Universal molecular cloning simulation method based on DNA sequence terminal base complementation

Abstract

The invention provides a universal molecular cloning simulation method based on DNA sequence terminal base complementation. The supported cloning methods are all molecular cloning methods based on DNA sequence terminal base complementation, and include but are not limited to restriction enzyme digestion and linking, Jinmen cloning, Gibson assembly and In-Fusion assembly. The supported linearization mode of the vector and the DNA insertion sequence comprises enzyme digestion linearization, PCR linearization, and enzyme digestion linearization after PCR, and the linear sequence is directly used. The system processes a vector and a DNA insertion sequence according to a selected linearization mode to generate a DNA sequence fragment with a blunt end or a protruding end, and then pretreats the blunt end or the protruding end of the sequence fragment according to a selected cloning method to generate a protruding end which can be used for final connection. And finally, carrying out connection simulation on the connectable tail end with the protrusion, and finally generating a simulation result. The simulation result lists all possible products and possible problems.

Description

Technical Field

[0001] The invention relates to a molecular cloning simulation method, in particular to a simulation of a molecular cloning method based on complementary bases at the ends of a DNA sequence. Background Art

[0002] Molecular cloning is a laboratory technique that involves isolating a specific DNA fragment (such as a gene), replicating it in vitro, and inserting it into a vector molecule (such as a plasmid or virus). This vector carrying the foreign DNA is then introduced into a host cell, where it is replicated or expressed.

[0003] In the past few decades, molecular cloning technology has experienced significant progress, from the initial restriction enzyme cloning to today's high-efficiency, high-precision gene editing technology. Restriction enzyme cloning relies on specific restriction enzymes to recognize and cut DNA sequences, producing fragments with complementary sticky ends or blunt ends, which can be reconnected with vector DNA under the action of DNA ligase to form recombinant DNA molecules. In recent years, more advanced assembly technologies, such as Gibson Assembly and In-Fusion cloning, have achieved efficient splicing of DNA fragments through enzymatic reactions without the need for restriction enzymes and ligases, greatly improving the flexibility and speed of cloning.

[0004] Existing molecular cloning design and simulation software can support fixed processes for single molecular cloning methods, but lacks a unified design and simulation method, and cannot flexibly set the molecular cloning process. It is also unable to display detailed details and provide error prompts for simulation results, especially abnormal results, which has a significant impact on the efficiency of molecular cloning design. Summary of the invention

[0005] In order to overcome the above technical defects, the first aspect of the present invention provides a universal molecular cloning simulation method, which can support all molecular cloning methods based on DNA sequence terminal base complementation, including restriction enzyme cutting and ligation, Golden Gate cloning, Gibson assembly, and In-Fusion assembly. Users can choose and switch freely between various methods.

[0006] The second aspect of the present invention provides a flexible and configurable DNA sequence linearization method, which can set different linearization methods for the vector and each inserted fragment, including enzyme linearization, PCR linearization, PCR first and then enzyme linearization, and direct use of linear sequences. By summarizing and abstracting various supported molecular cloning methods, this setting method can meet the settings of various supported molecular cloning methods.

[0007] The third aspect of the present invention provides a general molecular cloning simulation method, which comprises: Step S1: Select a cloning method, including but not limited to restriction enzyme digestion and ligation, Golden Gate cloning, Gibson assembly, and In-Fusion assembly; Step S2: Select a vector, which can be a circular plasmid or a linear DNA sequence; Step S3: Select the linearization method of the vector, which can be enzyme linearization, PCR linearization, PCR followed by enzyme linearization, or direct use of the linear sequence; Step S4: Select the inserted DNA sequence fragments, and any number of inserted fragments can be selected; Step S5: Select the linearization method of the inserted fragment. Different linearization methods can be selected for each fragment, such as enzyme linearization, PCR linearization, PCR followed by enzyme linearization, or direct use of the linear sequence. Step S6: generating blunt ends or ends with protrusions according to the linearization methods of the vector and the fragment; Step S7: pre-treating the blunt ends or protruding ends generated in S6 according to the cloning method to generate protruding ends that can be used for ligation; Step S8: Based on the rule of base complementarity, the ligation simulation is performed on the ligatable ends and the results are generated.

[0008] The fourth aspect of the present invention provides a method for displaying simulation results, including a graphical product schematic display and textual information prompts. The graphical product display includes products of successful connection, and also includes various byproducts when the results are abnormal, including ring-forming or non-ring-forming products. The textual information prompts include three levels of messages: successful messages, warning messages of possible risks, and error messages determined to be errors. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Flowchart of molecular cloning simulation for this application. DETAILED DESCRIPTION

[0010] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the application claimed for protection, but merely represents the selected embodiments of the present application. Based on the embodiments of the present application, each other embodiment obtained by those skilled in the art without making creative work belongs to the scope of protection of the present application.

[0011] Research has found that existing molecular cloning design and simulation software can support fixed processes for single molecular cloning methods, but lacks a unified design and simulation method, and is unable to flexibly set up the molecular cloning process. The simulation results, especially abnormal results, cannot be displayed in detail and error prompts cannot be given, which has a great impact on the efficiency of molecular cloning design.

[0012] Based on this, the embodiments of the present application provide a universal molecular cloning simulation method, which can use a unified method to simulate multiple molecular cloning technologies.

[0013] See also Figure 1 , Figure 1 This is a flowchart corresponding to a universal molecular cloning simulation method based on DNA base complementarity provided in this embodiment. The operation process of one embodiment is as follows: Step S1: Select Gibson assembly as the molecular cloning method; Step S2: Select a circular plasmid as a vector; Step S3: linearize the vector using PCR linearization and specify a pair of primers; Step S4: Select a DNA sequence fragment to be inserted; Step S5: selecting a PCR linearization method as the linearization method of the insert fragment, and specifying a pair of primers; Step S6: generating a result after PCR simulation, i.e., a vector fragment with blunt ends, according to the primers of the vector; generating a result after PCR simulation, i.e., an inserted DNA fragment with blunt ends, according to the primers of the inserted fragment; Step S7: The vector fragment and the insert fragment generated in step S6 are subjected to end treatment, and 15 bases at the 5' end of the fragment are removed to form a protruding end at the 3' end.

[0014] Step S8: Pair all four ends of the two fragments according to the rule of base complementarity, find all ends that can be paired, generate all possible products, and generate error information for the ends that cannot be paired.

[0015] It should be noted that the cloning method, linearization method, and number of inserted fragments selected in this example are only examples.

[0016] It should be noted that the embodiments of the present invention have better practicability and do not impose any form of limitation on the present invention. Any technician familiar with the field may use the technical content disclosed above to change or modify it into an equivalent effective embodiment. However, any modification or equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A general molecular cloning simulation method, characterized in that: Supports all molecular cloning methods based on DNA sequence terminal base complementation, including but not limited to restriction enzyme digestion and ligation, Golden Gate cloning, Gibson assembly, and In-Fusion assembly.

2. A universal molecular cloning simulation method as claimed in claim 1, characterized in that: The vector and each inserted fragment support different linearization methods, including restriction linearization, PCR linearization, PCR followed by restriction linearization, and direct use of linear sequences.

3. A universal molecular cloning simulation method as claimed in claim 1, characterized in that: Supports ligation or assembly of any number of insert fragments.

4. A universal molecular cloning simulation method as claimed in claim 1, characterized in that: Simulate all possible assembly outcomes, as well as all possible misconnections during assembly.

5. A universal molecular cloning simulation method according to any one of claims 1 to 4, characterized in that: Use the following molecular cloning simulation steps: Step S1: Select a cloning method, including but not limited to restriction enzyme digestion and ligation, Golden Gate cloning, Gibson assembly, and In-Fusion assembly; Step S2: Select a vector, which is a circular plasmid or a linear DNA sequence; Step S3: Select the linearization method of the vector, including restriction linearization, PCR linearization, PCR followed by restriction linearization, and direct use of the linear sequence; Step S4: Select the inserted DNA sequence fragment, supporting any number of inserted fragments; Step S5: Select the linearization method of the inserted fragment. Each fragment supports different linearization methods, including enzyme linearization, PCR linearization, PCR followed by enzyme linearization, and direct use; Step S6: generating blunt ends or ends with protrusions according to the linearization methods of the vector and the fragment; Step S7: pre-treating the blunt ends or protruding ends generated in S6 according to the cloning method to generate protruding ends for ligation; Step S8: Based on the rule of base complementarity, the connected ends are simulated and the results are generated.

Images