CN110066860A - DNA sequencing detects SMA short-cut method and process - Google Patents
DNA sequencing detects SMA short-cut method and process Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 19
- 238000001712 DNA sequencing Methods 0.000 title claims abstract description 12
- 102100021947 Survival motor neuron protein Human genes 0.000 claims abstract description 70
- 101000617738 Homo sapiens Survival motor neuron protein Proteins 0.000 claims abstract description 64
- 208000022074 proximal spinal muscular atrophy Diseases 0.000 claims abstract description 25
- 238000012217 deletion Methods 0.000 claims abstract description 21
- 230000037430 deletion Effects 0.000 claims abstract description 21
- 238000012216 screening Methods 0.000 claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 230000035772 mutation Effects 0.000 claims abstract description 12
- 101150081851 SMN1 gene Proteins 0.000 claims description 38
- 101150113275 Smn gene Proteins 0.000 claims description 24
- 238000012163 sequencing technique Methods 0.000 claims description 17
- 101150015954 SMN2 gene Proteins 0.000 claims description 14
- 201000010099 disease Diseases 0.000 claims description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 6
- 108700024394 Exon Proteins 0.000 claims description 3
- 230000002068 genetic effect Effects 0.000 claims description 3
- 230000001717 pathogenic effect Effects 0.000 claims description 3
- 230000002159 abnormal effect Effects 0.000 claims description 2
- 238000002567 electromyography Methods 0.000 claims description 2
- 235000013399 edible fruits Nutrition 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 10
- 238000011835 investigation Methods 0.000 abstract description 2
- 238000012408 PCR amplification Methods 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 210000002230 centromere Anatomy 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 108091035539 telomere Proteins 0.000 description 2
- 210000003411 telomere Anatomy 0.000 description 2
- 102000055501 telomere Human genes 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 101150050863 T gene Proteins 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 238000007838 multiplex ligation-dependent probe amplification Methods 0.000 description 1
- 208000018360 neuromuscular disease Diseases 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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- 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
- C12Q1/6869—Methods for sequencing
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Abstract
The present invention relates to the short-cut methods and detailed process of detection SMA.The main points of technical solution: PCR product of the DNA sequencing for zygote 6- exon 8 in SMN, SMN1 exon 7 and 8 for example homozygous deletions, the homozygous peak of+6T and the+245A on the 8th exon only on the 7th exon of SMN2；It can be used for the molecular background of screening identical SMN2 copy number and SMA phenodeviation；+ 245 on the 7th exon of SMN on+6 and the 8th exon are when there is set peak, and if phenotype is normally normal person, for example patient can just omit the point mutation of screening SMN1.Main application: it is clear gene level can be carried out to 98.6% patient；Can screening there is the molecular background of identical SMN2 copy number and SMA phenodeviation；Remaining only a few heterozygous deletion and point mutation SMA patient can screening SMN1 point mutation.It is very suitable to different medical unit, and is conducive to the further investigation to this interpretation of the cause, onset and process of an illness system.
Description
Technical field
Medical Genetics
Background technique
Duchenne-Arandisease (SMA) is the most common recessive hereditary neuromuscular disease, and SMN1 gene is SMA
Determine gene, can be caused a disease by micromutation in its gene delection and gene, SMN gene there are the copy of two very high homologies,
Telomere copy (SMN1) and centromere copy (SMN2), SMN2 is modifier, it is now recognized that the 7th exon of SMN1 gene with/
Or the 8th Exon deletion mutation be SMA morbidity the main reason for, there are about 95% I-III type SMA patients homozygous SMN1 the 7th
Exon and/or the 8th Exon deletion.SMN1 and SMN2 gene only have 5 nucleotide differences, be the 6th introne (- 45G →
A), the 7th exon (+6C → T), the 7th introne (+100A → G ,+214A → G), the 8th exon (+245G → A), at SMA
In body, polymerase chain reaction-restriction fragment length polymorphism (polymerase chain is previously used both at home and abroad
Reaction restriction fragment length polymorphism, PCR-RFLP) technology detection SMA SMN1
Gene delection.But the technology is only capable of detecting the disease roughly, cannot reflect the detail of ospc gene exception in detail.It is now domestic
Outer scientific research institution identifies homozygous deletion SMN1 gene patient, to sieve using the SMN1 gene copy number of MLPA technology detection patient
Heterozygous deletion SMN1 gene patient is selected, its SMN gene (including SMN1 and SMN2) is cloned by RT-PCR technology,
Because SMN1 and SMN2 gene are there are the difference of 5 nucleotide sequences, PCR-RFLP can identify positive gram containing SMN1 gene
Grand, by carrying out DNA direct Sequencing, the genome sequence Protocols in Molecular Biologies such as relatively, to detect SMN1 intragenic small prominent
Become.The detection process is cumbersome, and general medical unit is difficult to grasp.
We carry out PCR amplification sequencing for SMN gene extron 7 and 8 and its exon and introne intersection.Detection
SMN1 homozygous mutant gene also can be used for the molecular background that identical SMN2 copy number and SMA phenodeviation occurs in screening, to screening
Heterozygous deletion SMN1 gene patient out, can be by screening the intragenic micromutation of SMN1 to SMN1 gene sequencing.
We summarize and find out simplicity, general, comprehensively, economic SMA patient's detection method and process, because comprising more
SMN gene information can preferably establish genotype and phenotype correlativity, be very suitable to different medical unit, and be conducive to this
The further investigation of the interpretation of the cause, onset and process of an illness.
Summary of the invention
We carry out PCR amplification to zygote 6- exon 8 in SMN gene DNA, and pcr amplification product carries out sequencing analysis, because
There are the copy of two very high homologies, telomeres to copy (SMN1) and centromere copy (SMN2), SMN1 gene for the SMN gene of this disease
It is decided to be SMA and determines gene, for example homozygous deletion of SMN1 gene extron 7 and 8, patient only has on the 7th exon of SMN2 gene
The homozygous peak of+6T and the+245A on the 8th exon show that SMN gene only has SMN2 gene order, and without SMN1 gene order,
As patient has homozygous SMN1 gene the 7th and the 8th Exon deletion, because the I-III type SMA patient there are about 98.6% has homozygosis
The 7th exon of SMN1 and/or the 8th Exon deletion can define 98.6% such patient, while by SMN2 base
Because sequencing analysis can be also found that whether it has SMN2 genetic heterozygosis mononucleotide polymorphic site (single-nucleotide
Polymorphism, SNP), it can be used for the molecular background that identical SMN2 copy number and SMA phenodeviation occurs in screening, SMN base
Because+245 after sequencing on the 7th exon of SMN gene on+6 and the 8th exon are when there is set peak, if phenotype is normal
It is exactly normal person, if any SMA phenotype, then patient need to carry out SMN gene (including SMN1 and SMN2) cloning and sequencing and find SMN1 gene
Point mutation, such as with DNA sequencing detection SMN gene can just omit screening SMN1 gene point mutation situation.
DNA sequencing detects SMA method and can define to 98.6% such patient progress gene level, while by homozygosis
The SMN2 gene sequencing analysis of missing SMN1 gene patient can be also found that whether it has SMN2 gene SNP, and remaining only a few is miscellaneous
Close missing and point mutation SMA patient can also rough screening SMN1 gene point mutation situation, therefore this method relative ease is general,
Comprehensively, economical.
Detailed description of the invention
The sequencing discovery of Fig. 1 homozygous deletion SMN1 gene patient's SMN gene forward direction only has+6T on the 7th exon of SMN2 gene
Homozygous peak figure (figure A) ,+245A homozygosis peak figure (figure B) (arrow shows) on the 8th exon of SMN2；
There are heterozygosis 835-262C > T gene polymorphic positions for the 7th introne of Fig. 2 homozygous deletion SMN1 gene patient SMN2 gene
Point diagram (arrow shows)
The sequencing discovery of Fig. 3 normal person's SMN gene forward direction has the set peak figure (figure of+6T on the 7th exon SMN1+6C and SMN2
A), on the 8th exon the+245A of SMN1+245G and SMN2 set peak figure (figure B) (arrow shows)
Discovery is sequenced in patient's SMN gene forward direction that Fig. 4 heterozygous deletion SMN1 gene merges the point mutation of heterozygosis SMN1 gene
Have the set peak figure (figure A) of+6T and SMN1+6C on the 7th exon SMN2, on the 8th exon of backward sequencing+the 245A of SMN2 with
The set peak figure (figure B) of SMN1+245G, the 5th exon of SMN1 gene c.683T > A (p.Leu228X) heterozygous mutant is sequenced in forward direction
Figure (figure C) (arrow shows)
Specific embodiment
We carry out PCR amplification to zygote 6- exon 8 in SMN gene DNA, and pcr amplification product carries out sequencing analysis, always
Knot finds out DNA sequencing detection SMA short-cut method and process.
One, PCR amplification
1, primer sequence and synthesis
PCR amplification is carried out for zygote 6- exon 8 in SMN gene, is synthesized by Invitrogen company, each primer sequence
Column, see Table 1 for details for amplified fragments and annealing temperature.
Table 1, primer condition and product
2, PCR reaction system
React 25 μ l of total volume, comprising:
The ddH of HIGH PRESSURE TREATMENT is added2O to 25.0 μ l
The preparation of PCR reaction system operates on ice, is eventually adding Taq archaeal dna polymerase, dispenses after mixing.
3, PCR reaction condition
PCR amplification condition be 94 DEG C of initial denaturations 7min, 94 DEG C of 45sec, 56 DEG C of 45sec, 72 DEG C of 1min, recycle 30 times, most
Afterwards in 72 DEG C of extension 10min.
4, the inspection of pcr amplification product
PCR after reaction, takes 2 μ l of PCR product, with the pre- dye liquor of 6 × Loading buffer (in 1,/10 000 ratio
Gene finder dyestuff is added) 1 μ l adds to after mixing in 2% Ago-Gel loading hole, with the electrophoresis of 6-8V/cm
40 minutes.Gel scanning imagery in automatic gel imaging system, if electrophoresis banding pattern is clear, no miscellaneous band if, shows PCR amplification
Success.
Two, DNA sequencing detection SMA method and process
DNA sequencing is surveyed for the PCR product application double deoxidation chain termination method of zygote 6- exon 8 in SMN gene
Sequence (American AB I company 3730XL sequenator).The for example homozygous deletion of SMN1 gene extron 7 and 8, patient only have SMN2 gene
The homozygous sequencing peak of+6T and+245A on the 8th exon on 7 exons shows that SMN gene only has SMN2 gene order, and nothing
SMN1 gene order, as patient have homozygous SMN1 gene the 7th and the 8th Exon deletion, detect and successfully see Fig. 1.
It, can because there are about 98.6% I-III type SMA patients homozygous the 7th exon of SMN1 and/or the 8th Exon deletion
It is clear to 98.6% such patient progress gene level, while by can be also found that whether it has to the analysis of SMN2 gene sequencing
There is SMN2 genetic heterozygosis mononucleotide polymorphic site (single-nucleotide polymorphism, SNP), successfully detects
See Fig. 2, can be used for the molecular background that identical SMN2 copy number and SMA phenodeviation occurs in screening.
+ 245 after SMN gene sequencing on the 7th exon of SMN gene on+6 and the 8th exon when occurring covering peak,
If phenotype is normally exactly normal person, Fig. 3 is seen.For example patient's (abnormal electromyography and/or have clinical manifestation person) is using DNA sequencing
The point mutation situation of screening SMN1 gene can just be omited (because SMN1 gene is Disease-causing gene, heterozygosis by detecting all exons of SMN gene
Mutation has pathogenic meaning, can speculate pathogenic sites on SMN1 gene), tentatively screening it can go out heterozygous deletion merging heterozygosis SMN1
The patient of point mutation, successfully Fig. 4 is shown in detection.
Claims (3)
1.DNA sequencing is sequenced for the PCR product application double deoxidation chain termination method of zygote 6- exon 8 in SMN gene,
The for example homozygous deletion of SMN1 gene extron 7 and 8, patient only have on the 7th exon of SMN2 gene on+6T and the 8th exon+
The homozygous sequencing peak of 245A, shows that SMN gene only has SMN2 gene order, and without SMN1 gene order, as patient has homozygosis
It is clear can to carry out gene level to 98.6% I-III type SMA patient for SMN1 gene the 7th and the 8th Exon deletion.
2. being directed to the homozygous deletion patient of SMN1 gene extron 7 and 8, whether DNA sequencing SMN2 gene extron 1-8 can have found it
With SMN2 genetic heterozygosis mononucleotide polymorphic site, it can be used for screening and identical SMN2 copy number and SMA phenodeviation occur
Molecular background.
+ 245 after 3.SMN gene sequencing on the 7th exon of SMN gene on+6 and the 8th exon are when occurring covering peak, such as
Fruit phenotype is normally exactly normal person, and for example patient's (abnormal electromyography and/or have clinical manifestation person) detects SMN using DNA sequencing
All exons of gene can just slightly (because SMN1 gene is Disease-causing gene, heterozygous mutant has the point mutation situation of screening SMN1 gene
Cause a disease meaning, can speculate pathogenic sites on SMN1 gene), can preliminary screening go out heterozygous deletion to merge heterozygosis SMN1 gene point prominent
The patient of change.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111292804A (en) * | 2020-04-08 | 2020-06-16 | 北京智因东方转化医学研究中心有限公司 | Method and system for detecting SMN1 gene mutation by means of high-throughput sequencing |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058575A (en) * | 2017-05-27 | 2017-08-18 | 郑州大学第附属医院 | It is a kind of to be used to expand the PCR primer pair of SMN1 genes and the kit for detecting SMN1 point mutations |
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2018
- 2018-01-21 CN CN201810148922.5A patent/CN110066860A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058575A (en) * | 2017-05-27 | 2017-08-18 | 郑州大学第附属医院 | It is a kind of to be used to expand the PCR primer pair of SMN1 genes and the kit for detecting SMN1 point mutations |
Non-Patent Citations (3)
| Title |
|---|
| ELENA BUSSAGLIA等: "A frame-shift deletion in the survival motor meuron gene in Spanish spina muscular atrophy patients", 《NATURE GENETICS》 * |
| W.L. LIU等: "Molecular analysis of the SMN gene mutations in spinal muscular atrophy patients in China", 《GENETICS AND MOLECULAR RESEARCH》 * |
| 杨兰等: "Sanger测序对运动神经元存活基因1复合杂合突变型脊髓性肌萎缩症的诊断价值", 《中华医学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111292804A (en) * | 2020-04-08 | 2020-06-16 | 北京智因东方转化医学研究中心有限公司 | Method and system for detecting SMN1 gene mutation by means of high-throughput sequencing |
| CN111292804B (en) * | 2020-04-08 | 2021-11-26 | 北京智因东方诊断科技有限公司 | Method and system for detecting SMN1 gene mutation by means of high-throughput sequencing |
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Effective date of registration: 20190930 Address after: 550004 Department of Pediatrics, Affiliated Hospital of Guizhou Medical University, 28 Guiyi Street, Guiyang City, Guizhou Province Applicant after: Liu Weiliang Applicant after: He Zhixu Applicant after: Li Fang Applicant after: THE AFFILIATED HOSPITAL OF GUIZHOU MEDICAL University Address before: 550004 Department of Pediatrics, Affiliated Hospital of Guizhou Medical University, 28 Guiyi Street, Guiyang City, Guizhou Province Applicant before: Liu Weiliang Applicant before: He Zhixu Applicant before: Li Fang |
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