CN110452998A - The method for carrying out yellow crucian carp genetic analysis using mitochondrion DNA control area - Google Patents
The method for carrying out yellow crucian carp genetic analysis using mitochondrion DNA control area Download PDFInfo
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Classifications
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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
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
-
- 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
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
Abstract
The present invention provides a kind of method for carrying out yellow crucian carp genetic analysis using mitochondrion DNA control area, belong to gene engineering technology field, include the following steps: that lysate is added in yellow crucian carp musculature sample to be cracked, and is then separated supernatant, is obtained mitochondrial genomes DNA；Mitochondrial genomes DNA is carried out to the screening of target sequence；Yellow crucian carp genetic analysis is carried out from screening results；Wherein, lysate is the aqueous solution comprising NaOH, Tris-HCl, EDTA, NaGC and dimercaprol dimercaptopropanol；Target sequence include in mitochondrion DNA control area in whole or in part.The present invention easy can rapidly obtain high quality, high-purity, the mitochondrial genomes DNA that can be used as amplification template, and yield is high, then yellow crucian carp hereditary information and genetic diversity are analyzed using yellow crucian carp mitochondrion DNA control area, method is quick and precisely and simple and practical.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of to carry out yellow crucian carp something lost using mitochondrion DNA control area
The method for passing credit analysis.
Background technique
Molecular labeling is generally referred to as DNA marker, is based on inhereditary material inner nucleotide sequence variations
Genetic marker is widely used to the genetics research of marine organisms at present.Fish mitochondrial DNA (Mitochondrial
DNA, mtDNA) because having the characteristics that structure is simple, stringent matrilinear inheritance, lacking recombination and as important molecular labeling.
Mitochondrion DNA control area (Control region, CR, the also known as area D-loop) is located in mitochondrial DNA in one section of noncoding region
Between tRNA Pro and t RNA Phe gene, structure is slightly complicated, and evolutionary rate is fast, is easy more variation (such as base of accumulation
Replacement, insertion and missing etc.), be widely used in kind in a molecule genetics research.Even if the area mitochondrial DNA D-loop is
The maximum section of mtDNA variation, but equally exist relatively conservative segment.Control zone can be divided into 3 areas from composition
Section: i.e. termination sequence area (extended termination associated sequences, ETAS), central conserved region
(central conserved domain, CD) and conserved sequence area (conserved sequence blocks, CSBs).Its
Middle conserved sequence area and central conserved region are more conservative, and are mutated and are frequently experienced in this section of termination sequence area.Conserved sequence
Area is there are multiple conservative fragments such as CSB-F, CSB-E, CSB-D, CSB-1, CSB-2, CSB-3, these segments are in many mitochondrias
All be in DNA it is highly conserved, temporarily have jumping phenomenon, no evidence can be in terms of related gene function at present
Verify its function.It has analyzed and researched at present the mitochondrion DNA control area structures of a variety of fish, and to go out control zone each for summary and induction
The common form of conservative fragments.Length polymorphism phenomenon is also found in the fish mitochondrion DNA control area of part, controls section length
Polymorphism may be applied to kind of an interior or inter-species group and learn research.
Yellow crucian carp (Setipinna tenuifilis) is subordinate to Clupeiformes (Clupeiformes), Anchovy section
(Engraulididae), yellow crucian carp category, is distributed widely in Indian Ocean western part and northwest Pacific sea area, each sea area of coastal area of china is equal
It is distributed.Yellow crucian carp is lower floor fish in coastal waters, likes to inhabit in the more slow sea area of silt bottom, water flow.Yellow crucian carp is a kind of small-sized economy
Fish.Since the 1980s, as the traditional economies stock of fish amount such as little yellow croaker, scomberomorus niphonius, hairtail declines, yellow crucian carp
Quantity of the catch increases year by year.Since fishing pressure is excessive, downward trend has been presented in yellow crucian carp stock number.So how to yellow crucian carp resource
It is reasonably developed and used, and carries out the protection of resources and repair of science, it has also become urgent problem to be solved.And it utilizes
Mitochondrion DNA control area sequence studies the hereditary difference and differentiation degree of different Huang Ji groups, for the conjunction of yellow crucian carp resource
Reason utilizes and scientific conservation, provides important theoretical foundation.
Summary of the invention
It is an object of the present invention to provide it is a kind of quick and precisely and it is simple and practical using mitochondrion DNA control area into
The method of row Huang crucian carp genetic analysis, the detection method easy can rapidly obtain high quality, high-purity, can be used as expansion
Increase the mitochondrial genomes DNA of template, and yield is high.
The technical solution that the present invention is taken to achieve the above object are as follows: carry out yellow crucian carp using mitochondrion DNA control area and lose
The method for passing credit analysis, includes the following steps:
Lysate is added in yellow crucian carp musculature sample to be cracked, then separates supernatant, obtains chondriogen
Group DNA；
Mitochondrial genomes DNA is carried out to the screening of target sequence；
Yellow crucian carp genetic analysis is carried out from screening results；
Wherein, lysate is the aqueous solution comprising NaOH, Tris-HCl, EDTA, NaGC and dimercaprol dimercaptopropanol；According to
It is present in the feature in cell independently of except genomic DNA and with annular form according to mitochondrial genomes DNA, in the lysate
Each ingredient can play gain effect, yellow crucian carp musculature sample cell and intracellular protein are quickly split
Solution, denaturation treatment, release mitochondrial genomes DNA, subtract whole gene group DNA extractive process, and can be reduced the drop of DNA
Solution thus it is possible to which easy rapidly obtain high quality, high-purity, the mitochondrial genomes DNA that can be used as amplification template, and obtains
Rate is high；
Target sequence include in mitochondrion DNA control area in whole or in part.Control zone complete sequence is expanded to tie for control zone
Structure analysis, amplification control zone hypervariable region are used for length polymorphism analysis.
Preferably, final concentration 0.03-0.05M, EDTA of final concentration 0.3-0.4M, Tris-HCl of NaOH in lysate
The final concentration 6.0-10.0mM of final concentration 4.0-5.0M, NaGC, the final concentration 0.2-0.5mM of dimercaprol dimercaptopropanol.
Preferably, the OD of mitochondrial genomes DNA260/OD280Between 1.83-1.92, OD230/OD260In 2.12-2.33
Between.
Preferably, the concentration > 73.5ng/ μ L of mitochondrial genomes DNA.
Preferably, screening is implemented using target sequence described in PCR amplification.
It is furthermore preferred that control zone leading portion hypervariable region primer are as follows: HJ primer-F:5 '-CACGCACTTAGCTCCAAGCTA-
3'；HJ primer-R:5 '-GACGATACCAGAGTAGTGACG-3 '；
It is furthermore preferred that hypervariable region control zone second half section primer are as follows: HJH primer-F:5 '-
CAGTCTTCTCTATTCGTCTAG-3'；HJH primer-R:5 '-GTTAGCGAGCTTGATTGCTG-3 '.
It is furthermore preferred that PCR reaction system: 0.25 μ L of rTaq, 211 μ of μ L, primer-R of μ L, primer-F of DNA profiling
L, DNTP 2 μ L, 10 × PCRbuffer 2 μ L, 16.75 μ L of deionized water.
It is furthermore preferred that PCR amplification program are as follows: 95 DEG C of denaturation 5min；95 DEG C of 30s, 52 DEG C of 405s, 72 DEG C of 40s, 40 are followed
Ring；72 DEG C of extension 5min, 4 DEG C of preservations.
Compared with prior art, the invention has the benefit that
Each ingredient that the present invention obtains in mitochondrial genomes DNA lysate can play gain effect, to yellow crucian carp flesh
Meat tissue sample cell and intracellular protein quickly cracked, denaturation treatment, releases mitochondrial genomes DNA,
Whole gene group DNA extractive process is subtracted, easy can rapidly obtain high quality, high-purity, the line that can be used as amplification template
Mitochondrial genes group DNA, and yield is high；The present invention is using yellow crucian carp mitochondrion DNA control area to yellow crucian carp hereditary information and genetic diversity
Property is analyzed, quick and precisely and simple and practical.
There is provided present invention employs above-mentioned technical proposal a kind of utilizes mitochondrion DNA control area to carry out yellow crucian carp genetic analysis
Method, compensate for the deficiencies in the prior art, reasonable design, easy operation.
Detailed description of the invention
Fig. 1 is the purity and yield of 1 Mitochondria DNA of test example of the present invention.
Specific embodiment
To facilitate the understanding of the present invention, a more comprehensive description of the invention is given in the following sections with reference to the relevant attached drawings.In attached drawing
Give section Example of the invention.But the invention can be realized in many different forms, however it is not limited to this paper institute
The embodiment of description.On the contrary, purpose of providing these embodiments is keeps the disclosure of invention more thorough and comprehensive.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.
An embodiment of the present invention provides a kind of side that yellow crucian carp genetic analysis is carried out using mitochondrion DNA control area
Method includes the following steps:
S1: lysate is added in yellow crucian carp musculature sample and is cracked, supernatant is then separated, obtains mitochondria base
Because of a group DNA；
S2: mitochondrial genomes DNA is carried out to the screening of target sequence；
S3: yellow crucian carp genetic analysis is carried out from screening results；
Wherein, lysate is the aqueous solution comprising NaOH, Tris-HCl, EDTA, NaGC and dimercaprol dimercaptopropanol；According to
It is present in the feature in cell independently of except genomic DNA and with annular form according to mitochondrial genomes DNA, in the lysate
Each ingredient can play gain effect, yellow crucian carp musculature sample cell and intracellular protein are quickly split
Solution, denaturation treatment, release mitochondrial genomes DNA, subtract whole gene group DNA extractive process, and can be reduced the drop of DNA
Solution thus it is possible to which easy rapidly obtain high quality, high-purity, the mitochondrial genomes DNA that can be used as amplification template, and obtains
Rate is high；
Target sequence include in mitochondrion DNA control area in whole or in part.Control zone complete sequence is expanded to tie for control zone
Structure analysis, amplification control zone hypervariable region are used for length polymorphism analysis.
In one of the embodiments, in lysate final concentration 0.3-0.4M, Tris-HCl of NaOH final concentration 0.03-
The final concentration 6.0-10.0mM of the final concentration 4.0-5.0M of 0.05M, EDTA, NaGC, the final concentration of dimercaprol dimercaptopropanol
0.2-0.5mM。
The OD of mitochondrial genomes DNA in one of the embodiments,260/OD280Between 1.83-1.92, OD230/
OD260Between 2.12-2.33.
The concentration > 73.5ng/ μ L of mitochondrial genomes DNA in one of the embodiments,.
The specific steps of acquisition mitochondrial genomes DNA include: in one of the embodiments,
S11: by volume mixing cabezon musculature sample with lysate for 1:2-5, grinding, then transfer liquid
Into centrifuge tube, it is placed in 90-95 DEG C of water bath and is incubated for 10-20min, then ice bath 10-30min, in 10000-
15000rpm/min is centrifuged 5-10min, takes supernatant, obtains DNA crude product；Wherein, the final concentration 0.3-0.4M of NaOH in lysate,
The final concentration 6.0-10.0mM of the final concentration 4.0-5.0M of final concentration 0.03-0.05M, EDTA of Tris-HCl, NaGC,
The final concentration 0.2-0.5mM of dimercaprol dimercaptopropanol；Further, lysate further includes that DNaseI digestion step is added, and helps to disappear
Except the residual of core DNA；
S12: isopropanol being mixed with DNA crude product, centrifugation DNA, to remove removing protein, then and will contain DNA's
Precipitating mixed with cleaning solution I, be then centrifuged again with further such that albumen precipitation and remove the albumen in DNA.Then it takes
Clearly, cleaning solution II is added and dehydrated alcohol, -20 DEG C of placement 6-12h promotes DNA to precipitate, to remove the impurity such as the albumen in DNA.
Then with the cation in 70-80% ethyl alcohol removal DNA, mitochondrial genomes DNA after purification is obtained；Wherein, cleaning solution I contains
There are the phosphate buffer of final concentration of 0.08-0.10M and the potassium acetate of final concentration of 0.01-0.02M；Cleaning solution II contains end
The sodium acetate of concentration 1.4-1.8M and the sodium thioglycolate of final concentration 0.3-0.4mM.The cleaning solution can be played with lysate matches
Cooperation is used, and the impurity such as isolating protein, RNA, carbohydrate, salt ion and polyphenols more can be preferably removed, and improves what extraction obtained
Mitochondrial genomes DNA purity, while protecting the integrality of mitochondrial genomes DNA.Further, it with cleaning solution I and is washing
During washing liquid II cleaning DNA, further includes that RNase A digestion step is added, assist in removing the residual of RNA, extract to obtain line grain
Body DNA electrophoretic band is a clear, neat, uniform band, and clear background has no that loading wells nearby has macromolecular DNA and preceding
Situations such as holding RNA hangover.
Screening is implemented using target sequence described in PCR amplification in one of the embodiments,.
Control zone leading portion hypervariable region primer in one of the embodiments, are as follows: HJ primer-F:5 '-
CACGCACTTAGCTCCAAGCTA-3'；HJ primer-R:5 '-GACGATACCAGAGTAGTGACG-3 '；
Hypervariable region control zone second half section primer in one of the embodiments, are as follows: HJH primer-F:5 '-
CAGTCTTCTCTATTCGTCTAG-3'；HJH primer-R:5 '-GTTAGCGAGCTTGATTGCTG-3 '.
PCR reaction system in one of the embodiments: 0.25 μ L of rTaq, 21 μ L of μ L, primer-F of DNA profiling,
Primer-R1 μ L, DNTP 2 μ L, 10 × PCRbuffer 2 μ L, 16.75 μ L of deionized water.
PCR amplification program in one of the embodiments, are as follows: 95 DEG C of denaturation 5min；95 DEG C of 30s, 52 DEG C of 405s, 72 DEG C
40s, 40 circulations；72 DEG C of extension 5min, 4 DEG C of preservations.
It is described in detail below in conjunction with specific embodiment.Following embodiment, such as non-specified otherwise then do not include except can not
The miscellaneous other components in addition to inevitable impurity avoided.In embodiment if not otherwise indicated using drug and instrument, it is
This field conventional selection.Test method without specific conditions in embodiment, according to normal conditions, such as in document, books
The method that the condition or manufacturer are recommended is realized.
Embodiment 1:
A method of yellow crucian carp genetic analysis being carried out using mitochondrion DNA control area, is included the following steps:
S1: lysate is added in yellow crucian carp musculature sample and is cracked, supernatant is then separated, obtains mitochondria base
Because of a group DNA；
S11: yellow crucian carp sample picked up from coastal area of china 7 places (Dongying, Yantai, Qingdao, Nantong, temperature in 2005-2013 years
State, Xiamen, the North Sea), totally 202 tails are individual (table 1), in laboratory sample after Morphological Identification, take fritter musculature in
It is fixed in 95% alcoholic solution, it is placed in -20 DEG C of refrigerators and saves for use；
The yellow crucian carp sample information of table 1
| Group | Sampling position | Affiliated sea area | Sampling time | Sample number |
| DY | Dongying | The Bohai Sea | 2007.05 | 45 |
| YT | Yantai | The Huanghai Sea | 2008.04 | 23 |
| QD | Qingdao | The Huanghai Sea | 2007.04 | 30 |
| NT | Nantong | The Huanghai Sea | 2013.01 | 29 |
| WZ | Wenzhou | The East Sea | 2008.03 | 33 |
| XM | Xiamen | The East Sea | 2013.03 | 20 |
| BB | The North Sea | The South Sea | 2013.01 | 22 |
S12: by volume mixing cabezon musculature sample with lysate for 1:4, and grinding, then transfer liquid is extremely
It in centrifuge tube, is placed in 92 DEG C of water baths and is incubated for 15min, then ice bath 20min, be centrifuged 8min in 12000rpm/min, take
Supernatant obtains DNA crude product；Wherein, final concentration 0.04M, EDTA of final concentration 0.35M, Tris-HCl of NaOH in lysate
The final concentration 7.5mM of final concentration 4.6M, NaGC, the final concentration 0.3mM of dimercaprol dimercaptopropanol；
S13: by volume mixing isopropanol with DNA crude product for 1:2, is centrifuged 5min, centrifugation in 10000rpm/min
Precipitate DNA；Then cleaning solution I will be added in the precipitating containing DNA by volume for 1:1.5 to mix, sufficiently dissolution precipitating, sets ice
Upper incubation 60min is centrifuged 5min in 10000rpm/min, takes supernatant；Cleaning solution II will be added in supernatant for 1:2 by volume again
And dehydrated alcohol, -20 DEG C of placement 12h are centrifuged 5min in 10000rpm/min, discard ethyl alcohol, 75% ethyl alcohol is then added,
Attack centrifuge tube is suspended in DNA in 75% ethyl alcohol, is then centrifuged 5min in 10000rpm/min, discards 75% second
Alcohol obtains DNA after purification；Wherein, cleaning solution I contains the phosphate buffer of final concentration of 0.08M and final concentration of
The potassium acetate of 0.015M；Cleaning solution II contains the sodium acetate of final concentration 1.5M and the sodium thioglycolate of final concentration 0.37mM；
S2: it is divided to the amplification of mitochondrial genomes DNA control zone complete sequence to two sections of progress PCR amplifications, PCR amplification primer sequence
Column are shown in Table 2, and PCR amplification system is as follows:
PCR amplification program are as follows: 95 DEG C of denaturation 5min；95 DEG C of 30s, 52 DEG C of 405s, 72 DEG C of 40s, 40 circulations；72 DEG C of extensions
5min, 4 DEG C of preservations；2 μ L PCR products are taken to carry out 1.5% agarose gel electrophoresis (U=300V), using QIAquick Gel Extraction Kit to mesh
Band carry out recovery purifying, the PCR product of recovery purifying is carried out just using 3730 type DNA sequence analysis instrument of ABI Prism
Anti-chain sequencing；
2 joint sequence of table, PCR amplification primer sequence
| Primer | Primer |
| HJ primer-F | 5’-CACGCACTTAGCTCCAAGCTA-3’ |
| HJ primer-R | 5’-GACGATACCAGAGTAGTGACG-3’ |
| HJH primer-F | 5’-CAGTCTTCTCTATTCGTCTAG-3’ |
| HJH primer-R | 5’-GTTAGCGAGCTTGATTGCTG-3’ |
S3: yellow crucian carp genetic analysis is carried out from screening results；
Sequence is compared, edit and is analyzed using DNASTAR software package (DNASTAR, Inc., Madison, USA),
And manual synchronizing is aided with to result；It is for statistical analysis to repetitive sequence frequency using 2010 software of Microsoft Excel；
Repetitive sequence secondary structure is analyzed using 4.3 software of RNAstructure and calculates the free energy of its release；Use SPSS
18.0 softwares carry out Chi-square Test (Chi-square test) to repetitive sequence frequency, to calculate repetitive sequence between group two-by-two
The significance of difference of frequency.
According to the theory of Birky etc., different levels genetic diversity is calculated.Since there is no individuals by yellow crucian carp control zone DNA
Interior heterogeneity phenomenon, a internal genetic diversity Kb=0；Genetic diversity K between individual in groupc=1-Σ Xi 2, XiRefer to a certain
DNA type (difference in length) is in intragroup frequency；Overall genetic diversity Kt=1-Σ Yi 2, Yi refers to a certain DNA type (length
Difference) in all intraindividual frequencies.According to formula Cip=(mean Kc–Kb)/KtAnd Cpt=(Kt–mean Kc)/KtIt calculates and loses
Pass difference proportion between group in group.
After carrying out editor and manual synchronizing to yellow crucian carp Control region sequence, obtaining yellow crucian carp complete sequence length is 1192-1271bp,
Identify termination sequence area, central conserved region and conserved sequence area.Carried out extension terminate correlated series ETAS (TACAT and
ATGCA it) analyzes with 6 conservative fragments (CSB-D, E, F and CSB-1,2,3) and provides the characteristic sequence of each conservative fragments.To 202
Tail Huang crucian carp individual control zone hypervariable region sequence length is counted, and does not find that length heteroplasmy is existing in vivo in yellow crucian carp as the result is shown
As length polymorphism only occurs between individual.It is for statistical analysis to yellow crucian carp individual repetitive sequence frequency, find North Sea group
In 7 repetition rate highests, 6 repetition rate highests of other six clusters；Hereditary difference 79.77% caused by repetitive sequence
From in group between individual；There are extremely significant with other six clusters for North Sea group repetitive sequence frequency as the result is shown for Chi-square Test
Difference.Repetitive sequence analysis may be used for yellow crucian carp population genetic study as householder method.
Embodiment 2:
Difference from example 1 is that: extracting mitochondrial genomes DNA lysate further includes that 5mg/mL is added
DNaseI。
Embodiment 3:
Difference from example 1 is that: when extracting mitochondrial genomes DNA, clear with cleaning solution I and cleaning solution II
During washing DNA, the RNase A including final concentration 20ug/mL is added digests 60min step at 37 DEG C.
Embodiment 3:
With embodiment 2 the difference is that: during cleaning DNA with cleaning solution I and cleaning solution II, including be added eventually
The RNase A of concentration 20ug/mL digests 60min step at 37 DEG C.
Comparative example 1:
Difference from example 1 is that: mitochondrial genomes DNA, which is extracted, with lysate is free of dimercaprol dimercaptopropanol.
Comparative example 2:
Difference from example 1 is that: mitochondrial genomes DNA, which is extracted, with cleaning solution II is free of sodium thioglycolate.
Test example 1:
1. extracting the purity and concentration of mitochondrial genomes DNA
135V electrophoresis 20min, EB dyeing, with ultraviolet gel in 1 × TAE electrophoretic buffer with 0.8% Ago-Gel
Imaging system photograph.The pure of obtained DNA is extracted with NANODROP 1000TM UV/VIS Spectrophotometer measurement
Spend (OD260/OD280And OD230/OD260) and concentration.Wherein, OD260/OD280Between 1.8-2.0, OD230/OD260In 2.0-2.5
Between indicate to extract obtained DNA purity it is higher；OD260/OD280Illustrate between 1.8-2.0 DNA sample do not have protein and
RNA pollution, purity are good；OD260/OD280Illustrate to contain protein contamination, OD in DNA sample less than 1.8260/OD280It is said greater than 2.0
Contain RNA pollution or DNA fragmentation fracture in bright DNA sample；OD230/OD260Illustrate in DNA sample between 2.0-2.5 without more
The pollution of the impurity such as sugar, phenols and salt ion, purity are good.The purity for the DNA that embodiment 1-4, comparative example 1-2 are extracted and dense
Degree such as Fig. 1 (individual in DY group), it can be seen that embodiment 1 extracts the OD of obtained DNA260/OD280For 1.85, OD230/
OD260It is 2.18, embodiment 2 extracts the OD of obtained DNA260/OD280For 1.86, OD230/OD260It is 2.25, embodiment 3 is extracted
The OD of obtained DNA260/OD280For 1.89, OD230/OD260It is 2.31, embodiment 4 extracts the OD of obtained DNA260/OD280For
1.83、OD230/OD260It is 2.14, this illustrates that the DNA purity that embodiment 1-4 is extracted is higher；The DNA's that comparative example 1 is extracted
OD260/OD280Greater than 2.0, OD230/OD260Greater than 2.5, illustrate in DNA containing protein, RNA or DNA fragmentation fracture, polysaccharide,
The pollution of the impurity such as phenols and salt ion；The OD for the DNA that comparative example 1-2 is extracted260/OD280Less than 1.8, OD230/OD260It is greater than
2.5, illustrate the pollution containing impurity such as protein, RNA, polysaccharide, phenols and salt ions in DNA；Embodiment 1-4 is extracted
The concentration of DNA is greater than comparative example 1-2.The above result shows that the purity for the extraction mitochondrial genomes DNA that embodiment 1-4 is extracted
It is better than comparative example 1-2 with yield.It further relating to, each ingredient in lysate of the embodiment of the present invention can play gain effect,
Yellow crucian carp musculature sample cell and intracellular protein are quickly cracked, denaturation treatment, discharges mitochondrial base
Because of a group DNA, the mitochondrial genomes DNA of high quality, high-purity is obtained, and yield is high；The embodiment of the present invention with wash liquid can be with
Lysate plays mating reaction, more can preferably remove the impurity such as isolating protein, RNA, carbohydrate, salt ion and polyphenols, mention
Height extracts obtained mitochondrial genomes DNA purity.
The prior art of routine techniques dawn known to those skilled in the art in above-described embodiment, therefore herein no longer in detail
It repeats.
The above embodiments are only used to illustrate the present invention, and not limitation of the present invention, the ordinary skill people of this field
Member can also make a variety of changes and modification without departing from the spirit and scope of the present invention.Therefore, all equivalent
Technical solution also belong to scope of the invention, scope of patent protection of the invention should be defined by the claims.
Sequence table
<110>Zhejiang Ocean university
<120>method for carrying out yellow crucian carp genetic analysis using mitochondrion DNA control area
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
cacgcactta gctccaagct a 21
<210> 2
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gacgatacca gagtagtgac g 21
<210> 3
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<212> DNA
<213>artificial sequence (Artificial Sequence)
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cagtcttctc tattcgtcta g 21
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<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
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gttagcgagc ttgattgctg 20
Claims (9)
1. the method for carrying out yellow crucian carp genetic analysis using mitochondrion DNA control area, characterized by the following steps:
Lysate is added in yellow crucian carp musculature sample to be cracked, then separates supernatant, obtains mitochondrial genomes
DNA；
The mitochondrial genomes DNA is carried out to the screening of target sequence；
Yellow crucian carp genetic analysis is carried out from screening results；
Wherein, the lysate is the aqueous solution comprising NaOH, Tris-HCl, EDTA, NaGC and dimercaprol dimercaptopropanol；
The target sequence include in mitochondrion DNA control area in whole or in part.
2. the method according to claim 1 for carrying out yellow crucian carp genetic analysis using mitochondrion DNA control area, feature exist
In: the final concentration of final concentration 0.03-0.05M, EDTA of final concentration 0.3-0.4M, Tris-HCl of NaOH in the lysate
The final concentration 6.0-10.0mM of 4.0-5.0M, NaGC, the final concentration 0.2-0.5mM of dimercaprol dimercaptopropanol.
3. the method according to claim 1 or 2 for carrying out yellow crucian carp genetic analysis using mitochondrion DNA control area, special
Sign is: the OD of the mitochondrial genomes DNA260/OD280Between 1.83-1.92, OD230/OD2602.12-2.33 it
Between.
4. the method according to claim 1 or 2 for carrying out yellow crucian carp genetic analysis using mitochondrion DNA control area, special
Sign is: the concentration > 73.5ng/ μ L of the mitochondrial genomes DNA.
5. the method according to claim 1 for carrying out yellow crucian carp genetic analysis using mitochondrion DNA control area, feature exist
In: the screening is implemented using target sequence described in PCR amplification.
6. the method according to claim 5 for carrying out yellow crucian carp genetic analysis using mitochondrion DNA control area, feature exist
In: control zone leading portion hypervariable region primer are as follows: HJ primer-F:5 '-CACGCACTTAGCTCCAAGCTA-3 '；HJ
Primer-R:5 '-GACGATACCAGAGTAGTGACG-3 '.
7. the method according to claim 5 for carrying out yellow crucian carp genetic analysis using mitochondrion DNA control area, feature exist
In: the hypervariable region control zone second half section primer are as follows: HJH primer-F:5 '-CAGTCTTCTCTATTCGTCTAG-3 '；HJH
Primer-R:5 '-GTTAGCGAGCTTGATTGCTG-3 '.
8. the method according to claim 5 for carrying out yellow crucian carp genetic analysis using mitochondrion DNA control area, feature exist
In the PCR reaction system: rTaq 0.25 μ L, 2112 μ L of μ L, DNTP of μ L, primer-R of μ L, primer-F of DNA profiling,
10 × PCRbuffer, 2 μ L, 16.75 μ L of deionized water.
9. the method according to claim 5 for carrying out yellow crucian carp genetic analysis using mitochondrion DNA control area, feature exist
In: the PCR amplification program are as follows: 95 DEG C of denaturation 5min；95 DEG C of 30s, 52 DEG C of 405s, 72 DEG C of 40s, 40 circulations；72 DEG C of extensions
5min, 4 DEG C of preservations.
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