A method for sequencing small circular DNA molecules

Abstract

The application provides a method for sequencing small circular DNA molecules, comprising: constructing recombinant transposon fragments; enriching small circular DNA molecules; constructing recombinant small circular DNA molecules; and verifying positive clone vectors. Antibiotic resistance genes are randomly inserted into each small circular DNA molecule by using transposons, so that at least one antibiotic resistance gene is obtained in each recombinant small circular DNA molecule. Since an antibiotic is added to the culture medium in the subsequent culture process, only the clone vectors successfully transformed with the recombinant small circular DNA molecules can survive and reproduce in the culture medium, so that the antibiotic resistance gene becomes a "conserved sequence" of the recombinant small circular DNA molecule, and a corresponding PCR amplification can be designed for the antibiotic resistance gene. In addition, the antibiotic resistance gene sequence can be removed by using software when analyzing the sequence of the small circular DNA molecule.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene detection, in particular to a method for sequencing small circular DNA molecules. BACKGROUND

[0002] In the evolutionary process of secondary endosymbiosis of algae, the chloroplast genome of dinoflagellate evolved from a single large circular DNA molecule of 100-200kbp in size including 100-250 genes to multiple small circular DNA molecules of 0.4-10kbp in size including 0-2 genes. This change may be due to the splitting or recombination of the chloroplast genome. In the past, primers were generally designed according to the conserved sequences of small circular DNA molecules, and then PCR amplification was used to analyze the sequences of small circular DNA molecules, but this method is easy to miss some small circular DNA molecules without conserved sequences, so it cannot sequence all small circular DNA molecules of dinoflagellate; it is difficult to obtain complete information of small circular DNA molecules through PCR amplification and subsequent sequencing, splicing and other analysis. SUMMARY

[0003] The present application aims to provide a method for sequencing small circular DNA molecules to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0004] The first aspect of the present application provides a method for sequencing small circular DNA molecules, comprising the following steps:

[0005] Constructing a recombinant transposon fragment: inserting an antibiotic resistance gene into a transposon to obtain a recombinant transposon fragment for standby;

[0006] Enriching small circular DNA molecules: lysing target cells containing small circular DNA molecules, and collecting small circular DNA molecules of 0.4-10kbp in length through gel electrophoresis;

[0007] Constructing a recombinant small circular DNA molecule: inserting the standby recombinant transposon fragment into the enriched small circular DNA molecules through enzyme digestion, purifying to obtain a recombinant small circular DNA molecule;

[0008] Verifying positive clone vectors: transforming the constructed recombinant small circular DNA molecule into a cloning vector, screening through a culture medium containing an antibiotic to obtain a positive strain with an antibiotic resistance gene, extracting the plasmid of the positive strain, designing primers with the antibiotic resistance gene as a conserved sequence, and performing sequencing analysis.

[0009] The method for sequencing the small circular DNA molecule provided in the foregoing is performed by inserting an antibiotic resistance gene into the initially enriched small circular DNA molecule, and transforming the recombinant small circular DNA molecule with the antibiotic resistance gene into a cloning vector, so that the cloning vector acquires the ability to survive and reproduce in a culture medium containing the corresponding antibiotic, and the antibiotic resistance gene becomes a "conserved sequence" of the recombinant small circular DNA molecule, thereby enabling the design of a corresponding PCR amplification for the antibiotic resistance gene to analyze the sequence of the small circular DNA molecule.

[0010] Further, the target cell containing the small circular DNA molecule is a dinoflagellate.

[0011] Further, the enrichment is centrifugal collection of the dinoflagellate of Peridinium sp. cultured for five weeks. Amphidinium carterae The product of 0.4-10 kbp is obtained by alkaline lysis of the dinoflagellate and electrophoresis. By gel electrophoresis, the small circular DNA molecule of 0.4-10 kbp in length in the dinoflagellate cultured for a long time can be accurately obtained, facilitating subsequent insertion of the antibiotic resistance gene using a transposon.

[0012] Further, the antibiotic resistance gene is at least one selected from a kanamycin resistance gene, a bleomycin resistance gene, a neomycin phosphotransferase resistance gene, a hygromycin resistance gene, a paromomycin resistance gene, an erythromycin resistance gene, an ampicillin resistance gene, or a tetracycline resistance gene.

[0013] Further, the transposon is one selected from a piggyBac transposon, a piggyBat transposon, a Tc1 / mariner transposon, a SINE transposon, an Ac / Ds transposon, a Tn5058 transposon, a T2-Onc3 transposon, an EZ-Tn5 transposon, or a Tn4351 transposon. For example, the EZ-Tn5 transposon can be obtained by an EZ-Tn5 Pmod-3 vector (EZ-Tn5™ pMOD-3<R6K γori / MCS>). The EZ-Tn5 transposon includes two connection ports, a multiple cloning site, and a replication initiation site R6K γori The nucleotide sequence of the connection port is 5'-AGATGTGTATAAGAGACAG-3' (SEQ ID NO. 1).

[0014] Further, the purification is performed by adding a 25:24:1 volume ratio of phenol-chloroform-isoamyl alcohol mixture. The isoamyl alcohol reduces the bubbles generated during use. The phenol denatures the protein while inhibiting the degradation effect of RNase. The chloroform extraction accelerates the separation of the organic phase and the liquid phase, and removes the trace amount of phenol in the nucleic acid solution. The isoamyl alcohol reduces the bubbles generated during the protein denaturation operation. The upper layer after the use of phenol: chloroform: isoamyl alcohol extraction centrifugation is the aqueous phase containing RNA, the middle layer is the denatured protein and DNA, and the lower layer is the organic solvent phase. After standing, the product has a layered phenomenon, and when used, the product is shaken and then sucked with a pipette.

[0015] Further, the cloning vector is selected from one of E. coli, Agrobacterium, Bacillus subtilis, Saccharomyces cerevisiae, or Pichia pastoris. Specifically, TransforMax™ Ec100D™ pir + E. coli, which has a vector for replication containing R6Kγ conditional replication origin (R6K γori The EZ-Tn5 transposon can be adapted for use.

[0016] Further, the transformation is performed by electroporation, and the implementation conditions of the electroporation are 2.5 kV and 2 mm electroporation cup.

[0017] Further, the step of screening the positive strain of the antibiotic resistance gene includes: after the cloning vector transformed with the recombinant minicircle DNA molecule is cultured in an antibiotic-free medium for 1 hour, it is transferred to an antibiotic-containing solid plate medium for overnight culture, and the antibiotic corresponds to the antibiotic resistance gene.

[0018] The beneficial effects of the present application are that the antibiotic resistance gene is randomly inserted into each minicircle DNA molecule by using the transposon, so that at least one segment of the antibiotic resistance gene is obtained in each recombinant minicircle DNA molecule. Due to the addition of the related antibiotic in the subsequent culture medium, only the cloning vector successfully transformed with the recombinant minicircle DNA molecule can survive and reproduce in the culture medium, so that the antibiotic resistance gene becomes a "conserved sequence" of the recombinant minicircle DNA molecule, thereby enabling the design of a corresponding PCR amplification for the antibiotic resistance gene, and the use of software to exclude the antibiotic resistance gene sequence when analyzing the sequence of the minicircle DNA molecule. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is an electropherogram of enriched minicircle DNA molecules;

[0020] Figure 2 is a schematic diagram of the recombinant minicircle DNA molecule in Example 1. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts fall within the scope of the present application.

[0022] Embodiment 1: a method for sequencing small circular DNA molecules

[0023] (1) In order to accumulate the maximum amount of small circular DNA molecules, centrifugal collection of the cultured Peridiniopsis sp. for five weeks was performed. Amphidinium carterae

[0024] (2) The small circular DNA molecules obtained in step (1) were enriched by using a bacterial plasmid analysis method, i.e. alkaline lysis method, and electrophoresis was performed, and the results are shown in FIG. 1. The bands located at 1.5-3.0 kbp in the figure were cut and collected, which were the small circular DNA molecules. Figure 1

[0025] (3) The EZ-Tn5 transposon includes two connection ports, a multiple cloning site and a replication initiation site R6K γori ; the nucleotide sequence of the connection port is 5'-AGATGTGTATAAGAGACAG-3' (1). The EZ-Tn5 transposon was obtained by double digestion from the EZ-Tn5 Pmod-3 vector (EZ-Tn5 TM pMOD TM <R6K γori / MCS), and the sequence of the kanamycin resistance gene was inserted into the multiple cloning site of the EZ-Tn5 transposon to construct a recombinant transposon fragment.

[0026] (4) According to the reaction system in Table 1, the recombinant transposon fragment containing the kanamycin resistance gene was inserted into the small circular DNA molecules obtained in step (2) under the action of the EZ-Tn5 transposase to construct the recombinant small circular DNA molecules as shown in FIG. 2. Figure 2

[0027] Table 1: Reaction system for transposon insertion into small circular DNA molecules

[0028]

[0029] (5) The reaction was terminated by adding a reaction stop solution to terminate the insertion of the transposon.

[0030] (6) The recombinant small circular DNA molecules were purified by adding a phenol-chloroform-isopentyl alcohol mixture with a volume ratio of 25:24:1.

[0031] ​​​(7) Add 2.5 parts of 100% ethanol, 0.1 part of sodium acetate by volume ratio, adjust pH to about 4.8, and precipitate the recombinant mini-circle DNA molecule.

[0032] (8) Transform the recombinant mini-circle DNA molecule into E. coli cells (TransforMax EC100D pir + E. coli cells) under the electric transformation condition (2.5 kV, 2 mm).

[0033] (9) After the E. coli cells obtained after the electric transformation in step (8) are cultured in 300 mL of medium without antibiotics for 1 hour, they are transferred to solid plate medium containing antibiotics for overnight culture.

[0034] (10) Pick single colonies, extract plasmids, and design primers with kanamycin resistance gene inserted by EZ-Tn5 transposon as conservative sequences for sequencing analysis. The primers are shown in Table 2:

[0035] Table 2 Sequencing primer sequences

[0036]

[0037] Example 2, sequencing effect comparison test

[0038] The conventional sequencing method of PCR amplification according to known conservative sequences is used as a comparative example, combined with the sequencing method provided in Comparative Example 1.

[0039] (1) The sequence obtained in Example 1 is subjected to sequence alignment and splicing in Sequencher 5.1 software.

[0040] (2) According to the mini-circle DNA sequence fragment obtained in (1), primers are designed at the end of the sequence for PCR amplification and sequencing, and finally the complete mini-circle DNA molecule sequence is obtained.

[0041] (3) On the basis of the complete mini-circle DNA molecule sequence obtained, further sequence alignment is performed by Clustal X software.

[0042] Using the method provided in Example 1, the sequence of the recombinant mini-circle DNA molecule named A. carterae89 different small circular DNA molecules were successfully amplified and sequenced from CCAP 0512, and the detailed information of the 89 different small circular DNA molecules is shown in Table 3 and Table 4. Table 3 records the detailed information of 18 different small circular DNA molecules containing chloroplast genes, and Table 4 records the detailed information of 71 different small circular DNA molecules not containing chloroplast genes. In the conventional sequencing method according to the known conserved sequence for PCR amplification, the chloroplast gene is usually used as a conserved sequence to design PCR primers for amplification and sequencing.

[0043] Table 3 Detailed information of small circular DNA molecules containing chloroplast genes

[0044]

[0045] Table 4 Detailed information of small circular DNA molecules not containing chloroplast genes

[0046]

[0047] Table 5 lists the number of small circular DNA molecules sequenced by the present technical solution for CCAP 0512 and the number of small circular DNA molecules sequenced by some existing technologies. It can be clearly seen that the number of small circular DNA molecules that can be sequenced by the sequencing method provided by the present technical solution far exceeds that of the existing conventional method, proving that the method for sequencing small circular DNA molecules provided in Embodiment 1 can successfully amplify and sequence small circular DNA molecules without conserved sequences.

[0048] Table 5

[0049]

[0050] A. carterae CCAP1102 / 6 is also called Amphidinium operculatum , 16 small circular DNA molecules were obtained by PCR technology, and the specific reference is (Organisation and expression of the plastid genome of the dinoflagellate Amphidinium operculatum ; Minicircular plastid DNA in the dinoagellate Amphidinium operculatum ; Comparative analysis of dinoflagellate chloroplast genomes reveals rRNA and tRNA genes).

[0051] A. carteraeThe 21 minicircle DNA molecules of CS21 were isolated by PCR technique, reference (Empty minicircles and petB / atpA and psbD / psbE (cytb 559 α) genes in tandem Amphidinium carterae plastid DNA; Comparative analysis of dinoflagellate chloroplast genomes reveals rRNA and tRNA genes; Novel plastid genome minicircles in the dinoflagellate Amphidinium operculatum )。

[0052] A. carterae The 2 minicircle DNA molecules of CCMP1314 were isolated by PCR technique, reference (Evolution of Dinoflagellate Unigenic Minicircles and the Partially Concerted Divergence of Their Putative Replicon Origins).

[0053] It is apparent to those skilled in the art that the application is not limited to the details of the foregoing exemplary embodiments, and that the application can be implemented in other particular forms without departing from the spirit or essential characteristics of the application. Therefore, the embodiments should be considered in all respects as illustrative and not restrictive, the scope of the application being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. SEQUENCE LISTING <110> Southern Ocean Science and Engineering Guangdong Provincial Laboratory (Guangzhou); Shantou University <120> A method for sequencing minicircle DNA molecules <130> 2022 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <400> 1 agatgtgtat aagagacag 19 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <400> 2 attcaggctg cgcaactgt 19 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <400> 3 gtcagtgagc gaggaagcgg aag 23

Claims

1. A method of sequencing a small circular DNA molecule of Prorocentrum micans, characterized by, The method comprises the steps of: lysing Prorocentrum donghaiense to enrich small circular DNA molecules; inserting an antibiotic resistance gene into an EZ-Tn5 transposon to construct a recombinant transposon fragment; inserting the recombinant transposon fragment into the small circular DNA molecules, purifying to obtain recombinant small circular DNA molecules; transforming the recombinant small circular DNA molecules into a cloning vector, screening for antibiotic resistance gene positive strains, extracting plasmids, designing primers with the antibiotic resistance gene as a conserved sequence, and performing sequencing analysis.

2. The method of claim 1, wherein, The enrichment is achieved by alkaline lysis of Prorocentrum donghaiense and obtaining 0.4-10 kbp products by electrophoresis.

3. The method of claim 1, wherein, The antibiotic resistance gene is at least one selected from kanamycin resistance gene, bleomycin resistance gene, neomycin phosphotransferase resistance gene, hygromycin resistance gene, paromomycin resistance gene, erythromycin resistance gene, ampicillin resistance gene, or tetracycline resistance gene.

4. The method of claim 1, wherein, The purification is achieved by adding a 25:24:1 volume ratio of phenol-chloroform-isoamyl alcohol mixture for extraction.

5. The method of claim 1, wherein, The cloning vector is selected from one of Escherichia coli, Agrobacterium, Bacillus subtilis, Saccharomyces cerevisiae, or Pichia pastoris.

6. The method of claim 5, wherein, The transformation is performed by electroporation, and the implementation conditions of the electroporation are 2.5 kV and 2 mm shock cup.

7. The method of claim 1, wherein, The step of screening for antibiotic resistance gene positive strains comprises the following steps: after the cloning vector transformed with the recombinant small circular DNA molecules is cultured in an antibiotic-free medium for 1 hour, it is transferred to an antibiotic-containing solid plate medium for overnight culture, and the antibiotic corresponds to the antibiotic resistance gene.

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