A set of amplification universal primers for the whole genome of the family of trionychidae

Abstract

The application discloses a set of general primers for amplifying the whole genome of the mitochondrion of Trionychidae, and belongs to the technical field of molecular biology; the specific steps comprise the following steps: extracting the total DNA of the genome of Trionychidae, designing the general amplification primers of the whole sequence of the mitochondrial genome, and performing PCR amplification and sequencing on the mitochondrial genome DNA. The amplification primers for the whole genome sequence of the mitochondrion of Trionychidae are composed of 11 pairs of single-stranded oligonucleotides. The general amplification primers of the application can be used to quickly and efficiently amplify the whole sequence of the mitochondrial genome of various Trionychidae, and provide a favorable technical means for the identification of unknown germplasm of Trionychidae, the protection of species resources, and the research on the system evolution.

Description

Technical Field

[0001] The invention relates to the field of molecular biotechnology, in particular to a group of universal primers for amplifying the whole genome of mitochondria of the family Trionyx. Background Art

[0002] As a genetic system outside the cell nucleus, the mitochondrial genome has unique advantages such as integrity, polymorphism, autonomy, and small size. With the rapid development of molecular biology, the mitochondrial genome has become an ideal tool for studying evolution and the origin of species. There are several advantages to using organelle genomes for systematic evolutionary studies: (1) Organelle DNA has a large number of copies in the total DNA, making it easy to obtain through sequencing; (2) Organelle DNA is mostly maternally inherited, with little or no homologous recombination, and its gene structure and gene content are relatively conservative; (3) Organelle genome sequences are long, which can provide a large amount of taxonomic information, solving the difficulties in classifying and identifying closely related species.

[0003] Mitochondrial genome sequences are typically obtained using Sanger sequencing technology. Sequencing strategies typically fall into one of three categories: (1) isolating plastid and mitochondrial DNA (usually using cesium chloride density gradient centrifugation), then constructing a plasmid library and sequencing it using the WGS shotgun method (Reith et al., 1995); (2) constructing a species-specific BAC or Fosmid library, using organellar DNA fragments as probes to select fragments containing target clones from the library. This type of large-fragment library construction is expensive and time-consuming, and its accuracy is questionable (Cattolico et al., 2008); (3) using conserved sequences from plastid and mitochondrial sequences of closely related species to design primers, and using long-range PCR (Long PCR) or PCR for amplification and sequencing (Heinze, 2007). This is the most commonly used method, but it still suffers from issues such as high workload and long time consumption.

[0004] Testudinidae are a group of subaquatic reptiles with highly specialized body morphology and evolutionary status, making them excellent candidates for evolutionary and developmental studies. However, many species within this family are protected under first- and second-class national protection. However, species within this family share similar morphology, and many classifications rely on surface nodules and markings, as well as features of bony plates, making species identification difficult. Therefore, rapidly obtaining mitochondrial genome sequences from members of this family has significant implications for identifying unknown germplasm samples, species conservation, and phylogenetic research. Summary of the Invention

[0005] The present invention aims to provide a set of universal primers for amplifying the entire mitochondrial genome of the family Trionychidae. Specifically, these universal primers are designed using conserved regions of Trionychidae mitochondrial gene sequences to enable rapid amplification of the mitochondrial genomes of various Trionychidae species. Furthermore, the present invention divides the Trionychidae mitochondrial genome into distinct gene regions, enabling the amplification of sequence fragments from corresponding regions based on specific needs in practical applications.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a set of universal primers for amplifying the whole genome of mitochondria of the family Trionycii. The universal primer set for amplifying the whole genome comprises 11 pairs of amplification primers, and the sequences thereof are shown in SEQ ID NO: 1 to SEQ ID NO: 22.

[0008] The present invention also provides a method for amplifying the whole mitochondrial genome of the family Trionycii using universal primers, wherein the universal primers are used to amplify the target fragment using a 20 μl PCR system under PCR amplification conditions.

[0009] Preferably, the 20.5 μl PCR system specifically includes: 5 μl 10× Buffer, 8 μl dNTP Mix, 1 μl upstream primer, 1 μl downstream primer, 0.5 μl Taq enzyme, 0.5 μl DNA template, 4 μl MgCl2 2+ .

[0010] Preferably, the PCR amplification conditions specifically include: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 52-66°C for 2 minutes and 30 seconds, extension at 72°C for 5 minutes, amplification for 35-40 cycles; and finally extension at 72°C for 10 minutes.

[0011] The present invention discloses the following technical effects:

[0012] Using the primer sequences designed by the present invention, mitochondrial genomes of four species, namely, Chinese soft-shell turtle (Pelodiscus sinensis), Chinese soft-shell turtle (Pelochelys cantorii), mountain soft-shell turtle (Paleasteindachneri), and sand soft-shell turtle (Pelodiscus axenaria), were amplified, and complete mitochondrial genome sequences were obtained.

[0013] The universal amplification primers of the present invention can be used to quickly and efficiently amplify the full mitochondrial gene sequences of various turtles, providing a favorable technical means for the germplasm identification, species resource protection, and systematic evolution research of unknown turtles. DETAILED DESCRIPTION

[0014] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0015] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0016] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0017] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The present description and examples are intended to be illustrative only.

[0018] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0019] Example

[0020] 1. Extraction of mitochondrial genes from Trionyx species

[0021] Total genomic DNA is extracted from a tissue sample of a Trionychidae using conventional methods and stored at -20°C until ready for use. The tissue can be blood, scapula, muscle, or viscera. Fresh tissue is recommended, but slight degradation will not significantly affect the efficacy of the present invention.

[0022] 2. Design and synthesis of universal primers for the mitochondrial genome of Trionyx species

[0023] (1) Download all the published sequences of the mitochondrial genome of Trionychidae from Genbank database, and find out the conserved regions by multiple alignment of these sequences, and synthesize the universal primer sequences of the mitochondrial genome of Trionychidae according to Table 1;

[0024] Table 1 Universal primer sequences of the mitochondrial genome of Trionychidae

[0025]

[0026]

[0027]

[0028] (2) Using the 11 pairs of primers above, the extracted genome of Trionychidae as a template, PCR amplification is carried out, wherein the reaction system of PCR is prepared according to the reaction system in Table 2, and the PCR amplification is carried out according to the reaction program in Table 3.

[0029] Table 2 Reaction system of the universal primer of the mitochondrial genome of Trionychidae

[0030] Components Volume (μL) <![CDATA[Buffer(10×,Mg 2+ Free)]]> 5 dNTPMix (2.5 mM) 8 <![CDATA[Mg 2+ (25mM)]]> 4 Taq enzyme (5U / μL) 0.5 upstream primer (10 μM) § ]] 1 <![CDATA[下游引物(10μM) § ]]> 1 DNA (100 ng / μL) 0.5

[0031] §: The amplification effect is not ideal, and the volume of the primer can be doubled.

[0032] Table 3 Reaction program of the universal primer of the mitochondrial genome of Trionychidae

[0033]

[0034] Note: Ta: the annealing temperature corresponding to each pair of amplification primers in Table 1.

[0035] 3. Sequence determination and splicing of PCR amplification products

[0036] Sanger sequencing is performed on the amplification products to obtain the corresponding gene fragment sequences of the samples, and after obtaining all the gene fragment sequences, sequence splicing software such as DNAstar is used for sequence splicing, so that the complete mitochondrial genome of the test sample can be obtained.

[0037] The above-described embodiments are only descriptions of the preferred modes of the present application, and do not limit the scope of the present application, and various modifications and improvements of the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the protection scope determined by the claims of the present application. SEQUENCE LISTING <110> CHINA AQUATIC PRODUCTS RESEARCH AGENCY ZHUJIANG AQUATIC PRODUCTS RESEARCH INSTITUTE <120> A set of universal primers for amplifying the complete mitochondrial genome of Trionychidae <160> 22 <170> SIPOSequenceListing 1.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <400> 1 agtgaaaatg ccctaaaagt cacatc 26 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <400> 2 atacttattg ttgctagggg ctatgt 26 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <400> 3 aataacagat ggggtaagtc gtaaca 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <400> 4 gtgaagaagg ctacagcaat taagat 26 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <400> 5 gagttcagac cggagcaatc ca 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <400> 6 cagttcctgc gcctgtttca at 22 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <400> 7 ctacatggtt tgataagaag gggagt 26 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <400> 8 attggtgata ttgcgtcttg aaatcc 26 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <400> 9 cactacacca aacctgaacc aaagta 26 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <400> 10 gattgtgaat ggtgcttcgt agtattc 27 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <400> 11 cacacaacta tcaatgaaca tagcac 26 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <400> 12 tgaactgaaa ttgaatgatt ggaagt 26 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <400> 13 ctacaagcca tagaatacta cgaagcac 28 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <400> 14 ttcaagatca gttggtgatg attgattc 28 <210> 15 <211> twenty four <212> DNA <213> Artificial Sequence <400> 15 aatctcctta taaaccgaga aggt 24 <210> 16 <211> twenty two <212> DNA <213> Artificial Sequence <400> 16 agatttagtt cgtggtttgg ct 22 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <400> 17 atcattgcag gactactaat ctcatca 27 <210> 18 <211> 28 <212> DNA <213> Artificial Sequence <400> 18 atttcatcag atggagatgt tagatgga 28 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <400> 19 gtcaacgcca cagaataagc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <400> 20 attccggttt tggggatcgg 20 <210> twenty one <211> 26 <212> DNA <213> Artificial Sequence <400> twenty one gccaaccttg cagtactaac atgaat 26 <210> twenty two <211> 26 <212> DNA <213> Artificial Sequence <400> twenty two tctaatccca gtttgtgtct tagcga 26

Claims

1. A set of universal primers for amplifying the whole mitochondrial genome of the family Trionycii, characterized in that: The universal primer set includes 11 pairs of amplification primers, and the sequences thereof are shown in SEQ ID NO: 1 to SEQ ID NO:

22.

2. A method for amplifying the whole mitochondrial genome of a Trionycidae species, characterized in that: The universal primers according to claim 1 were used to amplify the target fragment using a 20 μl PCR system under PCR amplification conditions.

3. The method according to claim 2, characterized in that The 20 μl PCR system specifically includes: 5 μl 10× Buffer, 8 μl dNTP Mix, 1 μl upstream primer, 1 μl downstream primer, 0.5 μl Taq enzyme, 0.5 μl DNA template, 4 μl MgCl2 2 + .

4. The method according to claim 2, characterized in that The PCR amplification conditions specifically include: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 52-66°C for 2 minutes and 30 seconds, extension at 72°C for 5 minutes, amplification for 35-40 cycles; and finally extension at 72°C for 10 minutes.

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