CN107287333A - A kind of human mitochondrial total length haplotype is determined and Genetic Variation Analysis method - Google Patents
A kind of human mitochondrial total length haplotype is determined and Genetic Variation Analysis method Download PDFInfo
- Publication number
- CN107287333A CN107287333A CN201710654472.2A CN201710654472A CN107287333A CN 107287333 A CN107287333 A CN 107287333A CN 201710654472 A CN201710654472 A CN 201710654472A CN 107287333 A CN107287333 A CN 107287333A
- Authority
- CN
- China
- Prior art keywords
- total length
- haplotype
- determined
- sequence
- mitochondrial
- 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
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
- C12Q1/6869—Methods for sequencing
Abstract
Determined the invention discloses a kind of human mitochondrial total length haplotype and Genetic Variation Analysis method, this method is mainly expands the disposable template for obtaining homo mitochondrion gene group by primer, then mitochondria full length sequence is obtained by SMRT sequencing technologies, the sequence of acquisition is subjected to bioinformatic analysis again, so as to obtain the information that makes a variation.Advantage of the invention based on SMRT sequencing technologies, the mitochondria total length haplotype of the homo mitochondrion gene group of acquisition can be not only measured, and analyzed by sequence polymorphism etc., more more valuable mitochondrial inheritance variation information can be obtained, are had great importance to making accurate etiological diagnosis aspect applied to human diseases.Therefore, method of the invention is applicable to the demand determined in the measure demand of mankind institute mitochondrial relevant disease DNA total length haplotypes and medical jurisprudence research application to mitochondria total length haplotype.
Description
Technical field：
The invention belongs to technical field of molecular biology, and in particular to one kind obtains homo mitochondrion gene group and line grain
The measure of body total length haplotype and the method for analysis of variance.
Background technology：
Mitochondria is intracellular energy plants, and it has a set of DNA (mtDNA) of itself, is passed by the ovum of mother
Pass the next generation.In addition to red blood cell, it is present in each cell in human body, and mitochondrial major function is to provide
Energy-triphosphopyridine nucleotide (ATP) required for cell.Human mitochondrial DNA (mtDNA), altogether comprising 37 genes, this 37 bases
There are 22 coding transfer ribonucleic acids (tRNA), 2 encoding ribosomal ribonucleic acid (12S and 16S rRNA), 13 volumes because in
Code polypeptide.Mitochondria is related to many human diseases, and mitochondrial disease is due to that mitochondrial function is abnormal and lead
The some diseases of cause, the mitochondrial mutations occurred in ovum may cause maternal family's property disease.This mutation can cause sternly
The problem of weight, it is impacted it is most of be the high organ of energy requirement, such as muscle, brain, heart, be related to it is a series of extensively
Disease in Infants hereditary disease mitochondrial disease, current clinic can only select a small number of common chondriogen sites to be mutated and lacked
Examination is lost, positive rate is very low, and Most patients are difficult to obtain accurate etiological diagnosis.
Mitochondria is not only closely bound up with many human diseases, and in the research of mankind's maternal origin, medical jurisprudence, archaeology
Being studied in terms of all has important influence.In order to obtain more more valuable information, mitochondria full genome can be utilized
Group haplotype information is an important step.Traditional method is to expand a bit of, a bit of progress of mitochondrial genomes,
Then tens pairs of primers are needed in splicing sequencing, generation sequencing procedure, relatively time consuming laborious, the sequencing of two generations is needed with substantial amounts of small
Fragment is spliced, and be there is pseudogene, large fragment deletion or large fragment in mitochondrial genomes and repeated the used time, is sequenced with two generations
More can not accurately it be spliced.Therefore, applicant proposes a kind of support SMRT sequencing technologies to human mitochondrial total length list times
Type information is measured, the method for setting up mitochondria full-length genome analysis of variance.
The content of the invention：
The purpose of the present invention aims to provide a kind of based on SMRT sequencing technologies acquisition homo mitochondrion gene group and line
The measure of body total length haplotype and the method for analysis of variance.
The method of the present invention is mainly expands the disposable template for obtaining homo mitochondrion gene group, Ran Houtong by primer
Cross SMRT sequencing technologies and obtain mitochondria full length sequence, then the sequence of acquisition is subjected to bioinformatic analysis, so as to be become
Different information.
To achieve the above object, the present invention takes following technical scheme：
A kind of human mitochondrial total length haplotype is determined and Genetic Variation Analysis method, specifically includes following steps：
1) human mitochondrion complete genome DNA is extracted；
2) performing PCR amplification is entered by pair of primers and obtains mitochondria complete genome DNA template, i.e. PCR primer；
3) all PCR primers of acquisition are uniformly mixed to form DNA ponds；
4) DNA ponds are built into behind 20K libraries by SMRT sequencing technologies, surveyed on the sequenators of PacBio RS II
Sequence and sequence polymorphism analysis, you can obtain mitochondrial inheritance variation information.
The primer of above-mentioned PCR amplification is：
Forward primer sequence is：5 '-GTACCCTAACCCGTGCAAAGGTAGCATA-3 ',
Reverse primer is：5’-ACAGGTCCCTATTTAAGGAACAAGTGAT-3’.
The beneficial effects of the present invention are：The present invention is had based on SMRT sequencing technologies to be read long (up to more than 16k), surveys
Sequence result unsystematic error, the advantage such as repetitive sequence and high GC content sequence can be overcome, not only can be to the mankind of acquisition
The mitochondria total length haplotype of mitochondrial genomes is measured, and is analyzed by sequence polymorphism etc., can be obtained more
More valuable mitochondrial inheritance variation information, to making accurate etiological diagnosis aspect with important applied to human diseases
Meaning.Therefore, method of the invention be applicable to mankind institute mitochondrial relevant disease DNA total length haplotypes measure demand and
The demand determined in medical jurisprudence research application to mitochondria total length haplotype.
Brief description of the drawings：
Fig. 1 is the electrophoretogram of pcr amplification product in the embodiment of the present invention 1；
Fig. 2 is the sequence alignment comparison chart of sequence analysis preservation in the embodiment of the present invention 1.
Embodiment：
Technical scheme is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1
According to Qiagen full-length genome nucleic acid extraction kit operation instructions, human peripheral blood sample extraction genomic DNA；Always
The peripheral blood sample of 15 different peoples is gathered altogether.
1. peripheral blood sample DNA extraction：
1) EP centrifuge tubes are sequenced by sample number, and marked；
2) PK enzyme solutions 20ul are added into each 1.5ml EP pipes, peripheral blood sample 200ul is then respectively adding；
3) Proteinase K liquid 200ul is added into each EP pipes, EP lids are covered, vibrated 15 seconds in vortex oscillator, with
Brief centrifugation afterwards；
4) each EP pipes are put into suspension foam hole, are placed in 10-30min in 56 DEG C of water baths, during which can suitably overturn；
5) take out after each centrifuge tube, brief centrifugation, absolute ethyl alcohol 200ul is added into each pipe, vibrate mixing 15 seconds, instantaneously
Centrifugation；
6) the enzymolysis mixture in EP pipes is transferred on Q posts, 8000rpm, centrifuges 1min；
7) waste collection pipe, plus AW1 liquid 500ul, 8000rpm are changed, 1min is centrifuged；
8) waste collection pipe, plus AW2 liquid 500ul, 13000rpm are changed, 3min is centrifuged；
9) Q posts are transferred in the new 1.5ml centrifuge tubes marked, add 60ulAE liquid, be stored at room temperature after 10min,
8000rpm, centrifuges 1min；
10) Q posts are abandoned, by DNA indwellings in EP pipes.
2.PCR is expanded：
The primer of amplification is：
Forward primer sequence：5 '-GTACCCTAACCCGTGCAAAGGTAGCATA-3 ',
Reverse primer is：5’-ACAGGTCCCTATTTAAGGAACAAGTGAT-3’；
Wherein：Primer is synthesized by Beijing Qing Kexin industry Bioisystech Co., Ltd, and Taq enzyme is by Thermo Scientific
There is provided.
PCR amplification programs are：94 DEG C of 2min, 10cycles (94 DEG C of 10s, 68 DEG C of 17min), 25cycles (94 DEG C of 10s,
68 DEG C of 17min), 72 DEG C of 5min, 4 DEG C of preservations.
PCR reaction systems are as follows：
As shown in figure 1, the electrophoretogram of the pcr amplification product for the present embodiment.
3. build storehouse sequencing：
15 parts of PCR primers are uniformly diluted to 200ng/ul, then 10ul are taken from the PCR primer after every part of dilution, one
DNA ponds are uniformly mixed to form in individual 1.5ml EP pipes, then 20K libraries are built according to PacBio RS II normal process, on
Then sample is sequenced in a cell on the sequenators of PacBio RS II.
4. sequence analysis：
1) original sequence data that the sequenators of PacBio RS II are produced is corrected on SMRT analysis2.3,
Sequence after output calibration, wherein sequence original condition is as follows：
The original condition of sequencing result, wherein：Reads OfInsert are the copy number for completing sequencing, Read Bases
OfInsert is the total bases of sequencing, and Mean Read Length ofInsert are that length, Mean Read are read in average insertion
Quality ofInsert are that precision is averagely sequenced in Insert Fragment, and MeanNumber ofPass are sequencing depth；
2) the sequence inputting MEGA7 softwares after correction are compared, the sequential file after alignment is protected with fas forms
Deposit, as shown in Fig. 2 being the sequence alignment comparison chart (being only part sectional drawing) of preservation；
3) sequential files of fas forms is inputted into DNAsp V5 softwares, ensured before input in sequence without forbidden character, such as
" N, R " etc.；
4) sequence polymorphism analysis is carried out to sequence in file thereafter, it is as a result as follows：
Sequence length：16570
Total polymorphic position points：175
The different haplotype numbers of sequence：13
The various degree of haplotype：0.989
Diverse oligonucleotide degree：0.00246.
Embodiment described above is only a preferred embodiment of the present invention, and it can not be by technical scheme
In the application of the measure and Genetic Variation Analysis that limit or constrain in human mitochondrial relevant disease DNA total length haplotypes.This hair
It is bright to may apply to related any human mitochondrial disease or the medical jurisprudence based on mitochondria haplotype information and the origin of mankind grinds
In studying carefully and applying.
Claims (5)
1. a kind of human mitochondrial total length haplotype is determined and Genetic Variation Analysis method, it is characterised in that：Methods described is main
It is that the disposable template for obtaining homo mitochondrion gene group is expanded by primer, then obtains mitochondria by SMRT sequencing technologies
Full length sequence, then the sequence of acquisition is subjected to bioinformatic analysis, so as to obtain the information that makes a variation.
2. human mitochondrial total length haplotype according to claim 1 is determined and Genetic Variation Analysis method, its feature exists
In：Comprise the following steps：
1) human mitochondrion complete genome DNA is extracted；
2) performing PCR amplification is entered by pair of primers and obtains mitochondria complete genome DNA template, i.e. PCR primer；
3) all PCR primers of acquisition are uniformly mixed to form DNA ponds；
4) DNA ponds are built into behind 20K libraries by SMRT sequencing technologies, in being sequenced on the sequenators of PacBio RS II and
Sequence polymorphism is analyzed, you can obtain mitochondrial inheritance variation information.
3. human mitochondrial total length haplotype according to claim 2 is determined and Genetic Variation Analysis method, its feature exists
In：The primer of PCR amplification is：
Forward primer sequence is：5 '-GTACCCTAACCCGTGCAAAGGTAGCATA-3 ',
Reverse primer is：5’-ACAGGTCCCTATTTAAGGAACAAGTGAT-3’.
4. the human mitochondrial total length haplotype as described in claims 1 to 3 any claim is determined and Genetic Variation Analysis
The application of method, this method is applied to the measure demand and medical jurisprudence of mankind institute mitochondrial relevant disease DNA total length haplotypes
The demand determined in research application to mitochondria total length haplotype.
5. the human mitochondrial total length haplotype as described in claims 1 to 3 any claim is determined and Genetic Variation Analysis
The application of method, this method may be used on any human mitochondrial disease correlation or the medical jurisprudence based on mitochondria haplotype information
And in probing on Human origin and application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710654472.2A CN107287333A (en) | 2017-08-03 | 2017-08-03 | A kind of human mitochondrial total length haplotype is determined and Genetic Variation Analysis method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710654472.2A CN107287333A (en) | 2017-08-03 | 2017-08-03 | A kind of human mitochondrial total length haplotype is determined and Genetic Variation Analysis method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107287333A true CN107287333A (en) | 2017-10-24 |
Family
ID=60105389
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710654472.2A Pending CN107287333A (en) | 2017-08-03 | 2017-08-03 | A kind of human mitochondrial total length haplotype is determined and Genetic Variation Analysis method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107287333A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111172260A (en) * | 2020-03-17 | 2020-05-19 | 安吉康尔（深圳）科技有限公司 | Method and kit for detecting mitochondrial genome in human urine or blood sample |
| CN112481413A (en) * | 2021-01-13 | 2021-03-12 | 南京集思慧远生物科技有限公司 | Plant mitochondrial genome assembly method based on second-generation and third-generation sequencing technologies |
| CN114015749A (en) * | 2021-10-18 | 2022-02-08 | 浙江博圣生物技术股份有限公司 | Construction method of mitochondrial genome sequencing library based on high-throughput sequencing and amplification primer |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015017759A1 (en) * | 2013-08-02 | 2015-02-05 | Stc.Unm | Dna sequencing and epigenome analysis |
| CN105506103A (en) * | 2015-12-28 | 2016-04-20 | 天津中医药大学 | Mitochondria genome amplified universal primer mixture as well as design and amplification method thereof |
| CN106520945A (en) * | 2016-11-07 | 2017-03-22 | 江苏苏博生物医学股份有限公司 | Next generation sequencing platform-based noninvasive target mitochondrion sequencing method |
-
2017
- 2017-08-03 CN CN201710654472.2A patent/CN107287333A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015017759A1 (en) * | 2013-08-02 | 2015-02-05 | Stc.Unm | Dna sequencing and epigenome analysis |
| CN105506103A (en) * | 2015-12-28 | 2016-04-20 | 天津中医药大学 | Mitochondria genome amplified universal primer mixture as well as design and amplification method thereof |
| CN106520945A (en) * | 2016-11-07 | 2017-03-22 | 江苏苏博生物医学股份有限公司 | Next generation sequencing platform-based noninvasive target mitochondrion sequencing method |
Non-Patent Citations (4)
| Title |
|---|
| ANDREWS,R.M.等: "Homo sapiens mitochondrion, complete genome", 《GENBANK》 * |
| ROLF H. A. M. VOSSEN: "《Genotyping》", 8 November 2016 * |
| 张得芳等: "第三代测序技术及其应用", 《中国生物工程杂志》 * |
| 柳延虎等: "单分子实时测序技术的原理与应用", 《遗传》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111172260A (en) * | 2020-03-17 | 2020-05-19 | 安吉康尔（深圳）科技有限公司 | Method and kit for detecting mitochondrial genome in human urine or blood sample |
| CN112481413A (en) * | 2021-01-13 | 2021-03-12 | 南京集思慧远生物科技有限公司 | Plant mitochondrial genome assembly method based on second-generation and third-generation sequencing technologies |
| CN112481413B (en) * | 2021-01-13 | 2022-02-15 | 南京集思慧远生物科技有限公司 | Plant mitochondrial genome assembly method based on second-generation and third-generation sequencing technologies |
| CN114015749A (en) * | 2021-10-18 | 2022-02-08 | 浙江博圣生物技术股份有限公司 | Construction method of mitochondrial genome sequencing library based on high-throughput sequencing and amplification primer |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Pervez et al. | A comprehensive review of performance of next-generation sequencing platforms | |
| Parducci et al. | Ancient DNA from pollen: a genetic record of population history in Scots pine | |
| CN103608818B (en) | The antenatal ploidy identification device of Noninvasive | |
| Bocklandt et al. | Bionano genome mapping: high-throughput, ultra-long molecule genome analysis system for precision genome assembly and haploid-resolved structural variation discovery | |
| Lee et al. | Different evolutionary fates of recently integrated human and chimpanzee LINE-1 retrotransposons | |
| CN107077537A (en) | With short reading sequencing data detection repeat amplification protcol | |
| CN110656157B (en) | Quality control product for tracing high-throughput sequencing sample and design and use method thereof | |
| CN105969843A (en) | High-throughput sequencing detection method for gene copy number and gene mutation based on MLPA | |
| CN107287333A (en) | A kind of human mitochondrial total length haplotype is determined and Genetic Variation Analysis method | |
| KR20200010464A (en) | Methods and systems for digesting and quantifying DNA mixtures from multiple contributors of known or unknown genotypes | |
| CN110494562A (en) | PCR primer group for HLA gene and the sequencing approach using it | |
| CN108136389A (en) | Sample is to the automatic preparation in NGS libraries | |
| CN108060227A (en) | A kind of amplimer, kit and its detection method for detecting PAH gene mutations | |
| CN108728522A (en) | Drug Discovery detection method | |
| CN107365839A (en) | A kind of primer and its application for animal in deer family identification | |
| CN109280696A (en) | The method of SNP detection technique fractionation mixing sample | |
| CN111690733A (en) | Hormone-induced femoral head necrosis susceptibility gene panel | |
| CN105189781A (en) | Probability-directed isolation of nucleotide sequences (PINS) | |
| CN114875118B (en) | Methods, kits and devices for determining cell lineage | |
| CN110241460A (en) | A kind of immune group library method for screening the reaction of independent sample crossover | |
| EP3325697B1 (en) | Optimized clinical sample sequencing | |
| CN108441572A (en) | The identification method of DCIPThe chloroplast of maize cytoplasm type based on KASP technologies | |
| CN107937407B (en) | Specific human gene fragment, primer and probe for detecting specific human gene fragment and application of specific human gene fragment | |
| CN105648060B (en) | Method for detecting drug-resistant genes of non-diagnostic human pathogenic microorganisms | |
| KEKEÇ et al. | New generation genome sequencing methods |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171024 |