An automated online tool for primer design for plasmid point mutation modification
Patent Information
- Application Number
- CN202410851880.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-28
- Publication Date
- 2025-08-22
- Estimated Expiration
- 2044-06-28
AI Technical Summary
[0004]本发明的目的在于提供一种面向质粒点突变改造引物设计的自动化在线工具，以解决上述背景技术中提出的由于需要用户手动处理步骤繁多和引物设计的数据冗余复杂，会导致设计效率低下和错误率高的问题
[0063] 1. This automated online tool for designing primers for plasmid point mutation modification uses automated data processing technology and Primer3 software to automatically parse sequence data uploaded by users, efficiently and accurately design PCR primers suitable for plasmid point mutations, and generate mutant plasmid files containing primers.
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Figure CN118711661B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of genetic engineering, in particular to an automated online tool for designing primers for plasmid point mutation modification. Background Art
[0002] An automated online tool for designing primers for plasmid point mutation modification is an integrated bioinformatics platform designed to simplify and automate the plasmid point mutation primer design process. It uses automated data processing and primer design software to achieve efficient and accurate primer design and plasmid modification.
[0003] Existing primer design tools for plasmid point mutation modification can only perform a single primer design task. Due to the numerous manual steps required by the user and the complex and redundant primer design data, this leads to low design efficiency and high error rates. Therefore, an automated online tool for plasmid point mutation modification primer design was designed. Summary of the Invention
[0004] The purpose of the present invention is to provide an automated online tool for designing primers for plasmid point mutation modification, so as to solve the problems raised in the above background art, such as the need for users to manually process many steps and the redundant and complex data of primer design, which leads to low design efficiency and high error rate.
[0005] To achieve the above objectives, the present invention provides an automated online tool for designing primers for plasmid point mutation modification, comprising:
[0006] A data preprocessing unit reads and parses Excel files uploaded by users, removes duplicates and groups them to obtain plasmid file names, determines the mutation type of each sample and whether the plasmid file meets the requirements, and parses the plasmid file to determine the sequence and location of the target gene and resistance gene;
[0007] Also included is a single-point mutation primer design unit, which uses the Primer3 software package to generate PCR primers based on the input template sequence and the design parameters set by the user, and generates a mutation plasmid file containing the primers;
[0008] It also includes a double-point mutation primer design unit, which is used to process the primer design of double-point mutations and is divided into three double-point mutation primer design situations according to the mutation spacing;
[0009] Also included is a sequencing verification primer design unit, which is used to generate primers required for sequencing based on the designed mutation and plasmid sequence;
[0010] It also includes a recombinant plasmid map unit, which recombines the mutated new gene and primers into a new plasmid file through a Bio toolkit based on the primer design results and the PCR product sequence;
[0011] It also includes a data detection and collection unit, which is used to detect data generated during primer design and plasmid transformation, collect successful and failed design results, save error information, and generate a detailed detection report.
[0012] As a further improvement of the present technical solution, the data preprocessing unit includes a data parsing and verification module, a sequence extraction module and a data verification specification module;
[0013] The data parsing and verification module reads Excel content, removes duplicates and groups the template column, determines whether the mutation type of each sample conforms to the expected format, and checks whether the plasmid file is consistent with the specified one; if the mutation type conforms to the expected format, the sample passes the preliminary verification; if the mutation type does not conform to the expected format, the data detection and collection unit records an error message;
[0014] Among them, the sequence extraction module parses the .gb and .dna files according to different plasmid names, determines the direction of the target gene and the resistance gene and whether it crosses the zero point, and extracts the sequence and position data of the gene in a clockwise direction with the zero point as the center; if the direction and position of the target gene and the resistance gene are clear in the plasmid file and do not cross the zero point, the corresponding sequence information can be directly extracted; if the direction of the target gene and the resistance gene is unclear and crosses the zero point, the starting point and end point of the sequence extraction need to be redefined;
[0015] Among them, the data verification and standardization module is used to verify whether the base and position before mutation in the Excel file are consistent with the base at the same position in the plasmid, and convert the amino acid mutation into a nucleotide mutation form; if the base and position before mutation recorded in the Excel file are consistent with the base at the corresponding position in the plasmid file, the normalization processing of the mutation content will continue; if the base and position before mutation recorded in the Excel file are inconsistent with the base at the corresponding position in the plasmid file, the data detection and collection unit will record an error message.
[0016] As a further improvement of the present technical solution, the single point mutation primer design unit includes a design type determination module, a mutation content inspection module and a single point mutation primer design module;
[0017] Among them, the design type determination module is used to determine the number of mutations. If the multiple mutation identifier does not exist, it is determined to be a single mutation; if the multiple mutation identifier exists, it is determined to be a multiple mutation. If the mutation content format does not meet the requirements, the data detection and collection unit records the error information; the design type determination module is also used to determine whether the mutation type is "amino aci d". If the mutation type is "amino aci d", the mutation content is converted into a nucleotide mutation form and split into three new columns of data, including AAC, 1327, and ACC. If the mutation type is not "amino acid", the input nucleotide mutation form is directly used;
[0018] Among them, the mutation content inspection module is used to determine whether the base and position before mutation in Excel are consistent with the base at the same position in the plasmid. If the base and position before mutation recorded in the Excel file are consistent with the base at the corresponding position in the plasmid file, the verification is passed. If the base and position before mutation recorded in the Excel file are inconsistent with the base at the corresponding position in the plasmid file, the data detection and collection unit records an error message.
[0019] As a further improvement of the present technical solution, the single-point mutation primer design module combines the Python open source software package Primer3 with automated data preprocessing technology to generate upstream and downstream primer template sequences containing the mutation site based on the template sequence and design parameters input by the user, and designs the upstream and downstream primers using the Primer3 software package. The mutant plasmid file containing the primers is generated using the Bio toolkit. The specific steps involved in generating the mutant plasmid file containing the primers by the single-point mutation primer design module are as follows:
[0020] S2.1. Calculate the extension lengths on both sides of the mutation site based on the design parameters and template sequence entered by the user, and generate upstream and downstream primer template sequences containing the mutation site. The template sequence consists of three parts: the vector primer template sequence, the sequence between the vector primer template sequence and the target gene, and the partial sequence of the target gene containing the mutation site.
[0021] S2.2. Generate an upstream primer by calling the Primer3 software package based on the upstream primer template sequence and using the input primer design parameters;
[0022] S2.3. Based on the design parameters and template sequence entered by the user, locate the starting point of the upstream left primer on the vector primer template, add the length of the homology arm to obtain the new coordinates, extract the sequence from the starting point on the vector primer template to the new coordinates, and generate the downstream primer template;
[0023] S2.4. Generate downstream primers by calling the Primer3 software package and using the input primer design parameters;
[0024] S2.5. Based on the primer design results and PCR product sequences, use the Bio toolkit to generate a mutant plasmid file containing primer information and mutation sites.
[0025] As a further improvement of the present technical solution, the double-point mutation primer design unit (3) includes a double-point mutation design type module, a first double-point mutation primer design module, a second double-point mutation primer design module and a third double-point mutation primer design module;
[0026] Among them, the double-point mutation design type module is used to read the mutation spacing data and determine the corresponding primer design scheme according to the size of the mutation spacing; if the mutation spacing is between 0 and 21 bp, enter the first double-point mutation primer design module; if the mutation spacing is between 21 and 120 bp, enter the second double-point mutation primer design module; if the mutation spacing is greater than 120 bp, enter the third double-point mutation primer design module;
[0027] As a further improvement of the present technical solution, the first double-point mutation primer design module is used to process the design of double-point mutation primers with a spacing of 0 to 20 bp, generating a primer template containing two mutation sites. The specific steps involved in designing double-point mutation primers using the first double-point mutation primer design module are as follows:
[0028] S3.1.1. Generate an upstream primer template based on the vector primer template sequence, the upstream vector primer template sequence, the sequence between the target gene and the mutation site and its left and right extension sequences;
[0029] S3.1.2. Calculate the extension lengths to the left and right of the mutation site to ensure the mutation site is in the middle of the primer.
[0030] S3.1.3. Use the Primer3 software package to design upstream and downstream primers;
[0031] The second double-point mutation primer design module is used to process the design of double-point mutation primers with a spacing between 21 bp and 120 bp, generating a primer template containing two mutation sites. The specific steps involved in designing double-point mutation primers using the second double-point mutation primer design module are as follows:
[0032] S3.2.1. Select appropriate sequences for design based on the relationship between homology arm length and mutation spacing;
[0033] S3.2.2. If the length of the homology arm is equal to the distance between two mutations, the sequence of the homology arm is the sequence between the two mutations;
[0034] S3.2.3. If the homology arm length is less than the distance between two mutations, select the sequence with the same homology arm length between the two mutations;
[0035] S3.2.4. If the length of the homology arm is greater than the distance between two mutations, a sequence of the deletion length is taken from the tail of the target gene;
[0036] S3.2.5. Use the Primer3 software package to design upstream and downstream primers.
[0037] The third double-point mutation primer design module is used to process the design of double-point mutation primers with a spacing greater than 120 bp, generating a primer template containing two mutation sites. The specific steps involved in designing double-point mutation primers using the third double-point mutation primer design module are as follows:
[0038] S3.3.1. Calculate the extension lengths and coordinates on both sides of the two mutation sites to generate upstream and downstream primer templates;
[0039] S3.3.2. Extract the target gene sequence containing the first mutation and the extended sequence to the right of the second mutation.
[0040] S3.3.3. Extract the sequence containing the second mutation and the sequence following it in the target gene;
[0041] S3.3.4. Call the Primer3 software package to design upstream and downstream primers.
[0042] As a further improvement of this technical solution, the extension lengths on the left and right sides of the mutation site are determined by calculation in S3.1.2. The mathematical formula involved is as follows:
[0043] The formula for calculating the right extension length is:
[0044]
[0045] The formula for calculating the left extension length is:
[0046]
[0047] Wherein, the distance is the distance between two mutation sites; max_primer_length is the maximum length of the primer; mutation_length is the length of each mutation site; d right is the right extension length; d left The left side extension length.
[0048] As a further improvement of this technical solution, the sequencing verification primer design unit includes a template generation module, a sequencing primer design module and a result analysis module;
[0049] The template generation module extracts the region containing the mutation site in the plasmid sequence according to the design parameters and mutant plasmid sequence input by the user, and generates upstream and downstream primer template sequences for sequencing verification;
[0050] The sequencing primer design module uses the Primer3 software package to generate upstream sequencing primers and downstream sequencing primers according to the generated upstream and downstream sequencing primer design templates;
[0051] Among them, the result analysis module parses the sequencing primer design results generated by Primer3 to obtain the primer sequence and related information required for sequencing, including primer Tm value, primer GC content, primer length, primer pair score, primer position and primer secondary structure.
[0052] As a further improvement of the present technical solution, the recombinant plasmid map unit includes a primer design result acquisition module, a PCR product sequence generation module, a plasmid recombination module and a plasmid map generation module;
[0053] The primer design result acquisition module is used to read the primer design result file, obtain the sequence, Tm value, GC content, and primer position of the upstream and downstream primers, and use them for subsequent PCR product sequence generation and plasmid recombination processes;
[0054] The PCR product sequence generation module generates a PCR product sequence containing a mutation site by PCR amplification using upstream and downstream primer sequences;
[0055] The plasmid recombination module uses the PCR product sequence and the original plasmid sequence to generate a recombinant plasmid using the Bio toolkit through homologous recombination technology;
[0056] The plasmid map generation module generates a plasmid map using a plasmid drawing tool, and marks mutation sites, primer positions and other gene regions on the plasmid map.
[0057] As a further improvement of the present technical solution, the data detection and collection unit includes a primer design result detection module, a plasmid transformation process detection module, an error information collection module and a detection report generation module;
[0058] The primer design result detection module is used to monitor the primer design process, record the results of each design in real time, distinguish between successful and failed designs, collect successful primer sequences, Tm values, and GC content data, and record failed design parameters and reasons;
[0059] The plasmid transformation process detection module is used to monitor the plasmid recombination process, record the results of each recombination, collect the plasmid sequences and related information of successful recombination, and record the parameters and reasons for failed recombination;
[0060] The error information collection module collects error information from failure cases in the primer design and plasmid transformation process, including error causes, relevant parameters, and suggested modification plans;
[0061] The test report generation module is used to generate a detailed report containing success and failure cases based on the test results, including successful design results, failed cases and error information.
[0062] Compared with the prior art, the present invention has the following beneficial effects:
[0063] 1. This automated online tool for designing primers for plasmid point mutation modification uses automated data processing technology and Primer3 software to automatically parse sequence data uploaded by users, efficiently and accurately design PCR primers suitable for plasmid point mutations, and generate mutant plasmid files containing primers.
[0064] 2. This automated online tool for primer design for plasmid point mutation modification uses the BioPython toolkit to automatically reconstruct new plasmid files from primer design results and PCR product sequences, ensuring the integrity of the plasmid sequence and the accuracy of the mutation, while providing detailed plasmid maps for user verification and analysis. BRIEF DESCRIPTION OF THE DRAWINGS
[0065] Figure 1 It is a flowchart of the overall process of the present invention.
[0066] The meaning of each number in the figure is:
[0067] 1. Data preprocessing unit; 2. Single-point mutation primer design unit; 3. Double-point mutation primer design unit; 4. Sequencing verification primer design unit; 5. Recombinant plasmid map unit; 6. Data detection and collection unit. DETAILED DESCRIPTION
[0068] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0069] Example
[0070] See also Figure 1As shown, an automated online tool for designing primers for plasmid point mutation modification is provided, including:
[0071] Data preprocessing unit 1, which reads and parses the Excel file uploaded by the user, removes duplicates and groups them to obtain plasmid file names, determines whether the mutation type and plasmid file of each sample meet the requirements, and parses the plasmid file to determine the sequence and location of the target gene and resistance gene;
[0072] The data preprocessing unit 1 includes a data analysis and verification module, a sequence extraction module and a data verification specification module;
[0073] The data parsing and verification module reads the Excel content, removes duplicates and groups the template column, determines whether the mutation type of each sample conforms to the expected format, and checks whether the plasmid file is consistent with the specified one; if the mutation type conforms to the expected format, the sample passes the preliminary verification; if the mutation type does not conform to the expected format, the data detection and collection unit 6 records an error message;
[0074] Among them, the sequence extraction module parses the .gb and .dna files according to different plasmid names, determines the direction of the target gene and the resistance gene and whether it crosses the zero point, and extracts the sequence and position data of the gene in a clockwise direction with the zero point as the center; if the direction and position of the target gene and the resistance gene are clear in the plasmid file and do not cross the zero point, the corresponding sequence information can be directly extracted; if the direction of the target gene and the resistance gene is unclear and crosses the zero point, the starting point and end point of the sequence extraction need to be redefined;
[0075] Among them, the data verification and standardization module is used to verify whether the base and position before mutation in the Excel file are consistent with the base at the same position in the plasmid, and convert the amino acid mutation into a nucleotide mutation form; if the base and position before mutation recorded in the Excel file are consistent with the base at the corresponding position in the plasmid file, the normalization processing of the mutation content will continue; if the base and position before mutation recorded in the Excel file are inconsistent with the base at the corresponding position in the plasmid file, the data detection and collection unit 6 will record an error message.
[0076] It also includes a single point mutation primer design unit 2, which uses the Primer3 software package to generate PCR primers according to the input template sequence and the design parameters set by the user, and generates a mutation plasmid file containing the primers;
[0077] The single point mutation primer design unit 2 includes a design type determination module, a mutation content inspection module and a single point mutation primer design module;
[0078] Among them, the design type determination module is used to determine the number of mutations. If the multiple mutation identifier does not exist, it is determined to be a single mutation; if the multiple mutation identifier exists, it is determined to be a multiple mutation. If the mutation content format does not meet the requirements, the data detection and collection unit 6 records the error information; the design type determination module is also used to determine whether the mutation type is "amino acid". If the mutation type is "amino ac id", the mutation content is converted into a nucleotide mutation form and split into three new columns of data, including AAC, 1327, and ACC. If the mutation type is not "amino acid", the input nucleotide mutation form is directly used;
[0079] Among them, the mutation content inspection module is used to determine whether the base and position before mutation in Excel are consistent with the base at the same position in the plasmid. If the base and position before mutation recorded in the Excel file are consistent with the base at the corresponding position in the plasmid file, the verification is passed. If the base and position before mutation recorded in the Excel file are inconsistent with the base at the corresponding position in the plasmid file, the data detection and collection unit 6 records an error message.
[0080] The single-point mutation primer design module combines the Python open source software package Primer3 with automated data preprocessing technology to generate upstream and downstream primer template sequences containing the mutation site based on the template sequence and design parameters input by the user, and designs the upstream and downstream primers using the Primer3 software package. The mutant plasmid file containing the primers is generated using the Bio toolkit. The specific steps involved in generating the mutant plasmid file containing the primers by the single-point mutation primer design module are as follows:
[0081] S2.1. Calculate the extension lengths on both sides of the mutation site based on the design parameters and template sequence entered by the user, and generate upstream and downstream primer template sequences containing the mutation site. The template sequence consists of three parts: the vector primer template sequence, the sequence between the vector primer template sequence and the target gene, and the partial sequence of the target gene containing the mutation site.
[0082] S2.2. Generate an upstream primer by calling the Primer3 software package based on the upstream primer template sequence and using the input primer design parameters;
[0083] Examples of primer design parameters are as follows:
[0084] arg1={
[0085] "SEQUENCE_ID": sample name,
[0086] "SEQUENCE_TEMPLATE": template sequence,
[0087] "SEQUENCE_FORCE_LEFT_START":-1,
[0088] "SEQUENCE_FORCE_RIGHT_START": template length - 1,
[0089] "SEQUENCE_PRIMER_PAIR_OK_REG ION_LI ST":[[0,100,-1,-1]]
[0090] }
[0091] arg2={
[0092] "PRI MER_PI CK_ANYWAY":0,
[0093] "PRI MER_NUM_RETURN":1,
[0094] "PRI MER_OPT_SIZE": user-defined parameter,
[0095] "PRI MER_MIN_SIZE": user-defined parameter,
[0096] "PRI MER_MAX_SIZE": user-defined parameter,
[0097] "PRIMER_OPT_TM": user-defined parameters,
[0098] "PRI MER_MI N_TM": user-defined parameters,
[0099] "PRIMER_MAX_TM": user-defined parameter,
[0100] "PRI MER_MI N_GC": user-defined parameters,
[0101] "PRIMER_MAX_GC": User-defined parameter
[0102] }
[0103] S2.3. Based on the design parameters and template sequence entered by the user, locate the starting point of the upstream left primer on the vector primer template, add the length of the homology arm to obtain the new coordinates, extract the sequence from the starting point on the vector primer template to the new coordinates, and generate the downstream primer template;
[0104] S2.4. Generate downstream primers by calling the Primer3 software package using the input primer design parameters;
[0105] S2.5. Based on the primer design results and PCR product sequences, use the Bio toolkit to generate a mutant plasmid file containing primer information and mutation sites.
[0106] It also includes a double-point mutation primer design unit 3, which is used to process the primer design of double-point mutations, and is divided into three double-point mutation primer design situations according to the mutation spacing;
[0107] The double-point mutation primer design unit 3 includes a double-point mutation design type module, a first double-point mutation primer design module, a second double-point mutation primer design module and a third double-point mutation primer design module;
[0108] Among them, the double-point mutation design type module is used to read the mutation spacing data and determine the corresponding primer design scheme according to the size of the mutation spacing; if the mutation spacing is between 0 and 21 bp, enter the first double-point mutation primer design module; if the mutation spacing is between 21 and 120 bp, enter the second double-point mutation primer design module; if the mutation spacing is greater than 120 bp, enter the third double-point mutation primer design module;
[0109] The first double-point mutation primer design module is used to process the design of double-point mutation primers with a spacing of 0 to 20 bp, generating a primer template containing two mutation sites. The specific steps involved in designing double-point mutation primers using the first double-point mutation primer design module are as follows:
[0110] S3.1.1. Generate an upstream primer template based on the vector primer template sequence, the upstream vector primer template sequence, the sequence between the target gene and the mutation site and its left and right extension sequences;
[0111] S3.1.2. Calculate the extension lengths to the left and right of the mutation site to ensure the mutation site is in the middle of the primer.
[0112] S3.1.3. Use the Primer3 software package to design upstream and downstream primers;
[0113] The second double-point mutation primer design module is used to process the design of double-point mutation primers with a spacing between 21 bp and 120 bp, generating a primer template containing two mutation sites. The specific steps involved in designing double-point mutation primers using the second double-point mutation primer design module are as follows:
[0114] S3.2.1. Select appropriate sequences for design based on the relationship between homology arm length and mutation spacing;
[0115] S3.2.2. If the length of the homology arm is equal to the distance between two mutations, the sequence of the homology arm is the sequence between the two mutations;
[0116] S3.2.3. If the homology arm length is less than the distance between two mutations, select the sequence with the same homology arm length between the two mutations;
[0117] S3.2.4. If the length of the homology arm is greater than the distance between two mutations, a sequence of the deletion length is taken from the tail of the target gene;
[0118] S3.2.5. Use the Primer3 software package to design upstream and downstream primers.
[0119] The third double-point mutation primer design module is used to process the design of double-point mutation primers with a spacing greater than 120 bp, generating a primer template containing two mutation sites. The specific steps involved in designing double-point mutation primers using the third double-point mutation primer design module are as follows:
[0120] S3.3.1. Calculate the extension lengths and coordinates on both sides of the two mutation sites to generate upstream and downstream primer templates;
[0121] S3.3.2. Extract the target gene sequence containing the first mutation and the extended sequence to the right of the second mutation.
[0122] S3.3.3. Extract the sequence containing the second mutation and the sequence following it in the target gene;
[0123] S3.3.4. Call the Primer3 software package to design upstream and downstream primers.
[0124] In S3.1.2, the extension lengths on both sides of the mutation site are determined by calculation, and the mathematical formula involved is as follows:
[0125] The formula for calculating the right extension length is:
[0126]
[0127] The formula for calculating the left extension length is:
[0128]
[0129] Wherein, the distance is the distance between two mutation sites; max_primer_length is the maximum length of the primer; mutation_length is the length of each mutation site; d right is the right extension length; d left The left side extension length.
[0130] It also includes a sequencing verification primer design unit 4, which is used to generate primers required for sequencing according to the designed mutation and plasmid sequence;
[0131] The sequencing verification primer design unit 4 includes a template generation module, a sequencing primer design module and a result analysis module;
[0132] The sequencing verification primer design unit combines the Python open source software package Primer3 with automated data preprocessing technology to generate primer design templates for sequencing verification based on the mutation information and plasmid sequence entered by the user. The Primer3 software package is then called to design sequencing primers. The design results are analyzed to obtain the primer sequences required for sequencing and related information, including primer sequence, Tm value, GC content, length, primer pair score, position, and possible secondary structure. This ensures that mutation sites can be accurately verified in sequencing experiments, achieving efficient and accurate sequencing verification primer design.
[0133] The template generation module extracts the region containing the mutation site in the plasmid sequence according to the design parameters and mutant plasmid sequence input by the user, and generates upstream and downstream primer template sequences for sequencing verification;
[0134] The sequencing primer design module uses the Primer3 software package to generate upstream sequencing primers and downstream sequencing primers according to the generated upstream and downstream sequencing primer design templates;
[0135] The template generation module generates upstream sequencing primers and downstream sequencing primers based on the generated upstream and downstream sequencing primer design templates. The Python example is as follows:
[0136] import principal3
[0137] #Prepare upstream sequencing primer design template and design parameters
[0138]
[0139]
[0140] Among them, the result analysis module parses the sequencing primer design results generated by Primer3 to obtain the primer sequence and related information required for sequencing, including primer Tm value, primer GC content, primer length, primer pair score, primer position and primer secondary structure.
[0141] The system further comprises a recombinant plasmid map unit 5, which recombines the mutated new gene and primers into a new plasmid file through a Bio toolkit based on the primer design results and the PCR product sequence;
[0142] The recombinant plasmid map unit 5 includes a primer design result acquisition module, a PCR product sequence generation module, a plasmid recombination module and a plasmid map generation module;
[0143] The recombinant plasmid map unit combines primer design results and PCR product sequences, using the Bio toolkit to reconstruct the mutated new gene and primer sequences into a new plasmid file, generating a recombinant plasmid map. This unit first obtains the primer design results, including upstream and downstream primer sequences and related information. Then, based on the primer sequences, it generates a PCR product sequence containing the mutation site. The recombinant plasmid is generated through homologous recombination technology, and a plasmid map is generated using a plasmid drawing tool to intuitively display the mutation site and primer position, achieving efficient and accurate recombinant plasmid generation and visualization.
[0144] The primer design result acquisition module is used to read the primer design result file, obtain the sequence, Tm value, GC content, and primer position of the upstream and downstream primers, and use them for subsequent PCR product sequence generation and plasmid recombination processes;
[0145] The PCR product sequence generation module generates a PCR product sequence containing a mutation site by PCR amplification using upstream and downstream primer sequences;
[0146] The plasmid recombination module uses the PCR product sequence and the original plasmid sequence to generate a recombinant plasmid using the Bio toolkit through homologous recombination technology;
[0147] The plasmid map generation module generates a plasmid map using a plasmid drawing tool, and marks mutation sites, primer positions and other gene regions on the plasmid map.
[0148] The system also includes a data detection and collection unit 6, which is used to detect data generated during primer design and plasmid transformation, collect successful and failed design results, save error information, and generate a detailed detection report;
[0149] The data detection and collection unit 6 includes a primer design result detection module, a plasmid transformation process detection module, an error information collection module and a detection report generation module;
[0150] The primer design result detection module is used to monitor the primer design process, record the results of each design in real time, distinguish between successful and failed designs, collect successful primer sequences, Tm values, and GC content data, and record failed design parameters and reasons;
[0151] The plasmid transformation process detection module is used to monitor the plasmid recombination process, record the results of each recombination, collect the plasmid sequences and related information of successful recombination, and record the parameters and reasons for failed recombination;
[0152] The error information collection module collects error information from failure cases in the primer design and plasmid transformation process, including error causes, relevant parameters, and suggested modification plans;
[0153] The test report generation module is used to generate a detailed report containing success and failure cases based on the test results, including successful design results, failed cases and error information.
[0154] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely preferred examples of the present invention and are not intended to limit the present invention. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and improvements fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.
Claims
1. An automated online tool for designing primers for plasmid point mutation modification, characterized by: include: A data preprocessing unit (1) reads and parses the Excel file uploaded by the user, removes duplicates and groups to obtain plasmid file names, determines whether the mutation type of each sample and the plasmid file meet the requirements, and parses the plasmid file to determine the sequence and position of the target gene and the resistance gene; A single-point mutation primer design unit (2), wherein the single-point mutation primer design unit (2) uses the Primer3 software package to generate PCR primers according to the input template sequence and the design parameters set by the user, and generates a mutation plasmid file containing the primers; A double-point mutation primer design unit (3), wherein the double-point mutation primer design unit (3) is used to process the primer design of double-point mutations, and is divided into three double-point mutation primer design situations according to the mutation spacing; A sequencing verification primer design unit (4), wherein the sequencing verification primer design unit (4) is used to generate primers required for sequencing according to the designed mutation and plasmid sequence; A recombinant plasmid map unit (5), wherein the recombinant plasmid map unit (5) recombines the mutated new gene and primers into a new plasmid file based on the primer design result and the PCR product sequence through a Bio toolkit; A data detection and collection unit (6), the data detection and collection unit (6) is used to detect data generated during the primer design and plasmid transformation process, collect successful and failed design results, save error information, and generate a detailed detection report; The data preprocessing unit (1) includes a data analysis and verification module, a sequence extraction module and a data verification specification module; The data parsing and verification module reads the Excel content, removes duplicates and groups the template column, determines whether the mutation type of each sample conforms to the expected format, and checks whether the plasmid file is consistent with the specified one; if the mutation type conforms to the expected format, the sample passes the preliminary verification; if the mutation type does not conform to the expected format, the data detection and collection unit (6) records the error information; Among them, the sequence extraction module parses the .gb and .dna files according to different plasmid names, determines the direction of the target gene and the resistance gene and whether it crosses the zero point, and extracts the sequence and position data of the gene in a clockwise direction with the zero point as the center; if the direction and position of the target gene and the resistance gene are clear in the plasmid file and do not cross the zero point, the corresponding sequence information can be directly extracted; if the direction of the target gene and the resistance gene is unclear and crosses the zero point, the starting point and end point of the sequence extraction need to be redefined; The data verification and standardization module is used to verify whether the base and position before the mutation in the Excel file are consistent with the base at the same position in the plasmid, and convert the amino acid mutation into a nucleotide mutation form; if the base and position before the mutation recorded in the Excel file are consistent with the base at the corresponding position in the plasmid file, the normalization process of the mutation content is continued; if the base and position before the mutation recorded in the Excel file are inconsistent with the base at the corresponding position in the plasmid file, the data detection and collection unit (6) records an error message; The double-point mutation primer design unit (3) includes a double-point mutation design type module, a first double-point mutation primer design module, a second double-point mutation primer design module and a third double-point mutation primer design module; Among them, the double-point mutation design type module is used to read the mutation spacing data and determine the corresponding primer design scheme according to the size of the mutation spacing; if the mutation spacing is between 0 and 21bp, enter the first double-point mutation primer design module; if the mutation spacing is between 21 and 120bp, enter the second double-point mutation primer design module; if the mutation spacing is greater than 120bp, enter the third double-point mutation primer design module.
2. The automated online tool for designing primers for plasmid point mutation modification according to claim 1, characterized in that: The single point mutation primer design unit (2) includes a design type determination module, a mutation content inspection module and a single point mutation primer design module; The design type determination module is used to determine the number of mutations. If the multiple mutation identifier does not exist, it is determined to be a single mutation; if the multiple mutation identifier exists, it is determined to be a multiple mutation; if the mutation content format does not meet the requirements, the data detection and collection unit (6) records the error information; the design type determination module is used to determine whether the mutation type is "aminoacid". If the mutation type is "amino acid", the mutation content is converted into a nucleotide mutation form and split into three new columns of data, including AAC, 1327, and ACC. If the mutation type is not "amino acid", the input nucleotide mutation form is directly used; The mutation content checking module is used to determine whether the base and position before the mutation in the Excel file are consistent with the base at the same position in the plasmid. If the base and position before the mutation recorded in the Excel file are consistent with the base at the corresponding position in the plasmid file, the verification is passed. If the base and position before the mutation recorded in the Excel file are inconsistent with the base at the corresponding position in the plasmid file, the data detection and collection unit (6) records an error message.
3. The automated online tool for designing primers for plasmid point mutation modification according to claim 2, characterized in that: The single-point mutation primer design module combines the Python open source software package Primer3 with automated data preprocessing technology to generate upstream and downstream primer template sequences containing the mutation site based on the template sequence and design parameters input by the user, and designs the upstream and downstream primers using the Primer3 software package. The mutant plasmid file containing the primers is generated using the Bio toolkit. The specific steps involved in the single-point mutation primer design module to generate the mutant plasmid file containing the primers are as follows: S2.
1. Calculate the extension lengths on both sides of the mutation site based on the design parameters and template sequence entered by the user, and generate upstream and downstream primer template sequences containing the mutation site. The template sequence consists of three parts: the vector primer template sequence, the sequence between the vector primer template sequence and the target gene, and the partial sequence of the target gene containing the mutation site. S2.
2. Generate an upstream primer by calling the Primer3 software package based on the upstream primer template sequence and using the input primer design parameters; S2.
3. Based on the design parameters and template sequence entered by the user, locate the starting point of the upstream left primer on the vector primer template, add the length of the homology arm to obtain the new coordinates, extract the sequence from the starting point on the vector primer template to the new coordinates, and generate the downstream primer template; S2.
4. Generate downstream primers by calling the Primer3 software package using the input primer design parameters; S2.
5. Based on the primer design results and PCR product sequences, use the Bio toolkit to generate a mutant plasmid file containing primer information and mutation sites.
4. The automated online tool for designing primers for plasmid point mutation modification according to claim 3, characterized in that: The first double-point mutation primer design module is used to process the design of double-point mutation primers with a spacing of 0 to 20 bp, generating a primer template containing two mutation sites. The specific steps involved in designing double-point mutation primers using the first double-point mutation primer design module are as follows: S3.1.
1. Generate an upstream primer template based on the vector primer template sequence, the upstream vector primer template sequence, the sequence between the target gene and the mutation site and its left and right extension sequences; S3.1.
2. Calculate the extension lengths to the left and right of the mutation site to ensure the mutation site is in the middle of the primer. S3.1.
3. Use the Primer3 software package to design upstream and downstream primers; The second double-point mutation primer design module is used to process the design of double-point mutation primers with a spacing between 21 bp and 120 bp, generating a primer template containing two mutation sites. The specific steps involved in designing double-point mutation primers using the second double-point mutation primer design module are as follows: S3.2.
1. Select appropriate sequences for design based on the relationship between homology arm length and mutation spacing; S3.2.
2. If the length of the homology arm is equal to the distance between two mutations, the sequence of the homology arm is the sequence between the two mutations; S3.2.
3. If the homology arm length is less than the distance between two mutations, select the sequence with the same homology arm length between the two mutations; S3.2.
4. If the length of the homology arm is greater than the distance between two mutations, a sequence of the deletion length is taken from the tail of the target gene; S3.2.
5. Design upstream and downstream primers using the Primer3 software package. The third double-point mutation primer design module is used to process the design of double-point mutation primers with a spacing greater than 120 bp, generating a primer template containing two mutation sites. The specific steps involved in designing double-point mutation primers using the third double-point mutation primer design module are as follows: S3.3.
1. Calculate the extension lengths and coordinates on both sides of the two mutation sites and generate upstream and downstream primer templates; S3.3.
2. Extract the target gene sequence containing the first mutation and the extended sequence to the right of the second mutation. S3.3.
3. Extract the sequence containing the second mutation and the sequence following it in the target gene; S3.3.
4. Call the Primer3 software package to design upstream and downstream primers.
5. The automated online tool for designing primers for plasmid point mutation modification according to claim 4, characterized in that: In S3.1.2, the extension lengths on both sides of the mutation site are determined by calculation, and the mathematical formula involved is as follows: The formula for calculating the right extension length is: The formula for calculating the left extension length is: Wherein, the distance is the distance between two mutation sites; max_primer_length is the maximum length of the primer; mutation_length is the length of each mutation site; d right is the right extension length; d left The left side extension length.
6. The automated online tool for designing primers for plasmid point mutation modification according to claim 1, characterized in that: The sequencing verification primer design unit (4) includes a template generation module, a sequencing primer design module and a result analysis module; The template generation module extracts the region containing the mutation site in the plasmid sequence according to the design parameters and mutant plasmid sequence input by the user, and generates upstream and downstream primer template sequences for sequencing verification; The sequencing primer design module uses the Primer3 software package to generate upstream sequencing primers and downstream sequencing primers according to the generated upstream and downstream sequencing primer design templates; Among them, the result analysis module parses the sequencing primer design results generated by Primer3 to obtain the primer sequence and related information required for sequencing, including primer Tm value, primer GC content, primer length, primer pair score, primer position and primer secondary structure.
7. The automated online tool for designing primers for plasmid point mutation modification according to claim 1, characterized in that: The recombinant plasmid map unit (5) includes a primer design result acquisition module, a PCR product sequence generation module, a plasmid recombination module and a plasmid map generation module; The primer design result acquisition module is used to read the primer design result file, obtain the sequence, Tm value, GC content, and primer position of the upstream and downstream primers, and use them for subsequent PCR product sequence generation and plasmid recombination processes; The PCR product sequence generation module generates a PCR product sequence containing a mutation site by PCR amplification using upstream and downstream primer sequences; The plasmid recombination module uses the PCR product sequence and the original plasmid sequence to generate a recombinant plasmid using the Bio toolkit through homologous recombination technology; The plasmid map generation module generates a plasmid map using a plasmid drawing tool, and marks mutation sites, primer positions and other gene regions on the plasmid map.
8. The automated online tool for designing primers for plasmid point mutation modification according to claim 1, characterized in that: The data detection and collection unit (6) includes a primer design result detection module, a plasmid transformation process detection module, an error information collection module and a detection report generation module; The primer design result detection module is used to monitor the primer design process, record the results of each design in real time, distinguish between successful and failed designs, collect successful primer sequences, Tm values, and GC content data, and record failed design parameters and reasons; The plasmid transformation process detection module is used to monitor the plasmid recombination process, record the results of each recombination, collect the plasmid sequences and related information of successful recombination, and record the parameters and reasons for failed recombination; The error information collection module collects error information from failure cases in the primer design and plasmid transformation process, including error causes, relevant parameters, and suggested modification plans; The test report generation module is used to generate a detailed report containing success and failure cases based on the test results, including successful design results, failed cases and error information.