CN107858439A - A kind of specific primer in macaca thibetana mitochondria D loop areas and detection method and application - Google Patents
A kind of specific primer in macaca thibetana mitochondria D loop areas and detection method and application Download PDFInfo
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- CN107858439A CN107858439A CN201711068240.5A CN201711068240A CN107858439A CN 107858439 A CN107858439 A CN 107858439A CN 201711068240 A CN201711068240 A CN 201711068240A CN 107858439 A CN107858439 A CN 107858439A
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- 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
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- 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
Abstract
The invention discloses a kind of specific primer in macaca thibetana mitochondria D loop areas and detection method and application, primer sequence such as SEQ ID No:Shown in 1 and 2, performing PCR amplification is entered using the primer, amplification system is：2 μ L, 10 × PCR buffer solution of genomic DNA 5 μ L, 1.5mM Mg2+2 μ L, 1U/ μ L Taq enzyme 2.5 μ L, 0.2mM dNTPs 5 μ L, 10mM each 1 μ L of positive anti-primer, add ddH2O to 50 μ L；Amplification program is：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 90s, totally 30 circulations, last 72 DEG C extend 8min.The present invention devises the specific primer of amplification macaca thibetana mitochondria D loop areas full length sequence, can fast and accurately amplify complete D loop sequences.
Description
Technical field
The invention belongs to macaca thibetana genetic analysis technical field, and in particular to a kind of spy of macaca thibetana Mitochondrial D-loop Region
Specific primer and detection method and application.
Background technology
Macaca thibetana (Macaca thibetana), also known as big titi, Tibetan monkey, category Primates (Primates) monkey section
(Cercopithecidae) Macaca (Macaca), it is the distinctive non-human primate species in China, is distributed mainly on Central Asia heat
Sichuan, Guizhou, Anhui, Fujian and the zhejiang and other places of band-north subtropical.National II class of macaca thibetana category saves the wild animals, and is on the point of
Danger international trade of wildlife pact (CITES) is classified as the species of annex 2, in World Conservation Union (IUCN) endangered species
" nearly danger " species are classified as in Red List classification.In recent years because human activity is disturbed, wild macaca thibetana quantity drastically declines, according to
Estimate that current field quantity is no more than 8000, wild macaca thibetana protection of resources is extremely urgent.Present researcher starts to introduce
Wild macaca thibetana carries out domestication's breeding, by taming, breeding, has established fixed propagating population.In Laboratory Animal Standardization
In research, Genetic control is particularly significant, it is therefore desirable to establishes suitable monitoring method system and macaca thibetana stable breeding population is carried out
Genetic diversity is assessed, so as to avoid inbreeding from causing population genetic diversity to reduce and inbreeding depression phenomenon.
Macaca thibetana mitochondrial genomes are typically closed hoop duplex structure in 16540bp or so, can autonomous replication and
Transcription.Code area (13 protein coding genes, 2 ribosomal RNA genes, 22 transfer RNA bases including 37 genes
Cause) and 1 control zone (D-loop).D-loop areas are most important noncoding region on mtDNA, and evolutionary rate is compared with code area more
It hurry up, be that the colony for being adapted to affiliation nearer carries out the preferable molecular labeling of genetic variation and genetic differentiation research.D-loop areas part is complete
Portion's sequence is had been widely used in the genetic diversity and phyletic evolution research of animal monoid.But the macaca thibetana line of existing design
The primer and detection method in plastochondria D-loop areas can not obtain complete D-loop sequences, expect complete D-loop sequences
Also need, into the step of being attached cloning vector, to be then sequenced again, the sequence in sequencing procedure within the 100bp of front and back end
Inaccurate phenomenon occurs, while process is comparatively laborious, and it is unable to comprehensive assessment macaca thibetana group using part D-loop sequences
The genetic diversity of body.
The content of the invention
For above-mentioned deficiency of the prior art, the invention provides a kind of specificity of macaca thibetana Mitochondrial D-loop Region
Primer and detection method and application, can effectively solve the primer designed in the prior art and detection method can not obtain complete D-
Loop sequences, to expect that complete D-loop sequences also need, into the step of being attached cloning vector, to be then sequenced again,
Process is comparatively laborious and the problem of being unable to the genetic diversity of comprehensive assessment macaca thibetana colony using part D-loop sequences.
To achieve the above object, the technical solution adopted for the present invention to solve the technical problems is：
A kind of specific primer of macaca thibetana Mitochondrial D-loop Region, primer sequence are：
F：5’-CACTAGTCTCCCTAATCGAAAACAACCTACTC-3’(SEQ ID No:1)；
R：5’-GTGCGTGCTTGATGCTTGCTCCTCTTG-3’(SEQ ID No:2).
A kind of detection method of macaca thibetana Mitochondrial D-loop Region, comprises the following steps：
(1) extraction of macaca thibetana genomic DNA：
Macaca thibetana venous blood is gathered using EDTA anticoagulant tubes, is then extracted and hidden using poba gene group DNA extraction kit
Genomic DNA in chief of a tribe's monkey blood；
(2) in macaca thibetana mitochondrial Ca2+ control zone upstream and downstream conserved region CYTB genes and 12s ribosomal RNA
Specific primer is designed in region, and primer sequence is：
F：5’-CACTAGTCTCCCTAATCGAAAACAACCTACTC-3’(SEQ ID No:1)；
R：5’-GTGCGTGCTTGATGCTTGCTCCTCTTG-3’(SEQ ID No:2)；
(3) genomic DNA obtained by step (1) is entered into performing PCR amplification under the primer effect that step (2) designs, obtained
Mitochondrial DNA D-loop sequences；Wherein,
Amplification system is：The μ L (50ng) of genomic DNA 2,10 × PCR buffer solutions 5 μ L, 1.5mM Mg2+2 μ L, 1U/ μ L's
Taq enzyme 2.5 μ L, 0.2mM dNTPs 5 μ L, 10mM each 1 μ L of positive anti-primer, add ddH2O to 50 μ L；
Amplification program is：94 DEG C of pre-degeneration 5min；94 DEG C denaturation 30s, 60 DEG C annealing 30s, 72 DEG C extension 90s, totally 30
Circulation；Last 72 DEG C of extensions 8min.
The primer of above-mentioned design and method are used for population genetic diversity analysis, germplasm identification and the open country of macaca thibetana
Biological species protection etc..
The specific primer of macaca thibetana Mitochondrial D-loop Region provided by the invention and detection method and application, have following
Beneficial effect：
(1) present invention devises the specific primer of amplification macaca thibetana Mitochondrial D-loop Region full length sequence, to D-loop
And front and rear terminal sequence is expanded, single band is can obtain, illustrates that the primer specificity is good；Because the specific primer is located at D-
Each 100bp in loop front and back ends or so, removes this partial sequence after sequencing, obtain the D-loop sequences of complete and accurate, solve survey
The problem of inaccurate within the 100bp of front and back end in program process, while gained PCR fragment after performing PCR is entered using the primer and can obtain
D-loop areas full length sequence, can be directly used for being sequenced, it is not necessary to which the step of being attached cloning vector, the process is quick and precisely.
(2) primer provided by the invention and detection method can realize that mitochondrial DNA D-loop full length sequence is analyzed, can be more
Add the genetic structure and genetic diversity of reflection macaca thibetana colony comprehensively, can also realize to macaca thibetana Wild population and stable breeding kind
The genetic structure and genetic diversity of group is assessed, and the science of heredity management for wildlife conservation and stable breeding population provides theory
Instruct.
The method that macaca thibetana hereditary information analysis is carried out using mitochondria DNA D-loop control area carries out macaca thibetana heredity letter
Breath and analysis of genetic diversity, quick and precisely and simple and practical, the labeled primer that the present invention designs can be additionally used in the parent of macaca thibetana
The area researches such as edge relationship analysis, evolutionary analysis, species conservation, germplasm identification.
Brief description of the drawings
Fig. 1 is the DNA bands amplified using primer provided by the invention and detection method.
Embodiment
Embodiment 1
1st, the extraction of macaca thibetana genomic DNA
Macaca thibetana venous blood 2mL is gathered using EDTA anticoagulant tubes, then extracted using poba gene group DNA extraction kit
Genomic DNA in macaca thibetana blood, genomic DNA is detected through 1.2% agarose gel electrophoresis and ultraviolet spectrophotometry
Purity and concentration.
2nd, primer is designed
Upstream and downstream conserved region CYTB genes and 12s ribosomal RNA regions in macaca thibetana mitochondrial Ca2+ control zone
Specific primer is designed, primer sequence is：
F：5’-CACTAGTCTCCCTAATCGAAAACAACCTACTC-3’；
R：5’-GTGCGTGCTTGATGCTTGCTCCTCTTG-3’.
3rd, PCR is expanded
Amplification system is：The μ L (50ng) of genomic DNA 2,10 × PCR buffer solutions 5 μ L, 1.5mM Mg2+2 μ L, 1U/ μ L's
Taq enzyme 2.5 μ L, 0.2mM dNTPs 5 μ L, 10mM each 1 μ L of positive anti-primer, add ddH2O to 50 μ L；
Amplification program is：94 DEG C of pre-degeneration 5min；94 DEG C denaturation 30s, 60 DEG C annealing 30s, 72 DEG C extension 90s, totally 30
Circulation；Last 72 DEG C of extensions 8min.
Amplified production detects its size, purity and brightness with 1.5% agarose gel electrophoresis, as a result sees Fig. 1.
As shown in Figure 1, by electrophoresis detection, unicity band is can obtain, the band is without hangover, and brightness is moderate, each sample
Product stripe size is consistent, illustrates that primer specificity provided by the invention is good.
4th, it is sequenced
Sample is chosen：Macaca thibetana F1 generation 27, the F2 generations 27 in Sichuan Jinyang County source are chosen, Sichuan Ma Bianxian sources
Macaca thibetana F1 generation 24, F2 generations 24 (captive breedings).
Performing PCR amplification is entered using above-mentioned primer and detection method, pcr amplification product is directly sequenced：Returned using glue
Receive kit to be tapped rubber after purification, then send authorized company to carry out two-way direct Sequencing recovery product, sequencing primer is upper
Amplimer is stated, sequenator is the full-automatic DNA sequencer of the types of ABI 3730.Obtained sequence Vector NTI will be sequenced
The Assemble programs splicing of software, splicing sequence are compared with macaca thibetana mtdna sequence in GenBank, cut and draw
Thing and unnecessary sequence, and known macaca thibetana mitochondrial Ca2+ alignment, as a result unanimously.
The macaca thibetana mitochondrial DNA D-loop sequences surveyed are shown in SEQ ID No:3.
5th, analysis of genetic diversity
(1) sample is chosen：Choose F1 generation 27, the F2 generations 27 in Sichuan Jinyang source, the F1 generation 24 in Sichuan Ma Bian sources
Only, in F2 generations 24 (captive breeding), carry out statistical analysis.
(2) PCR amplifications, sequencing and sequence analysis：Using above-mentioned specific primer, performing PCR is entered to 102 macaca thibetana individuals
Amplification, amplified production are sequenced through glue reclaim Hou Song companies, sequence must be analyzed using Vector NTI softwares, obtain 102
Individual D-loop full length sequences.
(3) with the software statistics polymorphic site numbers of DnaSP version 5.10 and mutational site sum, haplotype number, Dan Bei
The various degree of type, diverse oligonucleotide degree etc., as a result such as following table：
As seen from the above table, Jinyang F2, Jinyang F1, horse side F2 and Ma Bian F1, individual sequence length in 4 populations, generation populations
Spend for 1091-1092bp, be the total length of macaca thibetana Mitochondrial D-loop Region, including 115 variant sites, 66 lists times
Type, the average various degree of haplotype (Hd) is 0.953, shows higher genetic diversity；The core of horse side 2 generation populations of population
Nucleotide polymorphism (Pi) is respectively 0.00477 and 0.00409, and polymorphism is declined slightly；And the core of 2 generation populations of Jinyang population
Nucleotide polymorphism is respectively 0.00230 and 0.00278, and polymorphism slightly rises, this that may be sampled with Jinyang F2 for colony
Body quantity is more relevant.
In summary, using specific primer provided by the invention and detection method, it can obtain macaca thibetana mitochondrial DNA
D-loop full length sequences, available for macaca thibetana analysis of genetic diversity.
Sequence table
<110>Institute of lab animals of People's Hospital, Sichuan Prov. of Sichuan Academy of Medical Sciences
<120>A kind of specific primer of macaca thibetana Mitochondrial D-loop Region and detection method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 32
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
cactagtctc cctaatcgaa aacaacctac tc 32
<210> 2
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gtgcgtgctt gatgcttgct cctcttg 27
<210> 3
<211> 1091
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
cattcctgta ctacacaagt tccacaaaca accctagcat aacttactca caatactact 60
atgtaattcg tgcattactg ctagccaaca tgtataatat atagtactat atatgcttaa 120
tcgtacataa cacatatcat tatatatcaa cttaacatcc ttgaagaaca tgcttacaag 180
caagtaacct agtaacaact tcaacagtag cacatgacat ggcccttcca aatcagtctc 240
ctctacccca tgaataccaa ccaaaccaat ccatgccagt cgtccatagt acattagatc 300
gttcatcgga catagcacat acctattaaa taatcctcct caccacggat gacccccctc 360
acttaggaat cccttgttca ccatcctccg tgaaatcaat atcccgcaca agagtgctac 420
tctcctcgct ccgggcccat aacccgtggg ggtagctatg cttgagctgt atccggcatc 480
tggttcttac ctcagggcca taacaaccaa gatcgcccac acgttcccct taaataagac 540
atctcgatgg atcacaggtc tatcacccta ttaaccagtc acgggagctt tccatgcatt 600
tggtatcttt tatctctggt ctgcacgcaa ccccatcgca gaatgctgac tcccaccaca 660
tcccgtcctg tatggacctg tctttgattc ctagtacata caaccattga tcgcacctac 720
gttcaatatt ctagctccac acggacatta gcaaggtgct atttaatcca tgcttgtagg 780
acataccaat aattatctta gccgacacca cccccaccga gccataaatc acaactatat 840
ctcgtcaaac ccccccaccc ccatctccga ccttcatcca aaacccactt ttgccaaacc 900
ccaaaaacaa aagttttaat atatccgatc agagcctgca ttttcatctt ttaggtgtgc 960
acaactccaa ctgccattcc ctcaactaac aaaaatttac ttcactaaac acccttcaca 1020
ccaacccaca acagaccctt ttcacacaac ccaaaagaac attgcacccc gtttatgtag 1080
cttaaatctg c 1091
Claims (3)
1. a kind of specific primer of macaca thibetana Mitochondrial D-loop Region, it is characterised in that primer sequence is：
F：5’-CACTAGTCTCCCTAATCGAAAACAACCTACTC-3’；
R：5’-GTGCGTGCTTGATGCTTGCTCCTCTTG-3’.
2. a kind of detection method of macaca thibetana Mitochondrial D-loop Region, it is characterised in that comprise the following steps：
(1) extraction of macaca thibetana genomic DNA；
(2) performing PCR amplification is entered using the primer described in claim 1：
Amplification system is：2 μ L, 10 × PCR buffer solution of genomic DNA 5 μ L, 1.5mM Mg2+2 μ L, the 1U/ μ L μ of Taq enzyme 2.5
L, 0.2mM dNTPs 5 μ L, 10mM each 1 μ L of positive anti-primer, add ddH2O to 50 μ L；
Amplification program is：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 90s, totally 30 circulate；
Last 72 DEG C of extensions 8min.
3. application of the method in the population genetic diversity analysis or germplasm identification of macaca thibetana described in claim 2.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110760598A (en) * | 2019-11-25 | 2020-02-07 | 云南大学 | Primer group for molecular biology research of golden monkey and application method thereof |
-
2017
- 2017-11-03 CN CN201711068240.5A patent/CN107858439A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| LI-JING ZHONG ET AL.: "Genetic diversity of two Tibetan macaque (Macaca thibetana) populations from Guizhou and Yunnan in China based on mitochondrial DNA D-loop sequences", 《GENES GENOM》 * |
| 姚永芳 等: "峨眉山藏酋猴mtDNA控制区序列变异及种群遗传多样性", 《四川动物》 * |
| 牛丽红 等: "基于藏酋猴线粒体基因组D-loop的遗传多样性分析", 《第十三届中国实验动物科学年会论文集》 * |
| 董高华: "藏酋猴线粒体基因组序列测定与进化分析", 《中国优秀硕士学位论文全文数据库基础科学辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110760598A (en) * | 2019-11-25 | 2020-02-07 | 云南大学 | Primer group for molecular biology research of golden monkey and application method thereof |
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Application publication date: 20180330 |