular/special requirement,
Suitable for the screening that c.492_493 insA is mutated of general hospital and molecular biosciences labs MMACHC gene.
Detailed description of the invention
C.492_493, Fig. 1: MMACHC gene locates sequencing peak potion and divides screenshot；Peak figure shows that c.492_493 MMACHC gene is located
Insertion mutation has occurred.
The gel electrophoresis figure of Fig. 2: digestion identification of M MACHC gene c.492_493 insA catastrophe；Wherein, M is
Marker, swimming lane 1-8 are band of the PCR product of sample 1-8 after digestion.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
First choice of the invention merges plasma homocysteine infants to 50 methylmalonic acidemias and has carried out MMACHC base
Because exon is sequenced, it was found that a new mutational site on MMACHC gene c.492_493 insA, by 50 just
The direct Sequencing of normal control group corresponding site is identified, it was demonstrated that it is new mutational site, and mutational site is c.492_493
The Sequencing chromatogram of insA is shown in Fig. 1.
After above-mentioned mutational site has been determined, inventor has further designed and developed detection MMACHC gene mutation position
Put the kit of c.492_493 insA.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
The test material that test material is this field routine is not specifically described used in the embodiment of the present invention,
It can be commercially available by commercial channel.
Embodiment 1: the detection MMACHC gene kit that c.492_493 insA is mutated
The composition of kit:
(1) (concentration of upstream primer and downstream primer is 10 μM to MMACHC gene-specific amplification primer, independent packaging
Or hybrid packed):
Upstream primer: 5 '-CAGGCTAGCTGCATGATTGA-3 ' (SEQ ID NO.1)；
Downstream primer: 5 '-GCTACTCGAAGGCAATTTC-3 ' (SEQ ID NO.2).
(2) PCR reaction solution: contain in the PCR reaction solution of every 10 μ L: archaeal dna polymerase 0.5U, dNTPs 1.0mM,
MgCl23mM。
(3) BmtI restriction endonuclease.
(4) digested liquid: l0 × Buffer.
(5) positive reference product and negative reference product: positive reference product are that c.492_493 insA is prominent containing MMACHC gene
Become the DNA fragmentation that digestion can occur；Negative reference product are that c.492_493 enzyme cannot occur for insA mutation without MMACHC gene
The DNA fragmentation cut.
MMACHC gene-specific amplification primer, PCR reaction solution, BmtI restriction endonuclease, digested liquid, positive reference in kit
Product and negative reference product are independent packaging.
The detection that c.492_493 insA is mutated of embodiment 2:MMACHC gene
Using embodiment 1 kit to MMACHC gene c.492_493 insA mutation detect, specific steps are such as
Under:
1. the extraction of sample DNA:
It is extracted using genomic DNA of the DNA extraction kit to sample to be tested, using the sample DNA of extraction as mould
Plate.
2.PCR amplification:
It is pressed using the MMACHC gene-specific amplification primer (shown in SEQ ID NO.1-SEQ ID NO.2) in kit
PCR amplification is carried out according to following system and program:
PCR reaction system (20 μ L):
PCR response procedures:
95 DEG C of initial denaturation 10min；Subsequent 94 DEG C of 15s, 62 DEG C of 20s, 70 DEG C of 25s, totally 30 recycle；4 DEG C of preservations.
Whether the size with 1% Ago-Gel detection PCR product is correct, as a result obtains target PCR product.
3. digestion is identified:
PCR product is subjected to digestion with restriction enzyme BmtI, digestion system is as follows:
By above-mentioned digestion system in 37 DEG C of heat preservation 4h.
Digestion products are splined on 3% agarose gel electrophoresis and are detected, and restriction enzyme BmtI can identify 5 ' GC
3 ' sequence of TAG/C, when c.492_493 insA mutation occurs for MMACHC gene, restriction enzyme BmtI can be produced PCR
Object segment is cut into two bands of 159bp and 113bp；And when not mutating, restriction enzyme site is not contained, cannot be cut, glue figure is aobvious
Show the band of only one 271bp.
As a result as shown in Fig. 2, sample 1 has mono- band of 271bp after digestion, illustrate c.492_ MMACHC gene does not occur
493 insA mutation.Sample 2-8 has two bands of 159bp and 113bp after digestion, illustrates c.492_ MMACHC gene occurs
493 insA mutation.
For the accuracy of verification result, PCR product is sequenced for we, sequencing result and digestion result complete one
It causes.This, which is sufficiently demonstrated, utilizes kit detection MMACHC gene reliability and standard that c.492_493 insA is mutated of the invention
True property.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
Sequence table
<110>it opens intelligent
<120>a kind of for detecting the kit of MMACHC gene mutation
<130> 2018
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence ()
<400> 1
caggctagct gcatgattga 20
<210> 2
<211> 19
<212> DNA
<213>artificial sequence ()
<400> 2
gctactcgaa ggcaatttc 19
Claims (8)
1. a kind of for detecting the kit of MMACHC gene mutation, which is characterized in that include: MMACHC base in the kit
Because of specificity amplification primer and BmtI restriction endonuclease；
The sequence of the MMACHC gene-specific amplification primer is as shown in SEQ ID NO.1-SEQ ID NO.2.
2. kit according to claim 1, which is characterized in that in the kit further include: PCR reaction solution, digestion
Liquid, positive reference product and negative reference product.
3. kit according to claim 2, which is characterized in that contain in the PCR reaction solution of every 10 μ L: archaeal dna polymerase
0.25-1U、dNTPs0.5-1.5mM、MgCl2 1-4mM。
4. kit according to claim 3, which is characterized in that contain in the PCR reaction solution of every 10 μ L: archaeal dna polymerase
0.5U、dNTPs 1.0mM、MgCl2 3mM。
5. kit according to claim 2, which is characterized in that the positive reference product are to contain MMACHC gene
C.492_493 the DNA fragmentation of digestion can occur for insA mutation.
6. kit according to claim 2, which is characterized in that the negative reference product are without MMACHC gene
C.492_493 the DNA fragmentation of digestion cannot occur for insA mutation.
7. utilizing the method for kit described in any one of claims 1-6 detection MMACHC gene mutation, which is characterized in that packet
Include following steps:
(1) sample to be tested DNA is extracted, as template；
(2) PCR amplification is carried out using MMACHC gene-specific amplification primer pair template, obtains pcr amplification product；
(3) pcr amplification product is subjected to digestion with BmtI restriction endonuclease, obtains digestion products；
(4) digestion products are subjected to gel electrophoresis, are determined according to gel electrophoresis result；If there is 159bp and 113b p two
Band then determines that sample to be tested contains the c.492_493 insA mutation of MMACHC gene；If there is mono- band of 271bp,
Without containing MMACHC gene, c.492_493 insA is mutated sample to be tested.
8. the method according to the description of claim 7 is characterized in that in step (2), the condition of PCR amplification are as follows: 95 DEG C of initial denaturations
10min；Subsequent 94 DEG C of 15s, 62 DEG C of 20s, 70 DEG C of 25s, totally 30 recycle.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811370750.2A CN109355371A (en) | 2018-11-18 | 2018-11-18 | It is a kind of for detecting the kit of MMACHC gene mutation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811370750.2A CN109355371A (en) | 2018-11-18 | 2018-11-18 | It is a kind of for detecting the kit of MMACHC gene mutation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109355371A true CN109355371A (en) | 2019-02-19 |
Family
ID=65345634
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811370750.2A Pending CN109355371A (en) | 2018-11-18 | 2018-11-18 | It is a kind of for detecting the kit of MMACHC gene mutation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109355371A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628892A (en) * | 2019-09-10 | 2019-12-31 | 上海浦东解码生命科学研究院 | Kit for detecting methylmalonic acidemia disease-causing gene mutation |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105297145A (en) * | 2015-11-06 | 2016-02-03 | 艾吉泰康生物科技(北京)有限公司 | Inherited metabolic disease screening method and reagent kit |
| CN106591273A (en) * | 2016-12-07 | 2017-04-26 | 中国人民解放军陆军总医院 | Gene new mutations relevant to IEM (Inborn Errors of Metabolism) and detection kit |
| CN108504730A (en) * | 2017-12-26 | 2018-09-07 | 北京金准基因科技有限责任公司 | MMACHC, MTHFR gene mutation detection kit |
-
2018
- 2018-11-18 CN CN201811370750.2A patent/CN109355371A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105297145A (en) * | 2015-11-06 | 2016-02-03 | 艾吉泰康生物科技(北京)有限公司 | Inherited metabolic disease screening method and reagent kit |
| CN106591273A (en) * | 2016-12-07 | 2017-04-26 | 中国人民解放军陆军总医院 | Gene new mutations relevant to IEM (Inborn Errors of Metabolism) and detection kit |
| CN108504730A (en) * | 2017-12-26 | 2018-09-07 | 北京金准基因科技有限责任公司 | MMACHC, MTHFR gene mutation detection kit |
Non-Patent Citations (10)
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628892A (en) * | 2019-09-10 | 2019-12-31 | 上海浦东解码生命科学研究院 | Kit for detecting methylmalonic acidemia disease-causing gene mutation |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hadd et al. | Targeted, high-depth, next-generation sequencing of cancer genes in formalin-fixed, paraffin-embedded and fine-needle aspiration tumor specimens | |
| CN104818320B (en) | Primer, probe, detection architecture and the kit of disposable detection lung cancer multiple gene | |
| US20210108256A1 (en) | Genomic alterations in the tumor and circulation of pancreatic cancer patients | |
| Malik et al. | A new era of prostate cancer precision medicine | |
| CN102586420B (en) | Method and kit for assaying breast cancer susceptibility genes | |
| CN111187839B (en) | Application of m5C methylation related regulatory gene in liver cancer prognosis prediction | |
| CN105861642B (en) | A kind of acipenser schrencki gender gap opposite sex molecular labeling and detection acipenser schrencki property method for distinguishing | |
| CN102586423B (en) | Method and kit for detecting susceptibility gene of colorectal cancer | |
| Sun et al. | An integrated epigenomic-transcriptomic landscape of lung cancer reveals novel methylation driver genes of diagnostic and therapeutic relevance | |
| CN105274094B (en) | SNP marker and its application | |
| CN104024426B (en) | The method for tracing of testing sample and detection kit in the secondary sequencing technologies of DNA | |
| CN106048008A (en) | EGFR gene ultralow frequency mutation detection kit | |
| Galindo-González et al. | Ion Torrent sequencing as a tool for mutation discovery in the flax (Linum usitatissimum L.) genome | |
| KR20200001159A (en) | DNA marker for discrimination of spicy taste degree in pepper pericarp and uses thereof | |
| CN107090494A (en) | The molecular labeling related to millet code grain number character and its detection primer and application | |
| Qiu et al. | Development of new PCR-based markers specific for chromosome arms of rye (Secale cereale L.) | |
| CN107164513A (en) | Mankind's polygenic mutation detection kit based on high-flux sequence method | |
| KR101778877B1 (en) | Markers for discrimination of Whangkeumbae and Minibae | |
| Liu et al. | Identification of medical plants of 24 Ardisia species from China using the matK genetic marker | |
| Ramani et al. | Factors impacting clinically relevant RNA fusion assays using next-generation sequencing | |
| CN107090450A (en) | The molecular labeling related to millet spike length character and its detection primer and application | |
| CN109355371A (en) | It is a kind of for detecting the kit of MMACHC gene mutation | |
| Silva et al. | Identification of SSR and RAPD markers linked to a resistance allele for angular leaf spot in the common bean (Phaseolus vulgaris) line ESAL 550 | |
| Chandra et al. | Smart breeding: molecular interventions and advancements for crop improvement | |
| BR112012014466B1 (en) | method for detecting a mutation using a DNA microarray showing a plurality of polynucleotide probes immobilized on the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20210511 Address after: 250000 No. 23976 Jingshi Road, Huaiyin District, Jinan City, Shandong Province Applicant after: JINAN CHILDREN'S HOSPITAL (QILU CHILDREN'S HOSPITAL OF SHANDONG University) Address before: No. 430, Jingshi Road, Huaiyin District, Jinan City, Shandong Province Applicant before: Zhang Kaihui |
|
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190219 |