阅读说明：本技术 一种环状dna的制备方法 (Preparation method of circular DNA ) 是由 高亚平 田晖 何筠 邓素华 伊戈尔·伊万诺夫 于 2019-10-12 设计创作，主要内容包括：本公开提供了一种环状DNA的制备方法,所述环状DNA的制备方法的具体步骤为：(1)首先合成需要成环的单链DNA分子,然后在合成的单链DNA分子的5’端做磷酸化修饰；(2)然后设计合成引导RNA分子,所述引导RNA分子的核苷酸序列与合成的单链DNA分子两端的核苷酸互补配对；(3)将合成的单链DNA分子和引导RNA分子进行退火,获得退火产物；(4)利用连接酶对得到的退火产物进行连接反应,得到连接反应产物；(5)利用核酸外切酶消化连接产物中未成环的线性核苷酸；(6)最后回收合成的单链环状DNA分子。所述制备方法简单易操作,且在方法中所使用的材料成本低,降低了制备环状DNA的成本。(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.)

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 ℃.

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.