CN110846381A - Primer combination for amplification of carp mitochondrial DNA fragment and application thereof - Google Patents
Primer combination for amplification of carp mitochondrial DNA fragment and application thereof Download PDFInfo
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Abstract
The invention discloses a primer combination for amplification of carp mitochondrial DNA fragments, which comprises 19 primer pairs shown as SEQ ID NO 1-SEQ ID NO 38. The invention also discloses a method, which comprises the following steps: 1) obtaining the total DNA of a carp sample to be detected; 2) respectively carrying out polymerase chain reaction by using the total DNA as a template and adopting primer pairs to obtain amplification products; 3) sequencing to obtain sequence information. The invention also discloses application of the primer combination in scientific research. The invention provides the specific sensitivity and high specificity primer, avoids the occurrence of nonspecific amplification, can conveniently, quickly and accurately amplify the mitochondrial DNA of the carp to obtain 19 fragments, and finally obtains the complete mitochondrial DNA of the carp after splicing, wherein the length of the complete mitochondrial DNA reaches about 16580 bp. The invention can provide great convenience for the research of the genetic material of the carp.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a primer combination for amplification of carp mitochondrial DNA fragments and application thereof.
Background
Carp (Cyprinus carpio L.) is cultivated in more than 100 countries and regions at present, and the annual yield is over 400 ten thousand tons, which is one of the most important aquaculture species. China is the largest carp breeding country and consumer country, and the annual output is 302 ten thousand tons, which accounts for 80% of the total world output. Carp as an important model species is widely applied to the research in the fields of environmental toxicology, developmental biology, physiology, immunology, evolutionary genomics and the like. Therefore, during the last decade, a large number of carp genomic resources have been extensively developed, including genetic markers, genetic maps, BAC databases, ESTs, and transcriptome sequences.
Therefore, the carp mitochondrial DNA sequence is amplified by using the polymerase chain reaction, and a scientific basis can be provided for the inheritance and evolution research of carps. The existing method for obtaining the carp DNA sequence mainly comprises primer amplification and sequencing, but has the biggest problems of poor primer sensitivity and specificity, frequent occurrence of phenomena of mismatch, nonspecific amplification or no band in amplification and the like. Therefore, the key to this technique is to amplify under suitable conditions with as few primer combinations as possible to obtain complete mitochondrial DNA fragments. There is a need for an amplification method with simplicity, convenience, high sensitivity, strong specificity and reliable results.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter.
The invention also aims to provide a primer combination for amplifying the carp mitochondrial DNA fragment. The invention avoids the occurrence of nonspecific amplification by designing a primer with specific sensitivity and high specificity. The invention uses as few primers as possible to amplify the complete carp mitochondrial DNA fragment, and provides convenience for the genetic research of carps.
Therefore, the technical scheme provided by the invention is as follows:
a primer combination for amplification of carp mitochondrial DNA fragments comprises 19 primer pairs, wherein the sequences of the 19 primer pairs are as follows in sequence: the first set of primer pairs shown as SEQ ID NO. 1 and SEQ ID NO. 2, the second set of primer pairs shown as SEQ ID NO. 3 and SEQ ID NO. 4, the third set of primer pairs shown as SEQ ID NO. 5 and SEQ ID NO. 6, the fourth set of primer pairs shown as SEQ ID NO. 7 and SEQ ID NO. 8, the fifth set of primer pairs shown as SEQ ID NO. 9 and SEQ ID NO. 10, the sixth set of primer pairs shown as SEQ ID NO. 11 and SEQ ID NO. 12, the seventh set of primer pairs shown as SEQ ID NO. 13 and SEQ ID NO. 14, the eighth set of primer pairs shown as SEQ ID NO. 15 and SEQ ID NO. 16, the ninth set of primer pairs shown as SEQ ID NO. 17 and SEQ ID NO. 18, the tenth set of primer pairs shown as SEQ ID NO. 19 and SEQ ID NO. 20, the eleventh set of primer pairs shown as SEQ ID NO. 21 and SEQ ID NO. 22, as shown in SEQ ID NO:23 and SEQ ID NO: a twelfth set of primer pairs as set forth in 24, as shown in SEQ ID NO:25 and SEQ ID NO:26 in a thirteenth group of primers shown in the specification, as shown in SEQ ID NO:27 and SEQ ID NO:28 in the sequence listing, a fourteenth set of primer pairs, as shown in SEQ ID NO:29 and SEQ ID NO:30 in a fifteenth group of primer pairs, as shown in SEQ ID NO:31 and SEQ ID NO:32, a sixteenth set of primer pairs shown in (b), as shown in SEQ ID NO:33 and SEQ ID NO:34 in the sequence listing, and a seventeenth primer set, as shown in SEQ ID NO:35 and SEQ ID NO:36 and the primer pair of the eighteenth group shown in SEQ ID NO:37 and SEQ ID NO:38 in the nineteenth group of primer pairs, wherein the position where the upstream primer in the latter primer pair is complementary to the mitochondrial DNA is located upstream of the position where the downstream primer in the former primer pair is complementary to the mitochondrial DNA. And the position of the complementary downstream primer shown as SEQ ID NO. 38 and the mitochondrial DNA is between the positions of the complementary primer pair shown as SEQ ID NO. 1 and SEQ ID NO. 2 and the mitochondrial DNA.
Preferably, in the primer combination for amplifying the carp mitochondrial DNA fragment, the 19 sets of primer pairs are respectively applied to polymerase chain reaction.
Preferably, in the primer combination for amplifying the mitochondrial DNA fragment of the carp, the template of the carp sample is the total DNA of the carp in the polymerase chain reaction.
Preferably, in the primer combination for amplifying the mitochondrial DNA fragment of the carp, the variety of the carp sample is Furui carp, yellow river carp, fancy carp or red purse carp.
A method for acquiring carp mitochondrial DNA sequence information by using the primer combination comprises the following steps:
1) obtaining the total DNA of a carp sample to be detected;
2) taking the total DNA extracted in the step 1) as a template, and respectively adopting the primers in the primer combination to carry out polymerase chain reaction to obtain an amplification product;
3) sequencing the amplification product, and then obtaining sequence information according to the amplification result.
Preferably, in the step 2), when the polymerase chain reaction is performed, 50mL of the reaction system comprises: DNA polymerase 2 units, 10 × polymerase buffer 5 μ L, 25mmol/L MgCl2mu.L of 5. mu.L, 1. mu.L of 10mmol/L dNTP, 2. mu.L of each 10. mu. mol/L primer pair, and 100-1000. mu.g of template DNA.
Preferably, in the method, after the polymerase chain reaction in the step 2), the amplification product is treated for 10-20 min under the condition of approximate vacuum state in the range of 666.61-1333.22Pa or below and 72 ℃, and then stored at 0-4 ℃.
Preferably, in the method, in step 1), the specific method for obtaining the total DNA of the carp sample to be tested comprises the following steps:
cutting a fin ray of the carp, and storing the fin ray in absolute ethyl alcohol;
and step two, soaking and washing the fin rays for more than 5 hours by using STE buffer solution, removing absolute ethyl alcohol, taking about 0.2g of fin rays, shearing, adding 0.5mL of STE buffer solution, 10mg/mL of proteinase K15 and SDS 40 mu L with the mass volume ratio of 10%, and carrying out water bath at 50 ℃ for 4-6 hours to remove proteins to obtain the total DNA.
The primer combination for carp mitochondrial DNA fragment amplification is applied to scientific research, particularly to the research of carp genetic materials.
The invention at least comprises the following beneficial effects:
the invention avoids the occurrence of nonspecific amplification by designing the primer with specific sensitivity and high specificity. The method can conveniently, quickly and accurately amplify the DNA of the carp with results to obtain 19 fragments, and the final length is 16580bp or so after the final length splicing is finished. The invention can provide great convenience for the research of the genetic material of the carp.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a schematic flow chart of a primer combination for amplification of carp mitochondrial DNA fragment according to the present invention;
FIG. 2 is a schematic diagram of the amplification structure of 19 pairs of primers in one embodiment of the present invention.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
As shown in FIG. 1, the present invention provides a primer combination for amplification of carp mitochondrial DNA fragment, comprising 19 primer pairs, wherein the sequences of the 19 primer pairs are as follows: the first set of primer pairs shown as SEQ ID NO. 1 and SEQ ID NO. 2, the second set of primer pairs shown as SEQ ID NO. 3 and SEQ ID NO. 4, the third set of primer pairs shown as SEQ ID NO. 5 and SEQ ID NO. 6, the fourth set of primer pairs shown as SEQ ID NO. 7 and SEQ ID NO. 8, the fifth set of primer pairs shown as SEQ ID NO. 9 and SEQ ID NO. 10, the sixth set of primer pairs shown as SEQ ID NO. 11 and SEQ ID NO. 12, the seventh set of primer pairs shown as SEQ ID NO. 13 and SEQ ID NO. 14, the eighth set of primer pairs shown as SEQ ID NO. 15 and SEQ ID NO. 16, the ninth set of primer pairs shown as SEQ ID NO. 17 and SEQ ID NO. 18, the tenth set of primer pairs shown as SEQ ID NO. 19 and SEQ ID NO. 20, an eleventh set of primer pairs shown as SEQ ID NO 21 and SEQ ID NO 22, a twelfth set of primer pairs shown as SEQ ID NO 23 and SEQ ID NO 24, a thirteenth set of primer pairs shown as SEQ ID NO 25 and SEQ ID NO 26, a fourteenth set of primer pairs shown as SEQ ID NO 27 and SEQ ID NO 28, a fifteenth set of primer pairs shown as SEQ ID NO 29 and SEQ ID NO 30, a sixteenth set of primer pairs shown as SEQ ID NO 31 and SEQ ID NO 32, a seventeenth set of primer pairs shown as SEQ ID NO 33 and SEQ ID NO 34, an eighteenth set of primer pairs shown as SEQ ID NO 35 and SEQ ID NO 36 and a nineteenth set of primer pairs shown as SEQ ID NO 37 and SEQ ID NO 38, wherein the upstream primer of the latter set of primer pairs is complementary to the mitochondrial DNA at a position on the downstream of the primer of the former set of primer pairs and complementary to the mitochondrial DNA at a position on the upstream of the downstream of the former set of the primer pairs And (4) swimming. And the position of the complementary downstream primer shown as SEQ ID NO. 38 and the mitochondrial DNA is between the positions of the complementary primer pair shown as SEQ ID NO. 1 and SEQ ID NO. 2 and the mitochondrial DNA.
Primer combination for amplified fragment
In one embodiment of the present invention, the 19 sets of primer pairs are preferably used in polymerase chain reaction respectively.
In one embodiment of the present invention, preferably, the template of the carp sample is total DNA of carp in the polymerase chain reaction.
In one embodiment of the present invention, preferably, the variety of the carp sample is a fury carp, a yellow river carp, a koi or a poached red carp.
A method for acquiring carp mitochondrial DNA sequence information by using the primer combination comprises the following steps:
1) obtaining the total DNA of a carp sample to be detected;
2) taking the total DNA extracted in the step 1) as a template, and respectively adopting the primers in the primer combination to carry out polymerase chain reaction to obtain an amplification product;
3) sequencing the amplification product, and then obtaining sequence information according to the amplification result.
In one embodiment of the present invention, preferably, in step 2), the polymerase chain reaction is performed in a 50mL reaction system comprising: DNA polymerase 2 units, 10 × polymerase buffer 5 μ L, 25mmol/L MgCl2mu.L of 5. mu.L, 1. mu.L of 10mmol/L dNTP, 2. mu.L of each 10. mu. mol/L primer pair, and 100-1000. mu.g of template DNA.
In one embodiment of the present invention, preferably, in step 2), after the polymerase chain reaction is finished, the amplification product is treated under a near vacuum condition of 666.61-1333.22Pa or below and at 72 ℃ for 10-20 min, and then stored at 0-4 ℃. The vacuum treatment after the PCR reaction can effectively further increase the extension time and efficiency of the PCR reaction so as to obtain more amplified DNA.
In one embodiment of the present invention, preferably, in step 1), the specific method for obtaining the total DNA of the carp sample to be tested includes the following steps:
cutting a fin ray of the carp, and storing the fin ray in absolute ethyl alcohol;
and step two, soaking and washing the fin rays for more than 5 hours by using STE buffer solution, removing absolute ethyl alcohol, taking about 0.2g of fin rays, shearing, adding 0.5mL of STE buffer solution, 10mg/mL of proteinase K15 and SDS 40 mu L with the mass volume ratio of 10%, and carrying out water bath at 50 ℃ for 4-6 hours to remove proteins to obtain the total DNA.
The primer combination for carp mitochondrial DNA fragment amplification is applied to scientific research.
The invention designs 19 pairs of upstream and downstream primers on the DNA of the carp, can amplify 19 fragments on the DNA of the carp, and has the length of 16580bp after the final length splicing is finished.
In order to make the technical solution of the present invention better understood by those skilled in the art, the following examples are now provided for illustration:
1. sampling: shearing a fin ray of the carp, and storing in absolute ethyl alcohol;
2. extraction of Total DNA
The tissue was washed with STE buffer for more than 5h to remove alcohol. About 0.2g of tissue was harvested, minced, and placed into a 1.5mL centrifuge tube, and 0.5mL STE buffer, 15 μ L proteinase K (10mg/mL) and 40 μ L SDS (10%) were added to each tube in a 50 ℃ water bath for 4-6 h. Protein was removed by standard phenol chloroform procedure.
STE buffer formulation 0.1mol/L NaCl, 10mmol/L Tris-Cl (pH8.0), 1mmol/L EDTA (pH8.0)
3. Primers and PCR amplification
The different regions of DNA were amplified with 19 pairs of primers, the primers used and the basic parameters were as follows:
primer combination for amplified fragment
The polymerase chain reaction adopts LA Taq DNA polymerase of TaKaRa company (Dalian), and the reaction system is as follows: LATaq DNA polymerase 2 units; 10 × LA Buffer (Mg)2+free)5μL；MgCl2(25mmol/L) 5. mu.L; 10mmol/L dNTP 1 mu L; 2. mu.L of each 10. mu. mol/L primer pair; 100-1000 mu g of template DNA; sterile deionized water was added to 50. mu.L. The PCR reaction was carried out for 30 cycles, each cycle consisting of 95 ℃ for 30s, annealing temperature 50s as indicated above, and 72 ℃ for 3 min. Pre-denaturation at 96 ℃ for 1min before the start of the cycle and re-extension at 72 ℃ for 15min after the end of the cycle. In order to enhance the specificity of the polymerase chain reaction amplification, the reaction solution is prepared on ice, and after the temperature of the PCR instrument is raised to 80 ℃, the centrifugal tube is placed into the polymerase chain reaction instrument for reaction;
4. after the completion of the electrophoretic polymerase chain reaction, 2. mu.L of the reaction solution was electrophoresed with 0.8% agarose gel (containing ethidium bromide), and the effect of amplification was observed under an ultraviolet lamp, as shown in FIG. 2;
5. product sequencing, namely sending a polymerase chain reaction product to Shanghai biological engineering GmbH for sequencing;
6. after the sequences are spliced to obtain 19 fragment sequences, DNAstar software is used for splicing the 19 fragments, and the final length is 16580bp or so after the length splicing is finished.
The method takes Furui carp, yellow river carp, fancy carp or Hebao red carp as samples, and mitochondrial genomes of the samples are obtained through amplification.
The number of modules and the processing scale described herein are intended to simplify the description of the invention. The use, modification and variation of the primer combination for amplification of carp mitochondrial DNA fragment of the present invention will be apparent to those skilled in the art.
As described above, the present invention avoids the occurrence of non-specific amplification by providing primers with specific sensitivity and high specificity. The method can conveniently, quickly and accurately amplify the mitochondrial DNA of the carp with a result to obtain 19 fragments, and the final length is the full length of the mitochondrial genome of the carp and reaches about 16580bp after the final length splicing is finished. The invention can provide great convenience for the research of the genetic material of the carp.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
SEQUENCE LISTING
<110> university of south Henan university
<120> primer combination for amplification of carp mitochondrial DNA fragment and application thereof
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Claims (9)
1. A primer combination for amplification of carp mitochondrial DNA fragments is characterized by comprising 19 primer pairs, wherein the sequences of the 19 primer pairs are as follows in sequence: the first set of primer pairs shown as SEQ ID NO. 1 and SEQ ID NO. 2, the second set of primer pairs shown as SEQ ID NO. 3 and SEQ ID NO. 4, the third set of primer pairs shown as SEQ ID NO. 5 and SEQ ID NO. 6, the fourth set of primer pairs shown as SEQ ID NO. 7 and SEQ ID NO. 8, the fifth set of primer pairs shown as SEQ ID NO. 9 and SEQ ID NO. 10, the sixth set of primer pairs shown as SEQ ID NO. 11 and SEQ ID NO. 12, the seventh set of primer pairs shown as SEQ ID NO. 13 and SEQ ID NO. 14, the eighth set of primer pairs shown as SEQ ID NO. 15 and SEQ ID NO. 16, the ninth set of primer pairs shown as SEQ ID NO. 17 and SEQ ID NO. 18, the tenth set of primer pairs shown as SEQ ID NO. 19 and SEQ ID NO. 20, the eleventh set of primer pairs shown as SEQ ID NO. 21 and SEQ ID NO. 22, as shown in SEQ ID NO:23 and SEQ ID NO: a twelfth set of primer pairs as set forth in 24, as shown in SEQ ID NO:25 and SEQ ID NO:26 in a thirteenth group of primers shown in the specification, as shown in SEQ ID NO:27 and SEQ ID NO:28 in the sequence listing, a fourteenth set of primer pairs, such as SEQ ID NO:29 and SEQ ID NO:30 in a fifteenth group of primer pairs, as shown in SEQ ID NO:31 and SEQ ID NO:32, a sixteenth set of primer pairs shown in (b), as shown in SEQ ID NO:33 and SEQ ID NO:34 in the sequence listing, and a seventeenth primer set, as shown in SEQ ID NO:35 and SEQ ID NO:36 and the primer pair of the eighteenth group shown in SEQ ID NO:37 and SEQ ID NO:38 in the nineteenth group of primer pairs, wherein the position where the upstream primer in the latter primer pair is complementary to the mitochondrial DNA is located upstream of the position where the downstream primer in the former primer pair is complementary to the mitochondrial DNA.
2. The primer combination for amplification of a mitochondrial DNA fragment of carp according to claim 1, wherein the 19 sets of primer pairs are respectively used in polymerase chain reaction.
3. The primer combination for amplification of mitochondrial DNA fragments of carp according to claim 2, wherein the template of the carp sample is total DNA of carp in the polymerase chain reaction.
4. The primer combination for amplification of a mitochondrial DNA fragment of a carp according to claim 3, wherein the variety of the carp sample is Furui carp, yellow river carp, Koi carp or Hepu red carp.
5. A method for obtaining carp mitochondrial DNA sequence information by using the primer combination according to claim 1, characterized by comprising the following steps:
1) obtaining the total DNA of a carp sample to be detected;
2) taking the total DNA extracted in the step 1) as a template, and respectively adopting the primers in the primer combination to carry out polymerase chain reaction to obtain an amplification product;
3) sequencing the amplification product, and then obtaining sequence information according to the amplification result.
6. The method of claim 5, wherein in step 2), when the polymerase chain reaction is performed, 50mL of the reaction system comprises: DNA polymerase 2 units, 10 × polymerase buffer 5 μ L, 25mmol/L MgCl2mu.L of 5. mu.L, 1. mu.L of 10mmol/L dNTP, 2. mu.L of each 10. mu. mol/L primer pair, and 100-1000. mu.g of template DNA.
7. The method of claim 5, wherein in the step 2), after the PCR is completed, the amplification product is treated under a near vacuum condition of 666.61-1333.22Pa or less at 72 ℃ for 10-20 min and then stored at 0-4 ℃.
8. The method according to claim 5, wherein the specific method for obtaining the total DNA of the carp sample to be tested in step 1) comprises the following steps:
cutting a fin ray of the carp, and storing the fin ray in absolute ethyl alcohol;
and step two, soaking and washing the fin rays for more than 5 hours by using STE buffer solution, removing absolute ethyl alcohol, taking about 0.2g of fin rays, shearing, adding 0.5mL of STE buffer solution, 10mg/mL of proteinase K15 and SDS 40 mu L with the mass volume ratio of 10%, and carrying out water bath at 50 ℃ for 4-6 hours to remove proteins to obtain the total DNA.
9. The use of the primer combination for amplification of a mitochondrial DNA fragment of carp according to claim 1 in scientific research.
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| CN112094926A (en) * | 2020-11-17 | 2020-12-18 | 水利部中国科学院水工程生态研究所 | Primer design and amplification method of mitochondrial genome complete sequence of gobiocypiocypris dabryi |
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