CN108048457B - Nested amplification primer for mitochondrial control region of Baijixi myna and amplification, cloning and sequencing methods - Google Patents
Nested amplification primer for mitochondrial control region of Baijixi myna and amplification, cloning and sequencing methods Download PDFInfo
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Abstract
The invention discloses a Baiding xi myna mitochondrial control region amplification nested primer and an amplification, cloning and sequencing method, belonging to the technical field of biology, wherein 6 groups of amplification nested primers are adopted to carry out 2 times of PCR amplification on a Baiding xi myna mitochondrial control region, the product of the first round of PCR can be directly sequenced, the fragment amplified by the second round of PCR is combined with an agarose gel recovery purification rapid cloning technology, so that the purity and the concentration of a sequencing template can be greatly improved, the heterogeneity in the sequencing process is eliminated, the problem of sequencing failure caused by a polynucleotide sequence is effectively solved, the workload of an operator is greatly reduced by combining direct sequencing with cloning sequencing, the secondary structure of the amplification nested primer is few, the conformation is stable, and the 3' tail ends of 6 nested primers avoid the introduction of A base, so that the risk of nonspecific amplification is greatly reduced.
Description
Technical Field
The invention belongs to the technical field of biology, relates to the technical field of amplification and cloning, and particularly relates to a nested amplification primer of a mitochondrial control region of Baijixi myna and an amplification, cloning and sequencing method.
Background
The Baiyingxi myna (Chaimarrorn leucocephalus) is used as an important wild bird, has a wide distribution range, and is beneficial to guiding the protection of the Baiyingxi myna wild resource by researching the biological diversity of the Baiyingxi by a molecular means.
The Baiding xi myna belongs to Bromus eye 40543in taxonomy, and has the advantages of wide variety, various forms and wide distribution; there is considerable controversy regarding the phylogenetic relationship of birds between or within genera of this family. The avian mitochondrial genome has the following advantages when phylogenetically analyzed compared to the nuclear genome: the molecular weight is small, the gene arrangement is compact, the maternal inheritance is strict, the tissue specificity is absent, the evolution rate is high, and the like, so that the mitochondrial DNA is widely used as a molecular marker for researching the phylogenetic relationship of birds. Wherein the mitochondrial control region (also known as D-loop) is the longest non-coding region in the mitochondrial genome and regulates replication and transcription initiation of mitochondrial DNA. Due to the large sequence change of the mitochondrial control region, the molecular marker can be used as an ideal molecular marker for population genetics to research the population characteristics of the Baiding xi myna.
A large number of polynucleotide tandem repeat sequences exist in a mitochondrial control region of the Baijixi myna, the effective reading length of a sequencing reaction is 600-800bp in the DNA sequencing process, and the repeated polynucleotide sequences can cause the change of a secondary conformation of DNA, so that the sequencing cannot be normally extended, signals fail to be early, and the sequencing reaction is interrupted to cause sequencing failure. Meanwhile, as the mitochondrial DNA is highly methylated, the PCR primer is possibly poor in specificity and is not suitable for sequencing, so that the sequencing fails and molecular data for research cannot be obtained. The two technical problems are great difficulties in developing the Muscicapidae bird mitochondrial control region research exemplified by Baiding xi myna, and the difficulties are mainly reflected in that: specific primers for amplification, an amplification method, a cloning method and a sequencing process.
Disclosure of Invention
According to the defects of the prior art, the technical problem to be solved by the invention is to provide a nested amplification primer of a mitochondrial control region of Baiding xi myna and an amplification, cloning and sequencing method, and in order to solve the technical problem, the technical scheme adopted by the invention is as follows:
an amplification nested primer of a leucotrichine myna mitochondrial control region, wherein the amplification nested primer comprises a specific nested primer shown in SEQ ID NO.1-6,
SEQ ID NO.1：ATCTCCAACTCCCAAAGCT；
SEQ ID NO.2：CAAACTGGGATTAGATACC；
SEQ ID NO.3：TCTCACGAGAACCGAGCTAC；
SEQ ID NO.4：ATCTTCCTCTTGACATGTCC；
SEQ ID NO.5：GGTATTTTCAACTAAACTACTT；
SEQ ID NO.6：AGGGTATGTACTCTCTGCATCG。
preferably, the amplification nested primer is synthesized by Anhui, a general biological systems, Inc. at a concentration of 5. mu. mol.
A method for amplifying and whitening a Yangxi myna mitochondrial control region by using the amplification nested primer comprises the following specific steps:
1) taking a sample: taking the muscles of the Baiding stream myna with the number K0017;
2) extracting genes: extracting total genes by adopting a phenol and chloroform extraction method, shearing muscle tissue with the number K0017, placing the sheared muscle tissue into a sterilized EP tube, adding a DNA extracting solution and RNaseA into the EP tube, and shaking uniformly in a water bath at the temperature of 37 ℃ for 1 h; adding protease K, shaking, and placing in 50 deg.C water bath until the muscle is completely digested; cooling to room temperature, adding saturated phenol with the same volume, and shaking until the water phase is mixed with the phenol phase to form emulsion; centrifuging, and extracting the upper water phase with phenol for 1-2 times; adding chloroform isoamyl alcohol with the same volume to the phenol extraction solution, shaking uniformly, performing centrifugal separation, then repeating chloroform extraction operation on the upper water phase for 1 time, taking the upper water phase, adding NaAC with the volume of 1/5 and precooled absolute ethyl alcohol with the volume of 2 times, and lightly shaking at room temperature and placing in a freezer with the temperature of-20 ℃ for freezing for 3 hours; centrifuging for 15min, removing supernatant, rinsing with circulating ethanol, centrifuging for 15min for 2 times, air drying the centrifugate, adding 1 × TE into the centrifugate to dissolve DNA, storing the solution in a freezer at-40 deg.C, and subjecting the DNA solution to agarose gel electrophoresis;
3) two times of PCR amplification: performing first PCR amplification, adding Dream-Taq buffer, an amplification nested primer 1, dNTPmix, Dream-Taq enzyme, extracted DNA, dimethyl sulfoxide and double distilled water into an EP tube, and performing pre-denaturation for 3min at 94 ℃; then, 36 cycles were performed, which included: denaturation at 94 ℃ for 30s, annealing and extension; finally, carrying out extension for 8min at 72 ℃, carrying out agarose gel electrophoresis detection, and sequencing the amplification products and corresponding amplification primers, wherein the group of the amplification nested primers 1 is the primers No.1 and No.4, the product number is B1-a, the group is the primers No.2 and No.3, the product number is B1-B, and each group is repeatedly amplified for 3 times;
and (3) performing second PCR amplification: adding Dream-Taq buffer, amplification nested primer 2, dNTPmix, Dream-Taq enzyme, a first PCR amplification product B1-a, dimethyl sulfoxide and double distilled water into an EP tube, and then performing pre-denaturation for 3min at 94 ℃; then, 36 cycles were performed, which included: denaturation at 94 ℃ for 30s, annealing and extension; and finally, carrying out extension at 72 ℃ for 8min, and then carrying out agarose gel electrophoresis detection, wherein the amplification nested primers 2 are primers No.5 and No.6, the product number is B2, and the amplification is repeated for 3 times.
Preferably, the annealing temperature is 55 ℃ and the annealing time is 30 s.
Preferably, the extension temperature of the first PCR amplification is 72 ℃, and the extension time is 90 s; the extension temperature of the second PCR amplification was 72 ℃ and the extension time was 45 s.
A cloning method of an amplified fragment prepared by the method for amplifying and whitening the Yangxi myna mitochondrial control region by adopting the amplification nested primer comprises the following specific steps:
1) purification of the second amplification product B2: cutting B2 fragment gel from agarose gel, adding Binding Solution to form Solution, transferring the Solution into adsorption column GC-3u of collection tube, standing at room temperature for 2min, centrifuging at 6000r/min for 1min, pouring out waste liquid, placing adsorption column GC-3u back into the collection tube, adding WA Solution, centrifuging at 12000r/min for 1min, placing adsorption column GC-3u back into the collection tube, adding Wash Solution, centrifuging at 12000r/min for 1min, repeating the operation for 1 time, placing adsorption column GC-3u back into the collection tube, centrifuging at 12000r/min for 1min at room temperature, opening adsorption column GC-3u, standing for 3-10min, placing adsorption column GC-3u into clean collection tube, suspending 25-30 μ L of elusion in the center of membrane, sealing, standing at 37 deg.C for 2min, 12000r/min for 1min, the liquid in the centrifugal tube is the purified B2 fragment;
2) fast cloning: adding Rapiliganation Mix, pGM-T Fast Vector, purified B2 fragment and double distilled water into an EP tube, gently shaking to Mix uniformly, centrifuging for 3-5s, putting the mixed solution into a PCR instrument for heat preservation and connection, wherein the heat preservation and connection procedure of the PCR instrument is as follows: reacting at 25 ℃ for 1min, reacting at 24 ℃ for 1min, reacting at 23 ℃ for 1min, reacting at 22 ℃ for 7min, adding a connecting product into DH5 alpha competent cells, shaking and mixing the connecting product evenly, placing the connecting product in an ice bath for 30min, a water bath at 42 ℃ for 90s and a water bath at 37 ℃ for 3min in sequence, then adding a preheated LB culture medium without antibiotics, performing shaking culture at 180r/min and 37 ℃ for 2h, mixing bacterial liquid evenly and adding the bacterial liquid onto an LB solid agar culture medium containing ampicillin, uniformly coating the cells, drying the surface of the plate, inverting the plate, performing culture at 37 ℃ for 16h, picking white bacterial colonies into EP (Ephedra) of the LB liquid culture medium containing ampicillin, and performing shaking culture at 200r/min and 37 ℃ for 3 h.
A method for sequencing a clone colony prepared by a cloning method of an amplified fragment prepared by the method for amplifying the leucoxi myna mitochondrial control region by using the nested amplification primers comprises the following specific steps: mixing Dream-Taq buffer, nested primers No.5 and No.6, dNTPmix, Dream-Taq enzyme, clone bacteria liquid, dimethyl sulfoxide and double distilled water uniformly, and marking as B2 clone; then pre-denatured at 94 ℃ for 6 min; and another 30 cycles comprising: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 45 s; and then carrying out extension for 8min at 72 ℃, carrying out agarose gel electrophoresis, and finally sequencing the positive clone bacterial liquid of a target strip under the condition that the universal primer is utilized to carry out bidirectional sequencing on the short segment in the plasmid of the bacterial liquid.
Preferably, the universal primer is T7 or SP 6.
Compared with the prior art, the invention has the beneficial effects that:
1. the amplification nested primers adopted by the invention have an overlap region of 298bp for the 2 pairs of amplification nested primers in the first round of PCR, and can be accurately spliced; the nested amplification primers of the second round of PCR are designed on two wings of the polynucleotide tandem repeat, the length of the polynucleotide tandem repeat structure can be strictly limited in the effective reading range of the sequencing reaction, the secondary structure is few, and the conformation is stable; the 3' ends of the 6 nested primers all avoided the introduction of A bases and greatly reduced the risk of non-specific amplification.
2. The invention adopts the optimized PCR amplification program, fully exerts the amplification activity of DNA polymerase, efficiently and stably amplifies complex mitochondrial control region sequences, can directly sequence the amplification product of the first round of PCR, can greatly improve the purity and concentration of a sequencing template by combining the fragment amplified by the second round of PCR with agarose gel recovery purification and rapid cloning technology, so as to eliminate the heterogeneity in the sequencing process, effectively overcome the problem of sequencing failure caused by polynucleotide sequences, and greatly reduce the workload of operators by combining direct sequencing with clonal sequencing.
3. In the cloning and connecting process, the PCR instrument is adopted to replace the conventional water bath for gradient temperature control, and the complex fragment and the vector are connected by prolonging the optimal enzyme activity temperature time, so that the success rate of connection is greatly improved.
Drawings
FIG. 1 is a flow chart of the PCR process and the sequencing method in the examples;
FIG. 2 is a photograph of PCR amplification bands in the examples.
1-3, a first PCR amplification band B1-a, 4-6, a first PCR amplification band B1-B, 7-9, a second PCR amplification band B2 and 10-12, and a positive amplification band of bacteria liquid PCR.
Detailed Description
The following embodiments are described in further detail to help those skilled in the art to more fully, accurately and deeply understand the inventive concept and technical solutions of the present invention.
It is to be clarified that the primers are synthesized by general biosystems (Anhui) Co., Ltd and the concentration of the primers is 5umol, dNTPmix used in the present invention is a commercially available product of Tiangen Biotechnology (Beijing) Co., Ltd., Dream-taq and its buffer are commercially available products of Sammer Feishel Co., Ltd., an agarose gel DNA recovery kit (centrifugal column type) is a commercially available product of Shanghai Jieli bioengineering Co., Ltd., and pGM-T Fast cloning kit is a commercially available product of Tiangen Biotechnology (Beijing) Co., Ltd. The rest of the used chemical reagents are conventional and commercially available analytical pure reagents.
An amplification nested primer of a leucotrichine myna mitochondrial control region, wherein the amplification nested primer comprises a specific nested primer shown in SEQ ID NO.1-6, wherein the sequence shown in SEQ ID NO. 1: ATCTCCAACTCCCAAAGCT, respectively;
SEQ ID NO.2：CAAACTGGGATTAGATACC；
SEQ ID NO.3：TCTCACGAGAACCGAGCTAC；
SEQ ID NO.4：ATCTTCCTCTTGACATGTCC；
SEQ ID NO.5：GGTATTTTCAACTAAACTACTT；
SEQ ID NO.6：AGGGTATGTACTCTCTGCATCG。
a method for amplifying and whitening a Yangxi myna mitochondrial control region by adopting an amplification nested primer comprises the following specific steps:
1) taking a sample: take the muscle of Baiding xi myna, code K0017, the information is as follows:
| numbering | Species (II) | Sample type |
| K0017 | Baiding xi myna | Muscle |
2) Extracting genes: extracting total genes by adopting a phenol and chloroform extraction method, taking 50mg muscle tissue with the number of K0017 white top stream myna, shearing, placing in a sterilized 2.0mL EP tube, adding 1mL DNA extracting solution and 4 mu L RNaseA with the concentration of 20mg/mL into the EP tube, shaking uniformly, and placing in a 37 ℃ water bath for 1 h; taking out the EP tube, adding 5 μ L of 20mg/mL proteinase K, shaking, placing in 50 deg.C water bath for about 3 hr until the muscle is completely digested, and shaking up and down every 10 min; taking out the EP tube, cooling to room temperature, adding saturated phenol with the same volume, and gently shaking up and down for 7 minutes until the water phase and the phenol phase are mixed into emulsion; centrifuging at 10 deg.C for 15min at 10000r/min, and taking out the upper water phase to another 20mLEP tube; repeating phenol extraction for 2 times; adding isovolumetric chloroform isoamyl alcohol into the phenol extraction solution, gently shaking up and down for about 7min until the mixture is uniformly mixed, centrifuging for 15min at 10 ℃ under 10000rPm, and taking out the upper aqueous phase to another 2.0mLEP tube; then, the chloroform extraction operation is repeated for 1 time on the upper aqueous phase, then the upper aqueous phase is taken, 1/5 volumes of 3mol/l NaAC and 2 volumes of precooled absolute ethyl alcohol are added, the EP tube is shaken lightly at room temperature and is placed in a freezer at the temperature of 20 ℃ below zero for freezing for 3 hours; taking out the EP tube from the freezer, centrifuging at 12000r/min at 4 ℃ for 15min, and discarding the supernatant; then adding 500 mu L of 75% ethanol for rinsing, centrifuging at 12000r/min at 4 ℃ for 15min, and removing the supernatant; adding 500 μ L of 75% ethanol, rinsing, centrifuging at 12000r/min at 4 deg.C for 15min, removing supernatant, and air drying the precipitate; and 100. mu.L of 1 XTE was added to dissolve the DNA; taking 2 mu of LDNA solution to carry out agarose gel electrophoresis; storing the total DNA in a freezer at-40 ℃;
3) two times of PCR amplification: for the first PCR amplification, 5. mu.L of Dream-Taq buffer, 4.4. mu.L of each of the corresponding amplification nested primers, 5. mu.L of dNTPmix, 0.25. mu.L of Dream-Taq enzyme and 4. mu.L of total extracted DNA, 1. mu.L of dimethyl sulfoxide were added to the EP tube and made up to 50. mu.L with double distilled water. One set of amplification nested primers is as follows: nos. 1 and 4, numbered B1-a; the other group is as follows: no.2 and No.3, numbered B1-B, each set was amplified 3 times in duplicate and the reaction procedure was: pre-denaturation at 94 ℃ for 3 min; then, 36 cycles were performed, which included: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 90 s; finally, extension is carried out for 8min at 72 ℃. Then, agarose gel electrophoresis is carried out, and B1-a and B1-B are sent to the company of Biotechnology engineering (Shanghai) for sequencing, wherein B1-a serves as a high-purity template during the second round of PCR amplification;
and (3) performing second PCR amplification: to an EP tube, 4.4. mu.L each of Dream-Taq buffer, amplification nested primers No.5 and No.6, 5. mu.L of dNTPmix, 0.25. mu.L of Dream-Taq enzyme, and 0.5. mu.L of first PCR amplification product B1-a, 1. mu.L of dimethyl sulfoxide were added, and the mixture was made up to 50. mu.L with double distilled water, amplification product No. B2, and amplification was repeated 3 times, in the following reaction sequence: pre-denaturation at 94 ℃ for 3 min; then, 36 cycles were performed, which included: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 45 s; finally, the product B2 obtained by the second round of PCR amplification is used for purification and rapid cloning after the extension for 8min at 72 ℃ and the subsequent agarose gel electrophoresis.
A method for cloning an amplified fragment prepared by amplifying and whitening a Yangxi myna mitochondrial control region by using an amplification nested primer comprises the following specific steps:
1) purification of the second amplification product B2: cutting B2 fragment gel from agarose gel, adding 400 μ LBinding Solution to form Solution, transferring the Solution to adsorption column GC-3u with 2ml collection tube, standing at room temperature for 2min, centrifuging at 6000r/min for 1min, taking out adsorption column GC-3u, pouring waste liquid from the collection tube, putting adsorption column GC-3u back into the collection tube, adding 500 μ L WA Solution, centrifuging at 12000r/min for 1min, putting adsorption column GC-3u back into the collection tube, adding Wash Solution, centrifuging at 12000r/min for 1min, repeating the operation for 1 time, putting adsorption column GC-3u back into the collection tube, centrifuging at 12000r/min at room temperature for 1min, opening the cover of adsorption column GC-3u, standing at room temperature for 5min, completely removing Wasol Solution, then putting adsorption column GC-3u into clean 1.5ml collection tube, suspending 30 μ L of Elution Buffer in the center of the membrane, covering the membrane with a cover, sealing, standing at 37 ℃ for 2min, and centrifuging at 12000r/min for 1min, wherein the liquid in the centrifuge tube is purified B2 fragment;
2) fast cloning: adding rapiLigation Mix5 mu L, pGM-T Fast Vector1 mu L and purified B2 fragment 1 mu L into an EP tube, supplementing the purified B2 fragment to 10 mu L with double distilled water, slightly flicking the centrifugal tube to Mix uniformly, centrifuging for 4s for a short time, putting the mixed solution into a PCR instrument for heat preservation and connection, wherein the heat preservation and connection procedure of the PCR instrument is as follows: reacting at 25 deg.C for 1min, at 24 deg.C for 1min, at 23 deg.C for 1min, and at 22 deg.C for 7min, and connecting for 10 min; adding the ligation product into 50 mu L of DH5 alpha competent cells, shaking gently and mixing uniformly, placing in an ice bath for 30min, water bath at 42 ℃ for 90s, ice bath for 3min without shaking a centrifuge tube during the ice bath, then adding 250 mu L of preheated LB culture medium without antibiotics into the centrifuge tube, carrying out shaking culture at 180r/min and 37 ℃ for 2h, mixing bacterial liquid uniformly, sucking 200 mu L of the bacterial liquid, adding the bacterial liquid into LB solid agar culture medium containing ampicillin, slightly and uniformly coating the cells with sterile glass beads, after the surface of the plate is dried, inverting the plate, carrying out culture at 37 ℃ for 16h, picking white bacterial colonies into EP containing 500 mu L of LB liquid culture medium containing ampicillin, and carrying out shaking culture at 200r/min and 37 ℃ for 3 h.
A method for sequencing a clone colony prepared by a cloning method of an amplified fragment prepared by a method for amplifying a leucogen myna mitochondrial control region by using an amplification nested primer comprises the following specific steps: 1.25 mu L of Dream-Taq buffer, 1.1 mu L of each of nested primers No.5 and No.6, 1.25 mu L of dNTPmix, 0.0625 mu L of Dream-Taq enzyme, 1 mu L of clone bacterial liquid and 1 mu L of dimethyl sulfoxide are mixed uniformly by supplementing 12.5 mu L of double distilled water, and the mixture is marked as B2 clone; then the colonies were pre-denatured at 94 ℃ for 6 min; and another 30 cycles comprising: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 55 s; and then extending for 8min at 72 ℃, then carrying out agarose gel electrophoresis, sending the positive clone bacterial liquid of the target band to the company of Biotechnology engineering (Shanghai) GmbH for sequencing, and carrying out bidirectional sequencing on the short fragment in the plasmid of the bacterial liquid by using a T7 or SP6 universal primer under the sequencing condition.
The PCR process and the sequencing method are shown in FIG. 1: in the figure, 1-3 are amplification bands of first PCR amplification B1-a, 4-6 are amplification bands of first PCR amplification B1-B, 7-9 are amplification bands of second PCR amplification B2, wherein a bright band is subjected to agarose gel recovery purification and rapid cloning, 10-12 are positive amplification bands of bacteria liquid PCR, wherein B1-a and B1-B can be directly sequenced, and B2 is subjected to clone sequencing; the fragment number, latin name and sequence length of the amplified species are as follows:
| fragments | Species (II) | Latin name | Length of sequence |
| B1-a | Baiding xi myna | Chaimarrornis leucocephalus | 1375bp |
| B1-b | Baiding xi myna | Chaimarrornis leucocephalus | 1295bp |
| Clone B2 | Baiding xi myna | Chaimarrornis leucocephalus | 692bp |
The present invention has been described in connection with the embodiments, and it is to be understood that the invention is not limited to the specific embodiments described above, and that various insubstantial modifications of the inventive concepts and solutions, or their direct application to other applications without modification, are intended to be covered by the scope of the invention. The protection scope of the present invention shall be subject to the protection scope defined by the claims.
SEQUENCE LISTING
<110> university of teacher's university in Anhui
<120> amplification nested primer of mitochondrial control region of Baiding stream myna and amplification, cloning and sequencing methods
<130> 2017.12.11
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 1
atctccaact cccaaagct 19
<210> 2
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 2
caaactggga ttagatacc 19
<210> 3
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 3
<210> 4
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 4
<210> 5
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 5
ggtattttca actaaactac tt 22
<210> 6
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 6
agggtatgta ctctctgcat cg 22
Claims (2)
1. An amplification nested primer of a mitochondrial control region of the Baiyinxi myna, which is characterized by comprising specific nested primers shown as SEQ ID NO.1-6,
SEQ ID NO.1：ATCTCCAACTCCCAAAGCT；
SEQ ID NO.2：CAAACTGGGATTAGATACC；
SEQ ID NO.3：TCTCACGAGAACCGAGCTAC；
SEQ ID NO.4：ATCTTCCTCTTGACATGTCC；
SEQ ID NO.5：GGTATTTTCAACTAAACTACTT；
SEQ ID NO.6：AGGGTATGTACTCTCTGCATCG。
2. the Baiding stream myna mitochondrial control region amplification nested primer of claim 1, wherein the amplification nested primer is synthesized by general biosystems, Inc. Anhui, at a concentration of 5 μmol.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101063170A (en) * | 2007-05-25 | 2007-10-31 | 中山大学 | Method for analyzing fish genetic information by using mitochondria DNA D-loop control area |
| CN101086014A (en) * | 2007-05-25 | 2007-12-12 | 中山大学 | Fish mitochondrion DNA control area amplification primer and its design method and uses |
| CN102409051A (en) * | 2011-10-18 | 2012-04-11 | 浙江海洋学院 | Marine fish mitochondrial genome control area amplification primer, as well as design and amplification methods |
| CN104531846A (en) * | 2014-12-11 | 2015-04-22 | 中国计量学院 | Amplification primer of mitochondrial genome control region of spider and reaction system of amplification primer |
-
2017
- 2017-12-13 CN CN201711332619.2A patent/CN108048457B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101063170A (en) * | 2007-05-25 | 2007-10-31 | 中山大学 | Method for analyzing fish genetic information by using mitochondria DNA D-loop control area |
| CN101086014A (en) * | 2007-05-25 | 2007-12-12 | 中山大学 | Fish mitochondrion DNA control area amplification primer and its design method and uses |
| CN102409051A (en) * | 2011-10-18 | 2012-04-11 | 浙江海洋学院 | Marine fish mitochondrial genome control area amplification primer, as well as design and amplification methods |
| CN104531846A (en) * | 2014-12-11 | 2015-04-22 | 中国计量学院 | Amplification primer of mitochondrial genome control region of spider and reaction system of amplification primer |
Non-Patent Citations (1)
| Title |
|---|
| 鸟类线粒体DNA控制区研究进展;黄族豪等;《安徽农业科学》;20090320(第8期);第3427-3428页 * |
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