CN106086014A - For expanding the universal primer of homopterous insect mitochondrial genome long segment - Google Patents
For expanding the universal primer of homopterous insect mitochondrial genome long segment Download PDFInfo
- Publication number
- CN106086014A CN106086014A CN201610539758.1A CN201610539758A CN106086014A CN 106086014 A CN106086014 A CN 106086014A CN 201610539758 A CN201610539758 A CN 201610539758A CN 106086014 A CN106086014 A CN 106086014A
- Authority
- CN
- China
- Prior art keywords
- primer
- universal primer
- long segment
- expanding
- mitochondrial genome
- 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
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The invention discloses a kind of universal primer for expanding the long segment covering homopterous insect mitochondrion full-length genome, this universal primer is made up of two pairs of primers, and its sequence is as shown in SEQ ID NO.1～SEQ ID NO.4.Using the universal primer of the present invention, can effectively amplify the long segment of many seed wings mesh species mitochondrial genome, contribute to shortening the cycle obtaining molecular data, the phylogeny research for China's homopterous insect provides important tool.
Description
Technical field
The present invention relates to two to the universal primer for expanding homopterous insect mitochondrial genome long segment.
Background technology
Wing mesh (Plecoptera) is also known as perlid, and such insecticide is distributed in polytype waters widely, is
Important water quality monitoring indicator organism, strengthens the sort research of this monoid, preferably can provide service for water quality environment monitoring.
From the perspective of systematics and theory of evolution, wing mesh is a crucial monoid.Although wing mesh phylogeny
It is widely studied, but remains a need for being proved by other more features.And the data obtained by Molecular tools will more be helped
In the sibship inferred between Chi Muge section and and other purpose sibships.Understand in depth homopterous insect origin,
Evolve and Phylogenetic Relationships, the evolutionary relationship rebuilding wing mesh phylogeny or even research Insecta is all had important meaning
Justice.This research being accomplished by homopterous insect carries out association area by quickly and accurately Molecular tools.
In recent years, along with molecular engineering and the fast development of high-throughput techniques, mitochondrial genome has become differentiation species
Hot research object with research spore relation.Therefore, utilize mitochondrial genome that homopterous insect is carried out system to send out
Educating analysis is a kind of preferably method.At present, during insecticide genetic research, the main flow side of mitochondrial genome research on a small scale
Method remains the primer walking of traditional PCR-based amplified production.The primer quantity that the method uses is more, amplification efficiency
Low, high to masterplate purity requirement, the longest, and lack enough specificitys, this will hinder Mitochondria In Developing Flight Muscle of Insects genome molecules
The Rapid Accumulation of data, and some insecticide is caused difficulty as studied the phylogeny of homopterous insect.Do not have in the world at present
There is the relevant report being specifically designed for expanding the universal primer of homopterous insect mitochondrial genome long segment.
Summary of the invention
It is an object of the present invention to provide the universal primer of a kind of homopterous insect mitochondrial genome long segment amplification, it can be expired
The demand of foot prior art.
A kind of universal primer for expanding homopterous insect mitochondrial genome long segment, by two pairs of its sequences of primer sets
As shown in SEQ ID NO.1～SEQ ID NO.4.
The invention also discloses the amplification method of a kind of homopterous insect mitochondrial genome long segment, be that employing is above-mentioned
Primer, carries out target fragment amplification by 25 μ l PCR system under specific PCR amplification condition.
25 described μ l PCR system are: 5 μ l 10 × buffer (+Mg2+), 2.5 μ l dNTP (2.5mM), 3 μ l upstreams are drawn
Thing (20 μMs), 3 μ l downstream primers (20 μMs), 0.2 μ l TaKaRa LA Taq enzyme (5U/ μ l), 1 μ l DNA profiling (30ng/ μ l),
10.3 μ l distilled waters.
Described PCR amplification condition is: 93 DEG C of denaturations 2min；92 DEG C of degeneration 10s, 54 DEG C of annealing 30s, 68 DEG C of extensions
8min, expands 20 circulations；92 DEG C of degeneration 10s afterwards, 54 DEG C of annealing 30s, 68 DEG C extend 8min, and each circulation increases 20s,
Expand 20 circulations；Last 68 DEG C extend 7min.
There is the characteristic of conservative region according to the mitochondrial genome of Insecta insecticide in the present invention, by comparison Genbank
The mitochondrial genome sequential design of middle different mesh insecticide goes out amplimer.Use the wing mesh mitochondrial gene group leader of the present invention
Fragment amplification primer, can amplify the long segment sequence of the most equal species of wing mesh effectively, has filled up wing mesh long segment
Not having the blank of general amplimer, insecticide that is the most equal to wing mesh and that belong to carries out large-scale long segment order-checking, will produce
Thing directly carries out high-flux sequence, effectively shortens the acquisition cycle of molecular data, for China's homopterous insect phylogeny and
Genetic Constitution of Population research provides important tool.The amplimer that the present invention relates to relatively passes by traditional method to be had bigger excellent
Gesture, is embodied in: this primer can effectively amplify the long segment of the nearly 16000bp of overall length, mitochondrial genome is completely covered complete
Long, and the effective amplification length of traditions of the past method is generally about 1000bp, and amplification efficiency is low, is unfavorable for the studies above
Data analysis.Utilize this primer that homopterous insect carries out large-scale phylogenetic evolution analysis, can significantly reduce and test into
This, shorten experimental period, improves the accuracy of experiment.
Accompanying drawing explanation
Fig. 1 is the primer of the present invention expanding effect figure to Chi Mu 4 section Mitochondria In Developing Flight Muscle of Insects long segment.1-2 is hook；3-4
For flat；5-6 is green；7-8 is thorn；M is Marker.
Fig. 2 is other primer expanding effect figure to Chi Mu 4 section Mitochondria In Developing Flight Muscle of Insects long segment in experimentation.1-2 is
Hook；3-4 is flat；5-6 is green；7-8 is thorn；M is Marker.
Detailed description of the invention
The amplimer screening technique of the wing mesh mitochondrial genome long segment of the present invention is the most in two steps: 1. with
Based on the mitochondrial genome sequence of the different mesh insecticides recorded in Genbank, find out relatively conservative sequence by comparison
Fragment carries out design of primers, and being simultaneously introduced degeneracy site increases primer versatility；2. by the primer of synthesis to homopterous insect line
Mitochondrial genes group expands, and detects its versatility in wing mesh and amplification efficiency；The most once designed multipair
Primer, shows that through experiment show the primer of the present invention is optimum.
Below in conjunction with example, invention is described further:
1. preparation of samples: sample all uses in depositing in Yangzhou University's application insecticide institute Specimen Room and deposits in 100%
The insecticide that the wing mesh of ethanol is the most equal, including hook section, flat section, green section, the homopterous insect of Ci Kedeng section.
2.DNA extracts: Axygen test kit extracts DNA, and concrete steps are with reference to description.
3. Data Source: Data Source in US National Biotechnology Information center (NCBI, http :/
www.ncbi.nlm.gov/)。
4. sequence alignment: utilize ClustalX software to be compared by the mitochondrial genome complete sequence of different mesh insecticides,
Find out than sequence area more conservative, that nucleotide variation degree is minimum.
5. primer synthesis: according to above-mentioned comparison result, utilize primer-design software Primer Premier 5 in base
The sequence area of degree of variation minimum designs 2 pairs of primers.Primer sequence is
Au:5’CGAGCWTACTTTACTTCAGCMACWATAATTA3’(SEQ ID NO.1)；
Ad:5’GCAAATARRAARTATCATTCATTCTGGTTGGAT 3’(SEQ ID NO.2)；
Bu:5’TACACCAAACAGGATCAAATAAYCCMWTAGG 3’(SEQ ID NO.3)；
Bd:5’GCTCCACARATTTCTRCATTGACCAAARAA3’(SEQ ID NO.4)。
6. primer amplification: above-mentioned amplimer is respectively applied to PCR and expands the sample gathered.
7. amplification detection: amplimer detects with the agarose gel electrophoresis of 1%.Result shows, this primer
Can effectively expand the mitochondrion long segment of homopterous insect.The DNA fragmentation total length utilizing these 2 pairs of primer amplifications to go out is about
16000bp, covers homopterous insect mitochondrial genome (total length is about 16000bp), and agarose gel electrophoresis detector bar
Band the most single (Fig. 1)；And when using other primer, the then a large amount of disperses of electrophoresis detection band, effect poor (Fig. 2).Therefore,
Primer of the present invention can amplify the mitochondrial genome long segment of the most equal species of wing mesh effectively, and sequencing result is calibrated
Really.
8. primer amplification condition: above-mentioned primer 25 μ l PCR system is: 5 μ l 10 × buffer (+Mg2+), 2.5 μ l dNTP
(2.5mM), 3 μ l forward primer (20 μMs), 3 μ l downstream primers (20 μMs), 0.2 μ l TaKaRa LATaq enzyme (5U/ μ l), 1 μ l
DNA profiling (30ng/ μ l), 10.3 μ l distilled waters.
Claims (4)
1. for expanding the universal primer of homopterous insect mitochondrion full-length genome long segment, it is characterised in that by two pairs of primers
Composition, its sequence is as shown in SEQ ID NO.1～SEQ ID NO.4.
2. an amplification method for homopterous insect mitochondrion whole genome sequence, is characterized in that using described in claim 1
Universal primer, carries out target fragment amplification by 25 μ l PCR system under PCR amplification condition.
3. method as claimed in claim 2, it is characterised in that 25 described μ l PCR system are: 5 μ l add Mg2+10 ×
Buffer, 2.5 μ l dNTP, 3 μ l forward primer, 3 μ l downstream primers, 0.2 μ l TaKaRa LA Taq enzyme, 1 μ l DNA profiling,
10.3 μ l distilled waters.
4. method as claimed in claim 2, it is characterised in that described PCR amplification condition is: 93 DEG C of denaturations 2min；92℃
Degeneration 10s, 54 DEG C of annealing 30s, 68 DEG C extend 8min, expand 20 circulations；92 DEG C of degeneration 10s afterwards, 54 DEG C of annealing 30s, 68
DEG C extend 8min, and each circulation increase 20s, expand 20 circulations；Last 68 DEG C extend 7min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610539758.1A CN106086014A (en) | 2016-07-08 | 2016-07-08 | For expanding the universal primer of homopterous insect mitochondrial genome long segment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610539758.1A CN106086014A (en) | 2016-07-08 | 2016-07-08 | For expanding the universal primer of homopterous insect mitochondrial genome long segment |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106086014A true CN106086014A (en) | 2016-11-09 |
Family
ID=57212750
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610539758.1A Pending CN106086014A (en) | 2016-07-08 | 2016-07-08 | For expanding the universal primer of homopterous insect mitochondrial genome long segment |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106086014A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111172159A (en) * | 2020-03-06 | 2020-05-19 | 佛山科学技术学院 | Bovine mitochondrial genome capture probe kit |
| CN111304196A (en) * | 2020-03-06 | 2020-06-19 | 佛山科学技术学院 | Buffalo animal mitochondrial genome capture probe kit |
| CN113151257A (en) * | 2021-03-09 | 2021-07-23 | 中南大学 | Amplification primer and method for whole genome sequence of Triphila corilaginella mitochondria |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946645A (en) * | 2015-07-24 | 2015-09-30 | 扬州大学 | Specific primers for amplifying plecoptera insect mitochondrion COI (cytochrome c oxidase I) gene |
-
2016
- 2016-07-08 CN CN201610539758.1A patent/CN106086014A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946645A (en) * | 2015-07-24 | 2015-09-30 | 扬州大学 | Specific primers for amplifying plecoptera insect mitochondrion COI (cytochrome c oxidase I) gene |
Non-Patent Citations (3)
| Title |
|---|
| QIAN YU-HAN等: "Mitochondrial Genome of the Stonefly Kamimuria wangi (Plecoptera: Perlidae) and Phylogenetic Position of Plecoptera Based on Mitogenomes", 《PLOS ONE》 * |
| 刘念等: "应用长PCR扩增蝗虫线粒体全基因组", 《动物学杂志》 * |
| 张乃心等: "双翅目昆虫线粒体基因组结构特点及其测序通用引物的设计和应用", 《昆虫学报A》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111172159A (en) * | 2020-03-06 | 2020-05-19 | 佛山科学技术学院 | Bovine mitochondrial genome capture probe kit |
| CN111304196A (en) * | 2020-03-06 | 2020-06-19 | 佛山科学技术学院 | Buffalo animal mitochondrial genome capture probe kit |
| CN113151257A (en) * | 2021-03-09 | 2021-07-23 | 中南大学 | Amplification primer and method for whole genome sequence of Triphila corilaginella mitochondria |
| CN113151257B (en) * | 2021-03-09 | 2022-07-01 | 中南大学 | Amplification primer and method for whole genome sequence of Triphila corilaginella mitochondria |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Singh et al. | CAAT box-derived polymorphism (CBDP): a novel promoter-targeted molecular marker for plants | |
| KR102026758B1 (en) | Molecular marker for identifying Perilla cultivars and Perilla wild species based on the information of chloroplast genomes and 45S nrDNAs sequence and uses thereof | |
| CN109517925A (en) | Flax SSR molecular marker and its application | |
| CN106086014A (en) | For expanding the universal primer of homopterous insect mitochondrial genome long segment | |
| CN103740702B (en) | A kind of SNP marker relevant to bay scallop heat tolerance and authentication method thereof and potential application | |
| KR101769874B1 (en) | Primer set for discriminating Allium cepa intra- or inter-species and uses thereof | |
| KR20160082292A (en) | Molecular Markers for Selecting radish genetic resources and use thereof | |
| CN104164433B (en) | For expanding the specific primer of lepidopteran insects mitochondrial COI gene | |
| Tamboli et al. | Phylogenetic analysis, genetic diversity and relationships between the recently segregated species of Corynandra and Cleoserrata from the genus Cleome using DNA barcoding and molecular markers | |
| CN104946645A (en) | Specific primers for amplifying plecoptera insect mitochondrion COI (cytochrome c oxidase I) gene | |
| Zhang et al. | Development and characterization of 109 polymorphic EST‐SSRs derived from the Chinese bayberry (Myrica rubra, Myricaceae) transcriptome | |
| KR101392305B1 (en) | Multiplex primer set for simultaneous diagnosing WSSV and HPV and uses thereof | |
| Rogers et al. | Direct PCR amplification from leaf discs | |
| CN106367498A (en) | Periplaneta americana microsatellite loci and application thereof | |
| KR102299258B1 (en) | Primer set for determining Prunus sp. and uses thereof | |
| KR102286871B1 (en) | Molecular marker for discriminating branchless watermelon cultivar and uses thereof | |
| KR101948389B1 (en) | Discriminating method of Codonopsis lanceolata and Adenophora triphylla using SSR marker | |
| KR101807623B1 (en) | Complete sequencing of chloroplast genome and nrDNA of Ledebouriella seseloides, Peucedanum japonicum and Glehnia littoralis-derived barcoding marker, DNA primer set for discrimination of origin and species and uses thereof | |
| KR101177787B1 (en) | SSR markers for discriminating of proso millet landraces and use thereof | |
| CN106967805B (en) | Tortoise microsatellite DNA marker based on high-throughput sequencing screening | |
| CN107022616B (en) | Quinoa binary InDel molecular marker and development method and application thereof | |
| CN105950618A (en) | Universal primer set for magnifying long segments of mitochondrial genomes of liriomyza mik insects | |
| CN108103203A (en) | Holotrichia parallela reference gene and its application | |
| Lv et al. | A transcriptomic approach to develop a novel set of low-copy nuclear gene primers for the sand whip grass, psammochloa villosa (TRIN.) bor (POACEAE), A dominant species from the inner mongolia plateau | |
| CN116516026B (en) | Molecular marker related to antibody titer of broiler chickens, detection method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20161109 |
|
| RJ01 | Rejection of invention patent application after publication |