CN110724728A - Preparation method of circular DNA - Google Patents
Preparation method of circular DNA Download PDFInfo
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- CN110724728A CN110724728A CN201910968892.7A CN201910968892A CN110724728A CN 110724728 A CN110724728 A CN 110724728A CN 201910968892 A CN201910968892 A CN 201910968892A CN 110724728 A CN110724728 A CN 110724728A
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
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- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The present disclosure provides a method for preparing circular DNA, which comprises the following specific steps: (1) firstly, synthesizing a single-stranded DNA molecule needing to be cyclized, and then carrying out phosphorylation modification on the 5' end of the synthesized single-stranded DNA molecule; (2) then designing and synthesizing a guide RNA molecule, wherein the nucleotide sequence of the guide RNA molecule is complementarily paired with the nucleotides at two ends of the synthesized single-stranded DNA molecule; (3) annealing the synthesized single-stranded DNA molecules and the guide RNA molecules to obtain an annealed product; (4) performing a ligation reaction on the obtained annealing product by using ligase to obtain a ligation reaction product; (5) digesting the non-circularized linear nucleotides in the ligation product by using exonuclease; (6) finally, the synthesized single-stranded circular DNA molecule is recovered. The preparation method is simple and easy to operate, the cost of the materials used in the method is low, and the cost for preparing the circular DNA is reduced.
Description
Technical Field
The disclosure relates to the technical field of nucleotide, in particular to a preparation method of circular DNA.
Background
Rolling circle replication (Loop-mediated isothermal amplification-LAMP) is a method for amplifying nucleic acids by using isothermal amplification enzymes with strand displacement capability using circular DNA molecules as templates. The method can perform sequence-specific amplification on a trace amount of nucleic acid template together with PCR (polymerase chain reaction), and is different in that the amplification reaction can be completed at a constant temperature in the rolling circle replication process, so that a PCR instrument with accurate temperature control is not needed, the method is a simple and economic nucleic acid amplification method, and single-stranded circular DNA molecules with different sequences can generate tandem repeat sequences with different lengths by rolling circle replication. A large number of random repetitive sequences exist in natural genes and proteins, and the repetitive sequences form a specific module sequence, so that the physiological specificity and activity of biological macromolecules of candidate genes can be increased rapidly. Such modules are widely distributed in nucleic acid sequences such as promoters/enhancers, etc., and facilitate recognition of target sequences by trans-acting factors, resulting in additive and/or synergistic effects. The construction of large libraries of random repeat sequences is one of the important strategies for studying structure-function relationships in vivo or in vitro. Random repeat sequences of different lengths and repeat times can be prepared by preparing single-stranded circular DNA molecules for rolling circle replication.
The currently commonly used single-stranded circular DNA template is mainly derived from single-stranded circular DNA M13mp18(7249bp) of virus and single-stranded circular DNA molecules (sscDNA) generated by an enzyme-linked reaction, wherein the sscDNA molecules are generally 34-120bp in length, but the virus culture process is slow and high in cost due to the use of the single-stranded circular DNA M13mp18, so that the price of M13mp18 is high, the Molecular weight (Molecular weight) of the single-stranded M13mp18 DNA is large, and the progress of a rolling circle replication reaction can be inhibited if the substrate concentration is too high. In the existing preparation method for producing single-stranded circular DNA molecules by enzyme-linked reaction, DNA (guide DNA) is mostly relied on as a guide molecule to complete the connection of target sequences, and the method can cause that trace guide DNA molecules are remained in a final product, so that subsequent DNA polymerase uses the guide DNA molecules as a primer to carry out rolling circle replication, thereby causing interference to result analysis.
Disclosure of Invention
The purpose of the present disclosure is to provide a method for preparing circular DNA, so as to achieve the purpose of reducing cost.
In order to realize the purpose, the technical scheme is as follows:
a preparation method of circular DNA comprises the following specific steps:
(1) firstly, synthesizing a single-stranded DNA molecule needing to be cyclized, and then carrying out phosphorylation modification on the 5' end of the synthesized single-stranded DNA molecule;
(2) then designing and synthesizing a guide RNA molecule, wherein the nucleotide sequence of the guide RNA molecule is complementarily paired with the nucleotides at two ends of the synthesized single-stranded DNA molecule;
(3) annealing the synthesized single-stranded DNA molecules and the guide RNA molecules to obtain an annealed product;
(4) performing a ligation reaction on the obtained annealing product by using ligase to obtain a ligation reaction product;
(5) digesting the non-circularized linear nucleotides in the ligation product by using exonuclease;
(6) finally, the synthesized single-stranded circular DNA molecule is recovered.
The reaction conditions of annealing in the step (3) are as follows: first at 85 ℃ for 5min, then at 25 ℃ for 3 h.
The reaction system annealed in the step (3) is a synthesized single-stranded DNA molecule: a guide RNA molecule: annealing buffer solution: the volume ratio of the water for removing the RNA enzyme is 1: 2: 1: 7.
the annealing buffer comprises: 100mM Tris-HCl, 10mM EDTA, and 1M NaCl, said Tris-HCl having a pH of 7.5.
The conditions of the ligation reaction in the step (4) are as follows: the reaction was first carried out at 16 ℃ for 16h and then at 65 ℃ for 20 min.
The steps areThe system of the ligation reaction in step (4) is a ligation buffer: annealing products: a ligase: h2The volume ratio of O is 3: 1: 2.5: 30.
the ligase is SplintR ligase or T4 DNA ligase.
The Exonuclease in the step (5) is a mixture of Exonuclease I Exonuclease and T7Exonuclease Exonuclease.
The reaction system of exonuclease digestion is the ligation product: exonuclease I Exonuclease: the volume ratio of the T7 Exonase Exonuclease is 7: 1: 2.
the conditions of exonuclease digestion in the step (5) are as follows: the reaction is carried out for 16h at 25 ℃.
The beneficial effects of this disclosure are: a method for producing a circular DNA is provided, which is simple and easy to handle, and in which a practical single-stranded circular virus DNA M13mp18 is not required, and the cost of the remaining materials is low, thereby reducing the cost for producing a circular DNA. Meanwhile, the preparation method of the circular DNA breaks through the limitation of a template sequence, does not need to add exogenous DNA in the preparation process, but adds RNA molecules synthesized at the tail end of single-stranded DNA molecules which are cyclized according to the requirement, reduces interference, utilizes exonuclease to digest non-cyclized linear nucleotides, further reduces the generation of byproducts, greatly increases the yield of the single-stranded circular DNA, and the single-stranded circular DNA prepared by the preparation method of the circular DNA disclosed by the invention can also be used for detecting whether DNA polymerase has the strand displacement capability.
Drawings
FIG. 1 is a diagram of gel electrophoresis of the annealing product, ligation product and exonuclease digestion product in example 1.
FIG. 2 is a gel electrophoresis image of the rolling circle replication product of example 2.
FIG. 3 is a gel electrophoresis of the products of example 3 at different extension reaction times.
FIG. 4 is a DNA polymerase chain displacement capability test chart according to example 4.
Detailed Description
The following steps are only used for illustrating the technical scheme of the disclosure and are not limited; although the present disclosure has been described in detail with reference to the foregoing steps, those of ordinary skill in the art will understand that: the technical solutions recorded in the foregoing steps may still be modified, or some or all of the technical features may be equivalently replaced; and such modifications or substitutions do not depart from the scope of the respective technical solutions of the steps of the present disclosure.
Example 1
A preparation method of circular DNA comprises the following specific operation steps:
(1) firstly synthesizing a single-stranded DNA molecule needing to be looped, and then carrying out phosphorylation modification on the 5' end of the synthesized single-stranded DNA molecule, wherein the nucleotide sequence of the single-stranded DNA molecule is SEQ ID NO. 1: 5' -pATTGGTCTACATGCATGCATGCATGCATGCATGCATGCATGCATGCATGCATGCATGCATGCATGCACCGCATGC;
(2) then designing and synthesizing a guide RNA molecule, wherein the nucleotide sequence of the guide RNA molecule is complementarily paired with two ends of the synthesized single-stranded DNA molecule, and the nucleotide sequence of the guide RNA molecule is SEQ ID NO. 2: 5'-UAGACCAAUGCAAUCCGUA-3', respectively;
(3) annealing the synthesized single-stranded DNA molecule and the guide RNA molecule to obtain an annealed product, wherein the system of the annealing reaction is shown in Table 1,
TABLE 1 annealing reaction System
| Reagent | System of |
| DNA(100uM) | 1uL |
| RNA(100uM) | |
| 10×buffer | 1uL |
| Rnase-free H2O | Adding to a total volume of 10ul |
Wherein the 10 × annealing buffer comprises: 100mM Tris-HCl (pH 7.5), 10mM EDTA and 1M NaCl, under the conditions of the annealing reaction: firstly reacting at 85 ℃ for 5min, then reacting at 25 ℃ for 3h, and after the reaction is finished, performing 15% polyacrylamide gel electrophoresis, wherein the result is shown in a lane 1 of a figure 1A;
(4) performing ligation reaction on the obtained annealing product by SplintR ligase to obtain a ligation reaction product, wherein the system of the ligation reaction is shown in Table 2,
TABLE 2 ligation reaction System
| Reagent | Volume of |
| |
3 |
| Annealing product (10. mu.M) | 1 |
| Ligase(10.5μM) | 2.5 |
| H2O | 30uL |
Wherein the conditions of the ligation reaction are as follows: 16h at 16 ℃; the reaction was terminated at 65 ℃ for 20min, and after completion of the reaction, 15% polyacrylamide gel electrophoresis was carried out, the results being shown in lane 2 of FIG. 1A;
(5) digesting the non-circularized linear nucleotides in the ligation product by using exonuclease, wherein the reaction system of exonuclease digestion is shown in Table 3,
TABLE 3 reaction System for exonuclease digestion
| Reagent | Volume of |
| Linking the reaction products | 7uL |
| Exonuclease I | 1uL |
| T7 gene 6exonuclease | 2ul |
Wherein the reaction conditions are as follows: reacting at 25 ℃ for 16h, and after the reaction is finished, performing 15% polyacrylamide gel electrophoresis to detect whether the target product and the linear molecule are completely digested, wherein the result is shown in fig. 1B, and the linear molecule can be seen to be completely digested;
(6) finally, using NEB MonarchTMPCR&The DNA clean Kit recovery Kit recovers the synthesized single-stranded circular DNA molecule.
Example 2
The single-stranded circular DNA molecule (sscDNA) prepared in example 1 was used for rolling circle replication experiments, which were performed by the following specific steps: firstly, designing a primer sequence, wherein the primer DNA sequence is SEQ ID NO. 3: 5'-aTAGACCAAT GCAATCCGTAc-3', respectively; then annealing reaction is carried out, and finally extension reaction is carried out by using Phi29DNA polymerase.
The system of the annealing reaction is shown in table 4:
TABLE 4 Rolling circle replication annealing reaction System
| Reagent | System of |
| sscDNA(2.5uM) | 1uL |
| Primer DNA (2.5uM) | 2ul |
| 10×buffer | 1uL |
| Rnase-free H2O | Adding to a total volume of 10ul |
Wherein the 10 × annealing buffer comprises: 100mM Tris-HCl (pH 7.5), 10mM EDTA and 1M NaCl, under the annealing conditions: 5min at 85 ℃; 25 ℃ and 3 h.
The system in which extension reaction was performed using Phi29DNA polymerase (Phi29 DNAP) was then set as a control, and the system is shown in table 5:
TABLE 5 System for Rolling circle replication extension reaction
Wherein Phi29DNA polymerase, purchased from NEB, cat # M0269S; the extension reaction conditions were 30 ℃ for 30 min.
And after the reaction is finished, detecting the extension product by adopting alkaline agarose gel electrophoresis, wherein the conditions of the alkaline agarose gel electrophoresis are 3V/CM and 3 h. The buffer for alkaline agarose gel electrophoresis was: 10x alkaline agarose electrophoresis buffer or 6 x alkaline gel loading buffer, wherein the 10x alkaline agarose electrophoresis buffer comprises: 500mM NaOH and 10mM EDTA; the 6 × basic gel loading buffer comprises: 300mM NaOH, 6mM EDTA, 18% (m/V) Ficoll-400 (Pharmacia), 0.15% (m/V) bromocresol green and 0.25% (m/V) xylene cyanide. After electrophoresis is finished, soaking the agarose gel in SYBR Gold dye solution, and slowly and horizontally oscillating for about 20 min; and then detected using an Azure Biosystems imager. As a result, as shown in FIG. 2, the control group (lane 1) had no extension product, while the experimental group (lane 2) produced an extension product of more than 48.5kbp, demonstrating that the DNA prepared in example 1 was circular DNA.
Example 3
Tandem repeat sequences with different lengths and concentrations can be generated by using the single-stranded circular DNA molecule prepared in example 1 as a template and setting different extension reaction times (0, 5, 10, 20 and 30min) according to the rolling circle replication method of example 2, wherein the system of the annealing reaction is shown in Table 1 of example 2, and the conditions of the annealing reaction are as follows: 5min at 85 ℃; at 25 ℃ for 3 h; the results of the extension reaction in the experimental group shown in table 5 in example 2 are shown in fig. 3, in which the reaction times in lanes 2-6 are 0, 5, 10, 20, and 30min, respectively, and it can be seen that the length and concentration of the extension product both increase with the extension time.
Example 4
Using the single-stranded circular DNA molecule prepared in example 1 as a template, it is possible to detect whether the DNA polymerase has a strand displacement ability or not by the rolling circle replication method in example 2 based on the polymerization ability of the DNA polymerase, wherein the system of the annealing reaction is shown in Table 1 of example 2, and the conditions of the annealing reaction are as follows: 5min at 85 ℃; at 25 ℃ for 3 h; wherein the extension reaction system is shown in Table 6:
TABLE 6 extension reaction System for detecting the polymerase chain Displacement capability of DNA
Wherein Phi29DNA polymerase, purchased from NEB, cat # M0269S; the extension reaction conditions were 30 ℃ for 30 min.
The gel electrophoresis of the product generated from the above reaction system is shown in FIG. 4, wherein lane 1 is a blank control group without DNA polymerase, lane 2 is Phi29DNA polymerase extension product, lane 3 is E.coli DNApol I, Large (Klenow) fragment, and it can be seen from FIG. 4 that the DNA polymerase with strand displacement ability can perform rolling circle replication by using circular DNA molecule to generate large single-stranded DNA product; however, since DNA polymerase having no strand displacement ability cannot produce a corresponding product, the method of using a single-stranded circular DNA molecule as a template and using the polymerization ability of DNA polymerase can be used to quickly and easily identify whether unknown DNA polymerase has a strand displacement ability.
SEQUENCE LISTING
<110> Shenzhen Qinghua university research institute; anthra Source Biotechnology (Shenzhen) Limited
<120> a method for producing circular DNA
<130>2019.10.12
<160>3
<170>PatentIn version 3.5
<210>1
<211>69
<212>DNA
<213> Artificial Synthesis
<400>1
attggtctac atgcatgcat gcatgcatgc atgcatgcat gcatgcatgc atgcatgcct 60
acggattgc 69
<210>2
<211>19
<212>RNA
<213> Artificial Synthesis
<400>2
uagaccaaug caauccgua 19
<210>3
<211>21
<212>DNA
<213> Artificial Synthesis
<400>3
atagaccaat gcaatccgta c 21
Claims (10)
1. A method for preparing circular DNA, which is characterized by comprising the following steps:
(1) firstly, synthesizing a single-stranded DNA molecule needing to be cyclized, and then carrying out phosphorylation modification on the 5' end of the synthesized single-stranded DNA molecule;
(2) then designing and synthesizing a guide RNA molecule, wherein the nucleotide sequence of the guide RNA molecule is complementarily paired with the nucleotides at two ends of the synthesized single-stranded DNA molecule;
(3) annealing the synthesized single-stranded DNA molecules and the guide RNA molecules to obtain an annealed product;
(4) performing a ligation reaction on the obtained annealing product by using ligase to obtain a ligation product;
(5) digesting the non-circularized linear nucleotides in the ligation product by using exonuclease;
(6) finally, the synthesized single-stranded circular DNA molecule is recovered.
2. The method for preparing circular DNA according to claim 1, wherein the annealing in step (3) is carried out under the following reaction conditions: first at 85 ℃ for 5min, then at 25 ℃ for 3 h.
3. The method for preparing circular DNA according to claim 1, wherein the reaction system for annealing in step (3) is a synthetic single-stranded DNA molecule: a guide RNA molecule: annealing buffer solution: the volume ratio of the water for removing the RNA enzyme is 1: 2: 1: 7.
4. the method for preparing circular DNA according to claim 3, wherein the annealing buffer comprises: 100mM Tris-HCl, 10mM EDTA, and 1M NaCl, said Tris-HCl having a pH of 7.5.
5. The method for preparing circular DNA according to claim 1, wherein the ligation reaction in step (4) is carried out under the following conditions: the reaction was first carried out at 16 ℃ for 16h and then at 65 ℃ for 20 min.
6. The method for preparing circular DNA according to claim 1, wherein the ligation reaction in step (4) is performed in a ligation buffer: annealing products: a ligase: h2The volume ratio of O is 3: 1: 2.5: 30.
7. the method for producing circular DNA according to claim 1, wherein the ligase is SplintR ligase or T4 DNA ligase.
8. The method for preparing circular DNA according to claim 1, wherein the Exonuclease in the step (5) is a mixture of Exonuclease I Exonuclease and T7Exonuclease Exonuclease.
9. The method for preparing circular DNA according to claim 8, wherein the reaction system of exonuclease digestion is ligation product: exonuclease I Exonuclease: the volume ratio of the T7 Exonase Exonuclease is 7: 1: 2.
10. the method for preparing circular DNA according to claim 1, wherein the conditions for exonuclease digestion in the step (5) are: the reaction is carried out for 16h at 25 ℃.
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| CN111793619A (en) * | 2020-06-04 | 2020-10-20 | 中国海洋大学 | Preparation method of single-stranded circular DNA |
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| CN111793619A (en) * | 2020-06-04 | 2020-10-20 | 中国海洋大学 | Preparation method of single-stranded circular DNA |
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