CN106498044A - A kind of quantitative detecting method of dusky white prawn mitochondrial genome copy number - Google Patents
A kind of quantitative detecting method of dusky white prawn mitochondrial genome copy number Download PDFInfo
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Abstract
The invention discloses a kind of quantitative detecting method of dusky white prawn mitochondrial genome copy number, comprises the steps：Select adenine nucleotide translocase gene (ANT) and ATP6 genes as dusky white prawn nuclear genome and the single copy gene of mitochondrial genome respectively, design is suitable to the primer of ANT and ATP6 genes real-time quantitative PCR reaction and Taqman fluorescence probe；The standard substance of ANT and ATP6 genes real-time quantitative PCR reaction are prepared, and by drawing standard curve (as shown in Figure of abstract), with standard curve as standard, quantitative and measuring and calculating are carried out to the copy number of the mitochondrial genome in sample to be tested cell.The present invention can accurate detection by quantitative dusky white prawn tissue samples cell Mitochondria genome copy number, high, simple to operate have the advantages that specificity, only need common fluorescent quantitative PCR apparatus just complete the quantitative of dusky white prawn mitochondrial genome copy number.
Description
Technical field：The invention belongs to technical field of molecular biology, is related to a kind of dusky white prawn mitochondrial genome and copies
The quantitative detecting method of shellfish number.
Background technology：
Existing research shows that mitochondrion is the organelle enlivened very much in biological life activity, in growth and development process
In, not only its form can occur certain change, and significantly change quantitatively can also occur.Substantially, in growth promoter
Different phase, organism are necessarily faced with the propagation of cell and apoptotic process, and the cyclic activity of this cell, also adjust
Intracellular mitochondrion activity.Current research shows, when the cell of living organism is under different situations, line grain therein
Body genome (mtDNA) copy number can also change, and this change is widely different, and an intracellular mtDNA is copied
Shellfish number can be from several to thousands of.This depends primarily on demand difference of the cell to energy metabolism, under normal circumstances, consumption
In histiocyte that can be high, mtDNA contents are higher, whereas a lower.Such as in isolated culture, in home mouse embryo fibroblasts
MtDNA quantity can reduce with the prolongation of incubation time；In disease and ageing process, cell Mitochondria genome is copied
Shellfish number can also be reduced.In addition, a lot of research shows, each side such as the sexual cell generation of organism, ontogeny
Change of the face along with mtDNA copy numbers.During the egg cell development of Mus, mtDNA in a cell even can be by
Step accumulation reaches 175000 copies, and if mtDNA number of copies is not enough, then can cause the sterile of ovum, it is seen that mtDNA
Copy number has important effect in growth and development process, determines mtDNA copy numbers and will be seen that the growth of bion is sent out
Situation is educated, disease is monitored and is detected, or even as the genetic assistant breeding that a new criterion for breeding is aquatic biological
Guidance and help are provided.
But how to determine mtDNA copy numbers is but a difficult problem, its reason is mitochondrial device very little, its gene
DNA is less for group, at the same one intracellular can have many set mitochondrial genome DNA, therefore by means of ultramicroscope or dye
The method of the physics such as color and chemistry quantitative counting difficult to realize.At present, developing into for Real-time quantitative PCR solves this problem
Thinking is provided, and such as mtDNA copy numbers in different quality human sperm's cell is solved the problems, such as using this technology.Its
In principle mainly by respectively finding a kind of single copy gene in Matrix attachment region and mitochondrial genome, then by preparing
The relative ratio of standard curve, quantitative analyses mitochondrial genome copy number and Matrix attachment region copy number；Due to a cell only
There is a Matrix attachment region, therefore this ratio may be considered the average of the mitochondrial genome DNA that has in a cell
Copy number.As in difference biology mtDNA and Matrix attachment region, single copy gene is had differences in species and DNA sequence, therefore
Above-mentioned mitochondria DNA copy number detection method has species specificity, needs to copy for different biological its distinctive mtDNA that develop
Shellfish number detection method.In aquatic biological, mtDNA copy numbers detection method is only had been reported that in minority Fish at present, has no ocean first
Shell apoplexy due to endogenous wind has the research of correlation.
Dusky white prawn (Exopalaemon carinicauda) is a kind of important marine economy shrimps, with breeding week
Phase short (3-4 month can breed for 1 generation), fast growth (in 1 year can many batches of cultivation) and environmental suitability extensively (extensively warm, wide salt and
Wide food) the advantages of, for Chinese prawn etc. just breeds the large-scale shrimps of a generation in 1 year, can observe at short notice
The growth characteristics of different growing periods dusky white prawn, therefore launch genetic breeding and the heredity of shell-fish basis with this shrimp as living model
Research in terms of is more and more.As " mitochondrion " of cellular energy factory, its genomic DNA (mtDNA) number with raw
The Physiology and biochemistry situation of thing and inherited characteristic are closely related, so if the quantitative detecting method of its copy number can be invented, so that it may
To follow the trail of corresponding physiological activity, it is also possible to study the inherited characteristic of Different Individual or family, the selection-breeding for new varieties provides skill
Art is instructed.
Content of the invention：
The present invention describes a kind of quantitative detecting method of dusky white prawn mitochondrial genome copy number, is quantitative analyses ridge
Mitochondrial genome copy number in tail white shrimp different tissues cell provides method, and main contents are selection dusky white prawn respectively
A single copy gene in nuclear DNA and mitochondrial genome, is suitable to corresponding base according to single copy gene sequential design
Primer and Taqman probes because of real-time quantitative PCR reaction；Each gene real-time quantitative PCR primer is expanded interval sequence fragment
It is transferred in plasmid, through amplification culture plasmid, extracts containing the copy for proceeding to plasmid after the plasmid of PCR fragment, measurement are extracted
Number, obtain for make fluorescence real-time quantitative PCR reaction in DNA profiling reference standard product；Anti- in same real-time quantitative PCR
Ying Zhong, carries out quantitative analyses with unknown sample and these standard substance as DNA profiling respectively, with the Ct value systems that standard substance PCR is expanded
Make standard curve, compared with the curve, it is possible to calculate the copy number of nuclear DNA and mitochondrion base in unknown sample
Copy number because of group DNA；As the genome of dusky white prawn is diploid, the therefore mitochondrial genome DNA in a cell
Quantity just can be obtained by equation below：Mitochondrial genome copy number=2 × mitochondrial genome in each cell is copied
Shellfish number/nuclear DNA copy number.
The present invention needs to be optimized screening to following experiment condition：
1st, the screening of single copy gene：Dusky white prawn nuclear genome single copy gene is from ANT (adenyltransferase)
With selected in two genes of Try (TYR), final determine that ANT genes are optimal candidate genes；Mitochondrion list copies base
Because being selected from ATP6, ND1, COII and D-loop, final determination ATP6 genes are optimal candidate genes.
2nd, primer screening：3 pairs of primers are respectively designed for ANT and ATP6 genes, verifies that each single copy gene is each really through PCR
The primer pair of fixed 1 pair of suitable real-time quantitative PCR reaction.The primer pair is short-movie section sequence, and the fragment length of PCR amplifications is in 90-
Between 130bp, such purpose is：PCR amplification efficiencies of standard substance and as synchronous as possible by the amplification efficiency of sample product are made, with
Just sensitivity and the accuracy of detection are improved.
3rd, the optimization of Taqman concentration and probe concentrations：The concentration for reacting middle probe in fluorescence real-time quantitative PCR is directly affected really
Result is tested, five Concentraton gradient 0.2ng/ μ l, 0.4ng/ μ l, 0.6ng/ μ l, 0.8ng/ μ l, 1ng/ μ l of probe are tested altogether,
The final optimum concentration for determining probe is 0.6ng//μ l.
The invention has the beneficial effects as follows：
The copy number of each cell mitochondrial genome in the examined sample tissue of present invention energy direct quantitative dusky white prawn,
So as to realize the quick detection of dusky white prawn Different Individual, tissue Mitochondria genome copy numbers；The inventive method specificity
Height, simple to operate, only need quantitative real time PCR Instrument just complete Different Individual, the quick detection of tissue Mitochondria genome.
Description of the drawings：
Fig. 1 is that the fluorescence real-time quantitative PCR for copying the making of ANT genes standard substance by dusky white prawn nuclear genome list is anti-
Answer standard curve：In figure, abscissa shows that the extension rate of standard substance, vertical coordinate show Ct values, and the point on figure bend is certainly
Under supreme Ct values when having indicated respectively that extension rate is 10,1,0.1,0.01,0.001 and 0.0001；
Fig. 2 is to copy the fluorescence real-time quantitative PCR that ATP6 genes standard substance make by dusky white prawn mitochondrial genome list
Reaction normal curve：In figure, abscissa shows that the extension rate of standard substance, vertical coordinate show Ct values, the point on figure bend
Ct values when having indicated respectively that extension rate is 10,1,0.1,0.01,0.001 and 0.0001 from bottom to top.
Specific embodiment：
The present invention can be realized by following operating procedure：
1st, (two are selected here according to dusky white prawn Matrix attachment region (nDNA) single copy gene：Adenine nucleotide translocase gene
(ANT) and trypsase gene (trypsin)) and mitochondrial genome (mtDNA) single copy gene (select four：ATP6、
ND1, COII and D-loop) sequence, the pcr amplification primer thing for being directed to each genetic fragment accordingly is designed, clip size is 90-
Between 130bp.React through Standard PCR, the expanding effect of primer designed by checking, and primer amplification of DNA fragments is sequenced with true
Fixed be whether corresponding gene purpose amplification region, according to the definition and the specificity of amplification of amplified band, and combine these
The quantitative stability features in the reaction of follow-up fluorescence real-time quantitative PCR of single copy gene, the gland on final choice Matrix attachment region
ATP6 genes in nucleotide-transferase gene (ANT) and mitochondrial genome as the present invention used in single copy gene (
The copy number of individual single copy gene represents the copy number of its place genomic DNA).From two above gene obtain for reality
When quantitative PCR analysis primer sequence be：Forward primer (ANT-F) sequence of ANT genes be 5 '-
GGGTTGAAAGAGGGAATG-3 ', reverse primer (ANT-R) sequence is：5’-CAAGCATTATATTTTGTACAGAC-3’；
Forward primer (ATP6-F) sequence of ATP6 genes be 5 '-TGCATGGTCCTCTCTCACGCT-3 ', reverse primer (ATP6-R)
Sequence is 5 '-AGTGAGACGAAGACAAGGGTGCC-3 '.
2nd, according to the DNA sequence of primer specificity amplification region, the Taqman probes for separately designing ANT and ATP6 are as follows：
It is 5 '-CCTTTTAGGGCCAGCCAACC-3 ' that ANT is 5 '-CTTACTACAGGGCATGTCGCTCC-3 ', ATP6；Deliver company,
On 5 ' end labellings of probe, TAMRA (a kind of fluorescent quenching group) on labellings is held in FAM (a kind of fluorescence group), 3 ' respectively.
3rd, prepared by plasmid standard template：The ANT gene primers in above-mentioned steps " 1 " are utilized respectively to (ANT-F and ANT-
R) and ATP6 gene primers to (ATP6-F and ATP6-R) by conventional PCR method expand dusky white prawn muscular tissue in extract
STb gene (containing nuclear DNA and mitochondrial genome DNA), carries out low melting-point agarose respectively the pcr amplification product for obtaining
Gel purified recovery；Illustrated according to pEASY-T3 support agents box, the corresponding fragment that reclaims be connected in plasmid vector,
Plasmid is transformed into E.coli DH5 α competent cells further, using the screening of blue white macula, identification, positive colony bacterium is obtained
Strain.Picking contains the monoclonal bacterium colony amplification culture of positive colony, extracts recombiant plasmid with plasmid extraction kit.Using nucleic acid
Protein assay (Nanodrop 2000) determines the concentration of the recombiant plasmid for extracting, and is diluted to 20ng/ μ l, further with double
Chain DNA copy number computer (http：//cels.uri.edu/gsc/cndna.html) by plasmid mass conversion into copy number
(copy number/μ l), this are nDNA the and mtDNA standard substance templates for being respectively used to the analysis of ANT and ATP6 gene quantifications.
Prepared by the standard substance for the 4th, being used for Real-time PCR Analysis：With without DNAase ddH2O respectively above-mentioned nDNA and
MtDNA standard substance template presses 10 times of gradient dilutions into 107、106、105、104、103、102、101(each gradient final volume is copy
1000μl).Respectively with this sample after diluting as DNA profiling, expand on fluorescence real-time quantitative PCR instrument, just can produce
The standard curve of corresponding gene, requires that the correlation coefficient of standard curve is reached between 0.997-0.999 herein, otherwise needs again
Make standard curve.
5th, the detection of unknown sample Mitochondria genome copy numbers：First the DNA concentration of unknown sample to be measured is uniformed
For 20ng/ μ l, then on same PCR Sptting plates, while the real-time quantitative PCR reaction of standard substance and unknown sample is executed,
Concrete pressing operates as follows：PCR reaction systems cumulative volume is 20 μ l, including 10 μ lPremix Ex TaqTM
(TaKaRa), the forward and reverse primers of 0.8 μ l, 0.4 μ l 2 × Rox Reference Dye, 2 μ l template DNAs, 1 μ l Taqman are visited
Pin (0.6ng/ μ l), finally uses ddH2O complements to 20 μ l cumulative volumes.Each DNA sample arranges 3 repetitions, each PCR Sptting plate
Upper 3 blanks (without any DNA profiling) of setting simultaneously.Response procedures are：95 DEG C of denaturations 20s；Subsequent 95 DEG C of degeneration
1s, 60 DEG C of extension 20s, totally 40 circulations.
6th, mtDNA cubages
According to the standard curve that plasmid standard is obtained, the mtDNA copy numbers in unknown testing sample and nDNA are copied
Number carries out quantitative respectively, as dusky white prawn genome is diploid, therefore can calculate averagely by following publicity that each is thin
The copy number of mtDNA in born of the same parents：MtDNA copy numbers/cell=2 × ATP6 copy numbers/ANT copy numbers.
Claims (4)
1. a kind of quantitative detecting method of dusky white prawn mitochondrial genome copy number, comprises the steps：1) select spine end white
In adenine nucleotide translocase gene (ANT) and mitochondrial genome in shrimp nuclear genome, ATP6 genes are used as respective genome
Single copy gene；2) fluorescence real-time quantitative PCR of nuclear genome and mitochondrial genome single copy gene is separately designed
The primer of amplified reaction and Taqman fluorescence probe；3) Matrix attachment region, the reality of mitochondrial genome single copy gene are made respectively
When quantitative PCR reaction standard substance and standard curve；4) respectively with DNA sample to be checked and Matrix attachment region, mitochondrial genome list
Copy gene standard substance are the template of real-time quantitative PCR reaction, carry out TaqMan quantitative fluorescent PCR reactions, according to standard substance system
Make standard curve, nuclear genome single copy gene and mitochondrial gene in sample to be checked is calculated according to standard curve further
The copy number of group single copy gene；5) the average mitochondrial gene in each cell of sample tissue to be checked is obtained according to equation below
Group copy number：Mitochondrial genome copy number/cell=2 × mitochondrion single copy gene copy number/Matrix attachment region list copy base
Because of copy number.
2. the quantitative detecting method of a kind of dusky white prawn mitochondrial genome copy number according to claim 1, its feature
The single copy gene for being selection is adenine nucleotide translocase gene (ANT) and the line in the nuclear genome of dusky white prawn respectively
ATP6 genes in mitochondrial genes group.
3. the quantitative detecting method of a kind of dusky white prawn mitochondrial genome copy number according to claim 1, its feature
It is that selected single copy gene is used for the primer pair of fluorescence real-time quantitative PCR analysis and is respectively：The forward primer of ANT genes
(ANT-F) sequence be 5 '-GGGTTGAAAGAGGGAATG-3 ', reverse primer (ANT-R) sequence be 5 '-
CAAGCATTATATTTTGTACAGAC-3’；Forward primer (ATP6-F) sequence of ATP6 genes be 5 '-
TGCATGGTCCTCTCTCACGCT-3 ', reverse primer (ATP6-R) sequence are 5 '-AGTGAGACGAAGACAAGGGTGCC-3 '.
4. the quantitative detecting method of a kind of dusky white prawn mitochondrial genome copy number according to claim 1, its feature
It is that selected single copy gene is used for the Taqman probes of fluorescence real-time quantitative PCR analysis and is respectively：ANT is 5 '-
CTTACTACAGGGCATGTCGCTCC-3 ', ATP6 are 5 '-CCTTTTAGGGCCAGCCAACC-3 '；And respectively the 5 ' of probe
End is modified through FAM, 3 ' ends are modified through TAMRA.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486959A (en) * | 2018-10-23 | 2019-03-19 | 浙江海洋大学 | The Variations of liver mtDNA copy number in Sepiella maindroni aging course |
| CN109913535A (en) * | 2017-12-12 | 2019-06-21 | 上海市刑事科学技术研究院 | The method of fluorogenic quantitative detection Matrix attachment region copy number and human mitochondria gene group copy number |
-
2016
- 2016-10-18 CN CN201610906439.XA patent/CN106498044A/en active Pending
Non-Patent Citations (3)
| Title |
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| GAO,H ET AL.: "Palaemon carinicauda adenine nucleotide translocase (ANT) mRNA, complete cds", 《GENBANK: KP892663.1》 * |
| SHEN,X ET AL.: "Exopalaemon carinicauda mitochondrion, complete genome", 《NCBI REFERENCE SEQUENCE: NC_012566.1》 * |
| 孙懿等: "荧光定量PCR精确检测人胚胎干细胞线粒体DNA拷贝数方法的建立", 《中国细胞生物学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109913535A (en) * | 2017-12-12 | 2019-06-21 | 上海市刑事科学技术研究院 | The method of fluorogenic quantitative detection Matrix attachment region copy number and human mitochondria gene group copy number |
| CN109486959A (en) * | 2018-10-23 | 2019-03-19 | 浙江海洋大学 | The Variations of liver mtDNA copy number in Sepiella maindroni aging course |
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Application publication date: 20170315 |