KR20030040908A - A Method and a Computer Program To Simulate Laboratory Gene Cloning Procedure Under Virtual Conditions for Generating Gene Clone Database. - Google Patents

Abstract

PURPOSE: A computer program for simulating a gene cloning process with the same as an actual experimental process and storing a result in a database, and a method for the same are provided to enable all persons specialized in molecular biology to easily and conveniently use, store, and manage the precious information. CONSTITUTION: A primer designer module designs and makes a primer in order to perform the DNA(Deoxyribo Nucleic Acid) PCR(Polymerize Chain Reaction). A PCR module makes the PCR product by checking an annealing state between the primer and a sequence. A DNA cutter module makes the DNA product generated when the sequence is cut by a restriction enzyme. A ligation module makes one sequence through the addition of two sequences by calculating overhang of the two sequences. A cut and ligation module realizes all cloning processes at one time by inputting a vector sequence object, an insert sequence object, and the restriction enzyme necessary for cloning. A sequence editor module edits the sequence in order to fit to a sequence object expression equation used by a cloning editor module. A sequence database stores the sequence object making all data generated by each process and inputted products as the object.

Description

Computer program and method for simulating the gene cloning process in the same way as the actual experiment process and storing the result in database form. {A Method and a Computer Program To Simulate Laboratory Gene Cloning Procedure Under Virtual Conditions for Generating Gene Clone Database.}

The present invention is one of the programs for genetic analysis, which simulates the genetic analysis and cloning work performed by the experimenter in the field of molecular biology using a computer, and stores the result in a database form through a similar process to the actual experiment. It is a program that allows you to do this.

In existing programs, the sequence of procedures required for cloning is arranged from the programmer's point of view, so that the actual user can read through hundreds of pages of manuals to perform the task. Genes that are manipulated and expressed also have limitations that cannot accurately express the actual form.

The present invention was invented by the following necessity which is felt desperately in the field when performing a cloning operation performed in a laboratory and a laboratory.

Firstly, users can easily and quickly use the computer to maximize the connection with their work.

Secondly, by recording and storing digital data of the experimenter's own clones and all of them, it is possible to accurately identify and manage the status of the numerous gene clones they possess.

Third, it was carried out by the necessity of facilitating research activities by enabling retrieval of recombinant gene retention status between experimenters (largerly between laboratories and institutes) and improving mutual exchange such as gene exchange.

In the case of university laboratories and corporate research institutes in Korea, researchers are often replaced by cycles within an average of five years. In the process, the predecessor's work is usually arranged in the form of a report, a takeover, or a dissertation and passed on to the successor, but the cloning work is not a record of the entire sequence of plasmids containing the specific genome. As a result, successors may not be able to reuse the cloning results of their predecessors and perform new cloning tasks or do completely different tasks, leading to the loss of national resources as well as the laboratory or laboratory.

Therefore, there is a need to secure a sequence of cloning results as a database and to obtain the sequence information. In addition, there is a growing demand for a program that can be easily used by experimenters who are not familiar with computing tasks.

In order to meet the needs of the experimental field as described above, the present invention allows researchers to simulate the gene cloning process in the same way as the actual experimental process and store the result in a database form, which is valuable for all who major in molecular biology. It was invented to enable easy and convenient use of information and to accurately store, store and manage information.

Accordingly, the present invention attempts to develop a gene expression method that corresponds to the modular concept of the experimental techniques necessary for the experimenter to perform the actual cloning operation, and expresses the characteristics of the gene to be manipulated. In other words, experimenters who are familiar with cloning can use modules in the program in the same process as manipulating genes and gene carriers (vectors). The entire sequence of this plasmid was calculated and recorded as if the result were a new plasmid.

1 is a view showing a cloning process consisting of a PCAL and a line according to the present invention.

2 is a view showing a cloning process consisting of a die cutter and ligation according to the present invention.

FIG. 3 is a diagram illustrating a primer designer during the process of FIG. 1.

4 is a diagram illustrating a sequence editor among cloning editors.

5 is a diagram for an overhang representation of a sequence.

6 is a diagram of a sequence file representation of ENDS, which is a sequence overhang representation.

7 is a view of the action of the SPM during the ligation process.

8 is a diagram for a sequence file representation of an SPM.

9 is a diagram for a method of determining whether or not ligation is possible when ligation is performed.

To achieve this goal, the Cloning Editor breaks down the actual cloning process into a series of steps and then modularizes it into seven modules:

First, Primer Designer, which designs and creates primers for DNA Polymerase Chain Reaction (PCR).

Second, the PAL that produces the PCR product by examining the alignment between the primer and the sequence.

Third, a DNA cutter that produces a DNA product produced when the sequence is cut with a restriction enzyme.

Fourth, ligation that calculates overhang of two sequences and combines them into one to create one sequence

Fifth, cut & ligation that enables all cloning process at once by inputting vector sequence object, insert sequence object and restriction enzyme required for cloning.

Sixth, a sequence editor that can edit a sequence to match the sequence object expression used in the cloning editor.

Seventh, a sequence object database that can store all the data created in each process and sequence objects that object the input products.

Consists of

This invention may be better understood by the following detailed description taken in conjunction with the accompanying drawings.

Referring to FIGS. 1, 2, 3, and 4, each drawing shows the cloning process in a pictorial manner, in which the corners with rounded rectangles are the processes of cloning, and the portions indicated with the angled squares are each process. Sequence object to be used as data. This process is the same as that performed in the laboratory.

1 illustrates a method of constructing a PCAL and a ligation in the cloning editor. The vector sequence object and the PC result sequence object form a new plasmid through ligation. If only one sequence is input when ligating, the license is executed with only one input sequence. This method is called self ligation.

Referring to FIG. 2, a method of constructing a die cutter and a ligation in the cloning editor is described. When a sequence to be ligated is not prepared, a process of creating a vector sequence (hereinafter referred to as vector) and an insert sequence (hereinafter referred to as insert) is described. It is explained. This step can be omitted if the vectors and inserts are ready in advance. The vector cuts the prepared sequence vector and insert with the appropriate restriction enzyme in the DNA cutter to make the overhang that can be ligated to the vector insert and then perform the ligation. Also, the process of FIG. 2 may be performed at once with cut and ligation.

Referring to FIG. 3, a process of making an insert in FIG. 2 is described in detail, and may be omitted if an insert is already prepared. A primer is needed to amplify a specific part of the sequence in the insert using the PCAL. Create a primer that is available in the primer designer. The primers and the template sequence object are then subjected to the PSA process to generate a cut insert ready for the DNA cutter.

Referring to FIG. 4, a sequence editor capable of editing a sequence object required in a cloning process has been described. The sequence editor is a tool for calling, modifying, editing, and storing a sequence object as a sequence object.

Integrating all of these steps, cloning requires vectors and inserts, which require a PCR process to amplify a specific range of sequences in the insert, and using primer designers to create the primers needed for this process. To produce This process can be omitted if you have a primer already made or do not need it. When the PAL comes out of a blunt sequence, it cuts through the die cutter to create a cut insert for ligation. When the vector has an overhang or a blunt cut vector from the DNA cutter, a ligation process creates a new plasmid.

In order to implement this process, the program of each process objectizes the sequence, which is the data coming and going between the programs, in order to maintain its independence from the program of the previous process. Here, the representation of the physically present molecules in the laboratory is called a sequence object.

In addition, in the present invention, the sequence object representation format is a representation of a format that can be input and output from each module of the cloning editor, and the example can be seen in FIGS. 6 and 8. The sequence object representation can express the exact form of the gene used in the cloning editor by expressing an overhang of a sequence that cannot be represented by another sequence representation format.

All programs in each process are configured to load and save sequence objects represented in this sequence object representation format. However, the form of sequence representation (Genbank, EMBL, FASTA ...) used by other software is not enough to represent the exact state of the molecule in the laboratory, so we devised a new sequence object representation format that includes the existing sequence object representation format. We added ENDS (sequence object overhang, blunt display) and SPM (sequence object reference position display). 6 and 8, ENDS and SPM are added to the newly designed object expression format.

In addition, the sequence object types handled in each process have two types, linear or circular. However, the prototype has only the same representation as that of the sequence object, although the representation of the sequence object is linear. The problem with computing these circular sequence objects is that the last part of the sequence represented by the circular sequence object is connected to the beginning of the sequence, so some additional steps are required, unlike when dealing with linear sequence objects. In the present invention, when the input sequence object of each component program is circular, if the sequence of the maximum operation unit of the program is removed from the beginning of the sequence object and added to the end of the sequence, the sequence object is processed in the same manner as the linear sequence object. can do. The result of having the position larger than the length of the sequence object in the result calculated in the same way as this linear sequence object is the mapping of the position of the circular sequence object to the remaining position divided by the length of the circular sequence object. Is converted to the result.

The maximum unit of operation of each program is shown in Table 1.

Restriction Enzyme Program Longest recognition sequence length among the restriction sequences applied PCR Length of the longest primer among the primers entered Primer designer Sequence length of maximum length configurable with given Tm value or length of given primer

Also, in order to display the exact state of the sequence object in the die cutter and ligation, the overhang state of the sequence object must be represented. Referring to FIG. 5, in order to express an overhang, an expression of an overhang is represented by a difference between 5 ′ and 3 ′ of a sequence object. If the position of 5 'is outward than 3' at each position, it becomes the state of + and the position of 3 'is out of the position of 5' at each position. Becomes-. In addition, the number is represented by the position difference between 5 'and 3'. If the sequence object's 5 'and 3' overhang states are known, concatenate them with two comma delimiters (..) to form "(overhang at the beginning of the sequence object) .. (overhang at the end of the sequence object)". Express as This is called ENDS.

In this case, when the sequence is cut with the restriction enzyme BamHI, the truncated sequence has an overhang state of +4 .. + 4, and the field representing the overhang as shown in (1) of FIG. ) To represent a sequence object. In this case, if the blunt is specified, the ENDS is expressed as 0..0 to express the blunt.

In addition, primer designers and DNA cutters receive input restriction enzymes. HindIII, Hind3, hind3, hindiii… Likewise, the same restriction enzyme name is mixed with roman numerals, arabic numerals, and upper and lower case letters, which makes it necessary to distinguish between uppercase and lowercase letters.

In order to solve this problem, the name of the restriction enzyme is expressed in Roman numerals, and I, II, III, IV, V, I, ii, iii, iv, and v are used for the restriction enzyme. It is recognized as the same meaning as, 3,4,5, and it is not case sensitive. This means that if the restriction enzyme number is based on uppercase Roman letters, and if the restriction enzyme contains a number, change the part to the corresponding Roman numeral, and the restriction enzyme name will be separated regardless of Roman and Arabic numerals. It is possible. For example, if the input hind3 comes in, there is a number '3' in the input, so the restriction enzyme is selected by searching the restriction table for hindIII, which has been converted to Roman numerals, and the existing input hind3.

In addition to the normal sequence character ATGC, the restriction sequence of restriction enzymes contains a complex character commonly called an Ambiguity IUPAC code. The mixed IUPAC codes are shown in Table 2. In order to recognize such mixed IUPAC codes, the present invention borrows the concept of aggregation. Taking mixed IUPAC code R as an example, R means A, G. Therefore, if you expand R ==> {A, G, R} and search on the target sequence, you can see whether it is included in the R representation. Similarly, Table 3 can be obtained by applying the remaining mixed IUPACs.

Now, if the recognition sequence of the restriction enzyme is extended according to Table 3, and the sequence corresponding to the expanded sequence set is searched in the input sequence object, the recognition sequence of the restriction enzyme composed of the corresponding mixed IUPAC code can be also retrieved. In the present invention, the concept of a set is processed in a program using a regular expression search method for recognition of mixed IUPAC codes.

Expansion of the recognition sequence For example, the restriction enzyme 'Bsp1286I' has a recognition sequence of "GDGCHC". When expanded according to Table 3, it becomes "{G} {A, G, T, D} {G} {C} {A, C, T, H} {C}". Expressing this extended sequence as a regular expression results in "[G] [AGTD] [G] [C] [ACTH] [C]", which performs a regular expression search on the target sequence object.

Primer Designer requires primers to do the PCR. The primer is a program that automatically creates primers. Basically, the primer length for TPM value or sequence object and primer is annealed for the user specified region. Can be created by There are several ways to automatically determine the area for this PCAL.

Methods for automatically determining such areas include searching CDS in a feature table of a sequence object, searching a coding sequence of a sequence object, including a longest ORF, longest ORF include with out start or stop codon, and full length. The method of searching the coding sequence of a sequence object is to find OARF in a program in a given sequence and set its range. Longest ORF is a start codon and end codon in a sequence object. The longest ORF include without start or stop codon is similar to the Longest ORF method, but if there is no start codon or end codon, the assumption is that there is a codon at the end of the sequence object, at the beginning of the sequence. This part is also a way to recognize OAL.

In addition, it can be designed by adding a linker sequence desired by the user at the 5 'position of the designed primer. When the recognition sequence of the desired restriction enzyme is added to the added linker sequence, the DNA product is cut into the DNA cutter using the restriction enzyme, and the sequence object can be used for ligation.

To check if a primer designed in the primer designer is a primer suitable for writing to a PC, there is a set of predetermined restriction enzymes in the internal sequence in which the created primers are annealed and a recognition sequence that is cut by a specific restriction enzyme specified by the user. Paper and designed primers can be annealed to internal sequences to determine if they are not suitable for primers. In this case, the condition to be annealed inside is checked by giving a value lower than the Tm value (default 54) when designing the primer.

These primers can be designed with primer designers or pre-made primers to get the product. The PCR program checks the binding between the primer and the template sequence from the 3 'position of the primer sequence object, and recognizes that the primer is bound to the template sequence if the Tm value of the primer sequence completely corresponding to the template sequence is greater than the Tm value specified by the user. . The sequence object resulting from the PCAL in the PC program produces a blunt form of the object without an overhang. It is also possible to add the sequence 'A' at position 3 'of the PD product made for T-vector cloning.

In the DNA cutter, the sequence object, such as an insert sequence or a vector sequence from a PSA product, is received, the sequence object is cut with a given restriction enzyme, and an overhang according to the restriction enzyme is added to the later work. At this time, if the sequence object input to the die cutter is circular, SPM (Standard Position Marker) is added to the sequence object start position (position 1).

Referring to FIG. 7, when a vector and an insert are prepared and a new plasmid is prepared by ligation, the ligation determines whether the ligation is possible by judging an overhang as shown in FIG. 9 even if the ENDS items of the vector and the insert are prepared. Check and perform the ligation. If there is no ENDS item, the sequence object is recognized as a blunt and blunt ligation is performed. If the ligated sequence object is circular and the SPM exists, move the sequence object relative to the starting position of the SPM and remove the SPM to correct the position of the vector. This corrects the position of the sequence object containing the SPM.

At this time, if only one input sequence object of the ligation is input, not two, the ligation is attempted with one of the input sequence objects. This is called self ligation.

As described above, the present invention moves the cloning process from the laboratory to the computer as it is, which is easily used by biologists, is easy to use, and separates the data from the work process by objectizing each moving data (sequence object). Can represent the change of the sequence appearing in the actual experiment as it is.

As a result of the present invention, the experimenter can operate the program consisting of each module to obtain the result as a complete sequence on the computer as if the gene is actually cloned into the vector. In order to use the present invention, it is enough to try a simple manual showing the actual process, and since all of the process shows the cloning operation as it is, it has the advantages of being familiar and comfortable to use.

In addition, since the process can be written naturally, the complete sequence object can be recorded in the output, reducing the cost of reproducing the genes and their carriers (vectors) that accumulate in each laboratory or lab. It has opened the way for sharing and reuse among people. In other words, by minimizing human material waste and maximizing research efficiency, it is possible to open a way to reconsider national competitiveness.

Claims (26)

A method of applying a laboratory's gene cloning process in the same way as a laboratory in software. The method according to claim 1, wherein the genetic process of the software is composed of PCL and ligation as shown in FIG. The method of claim 1, wherein the gene cloning process of the cloning software comprises a DNA cutter and a ligation as shown in FIG. 2. The method according to claim 3, wherein the clone software comprises a cut and ligation that performs the ligation with the die cutter during the gene cloning process. The method of claim 1, wherein the process of automatically designing the primers required for the PCR during the gene cloning process of the cloning software comprises a primer designer as shown in FIG. 3. In each of the constituent modules constituting the gene cloning, the sequence of genes (die, RNA, plasmid, primers, restriction enzyme) that are handled in the laboratory, the input data of each process, is recognized as one object, and each sequence is cloned. How to treat independently, regardless of the process. The method of claim 6, wherein the overhang and blunt information of the sequence object is added to the sequence object. The method of claim 7, wherein the sequence object including the overhang and blunt information is represented by the sequence object according to the sequence object representation format. The method of claim 6, wherein a Standard Position Marker (SPM) is added to the sequence object to mark the beginning of the sequence object. The method of claim 9, wherein the sequence object including the sequence object positional reference indication is represented according to a sequence object representation format. Primer designer using the CDS region search, Longest ORF region, and sequence-wide region search of the feature table of the sequence object as a method of automatically determining the region for generating the primer for the PC. The method of claim 11, wherein the primer is designed by adding a linker sequence to the 5 ′ position of the primer produced by the primer designer. The function of claim 11, wherein a restriction enzyme that recognizes a primer made by a primer designer can determine the template sequence and indicate whether the primer sequence is suitable. 12. The function of claim 11, wherein each primer designed to bind at both ends of the template sequence is bound to at least two positions in the template sequence to indicate that undesired PD products may occur when the PC is to be seeded. The PCR program checks the annealing state between the template sequence and the primer. If the T's Tm value of the sequence is higher than the value specified by the user from 3 'to 3, To determine the state of engagement of the. The method of claim 15, wherein the PCL program blunts the PA product sequence object. 16. The method of claim 15, wherein the PCL program adds a 3 '"A" overhang at both ends of the PCL product sequence object. DNA cutter program for generating an ENDS that displays overhangs and blunts in a result sequence. 19. The DNA cutter of claim 18, wherein the result of executing the program is a sequence object. Ligation program characterized in that the recognition of the overhang, blunt marked ENDS in the sequence object automatically determines whether or not. 21. The method of claim 20, wherein if the shape of the sequence object is circular and the sequence object position indication is the result of the ligation program, the method maintains the shape of the sequence object by switching the sequence object based on the sequence object position indication. 21. The method of claim 20, wherein when only one linear sequence object is input to the ligation, a self ligation is performed by ligating both ends of the input sequence object to a circle. Gene Cloning Software A method of calculating when the input sequence of each process is circular, copying a sequence of at least the maximum operation unit of each operation from the beginning of the sequence object and adding it to the end of the sequence, and then copying the sequence object in the same manner as the linear method. After processing, the result is divided by the length of the circular sequence and the remainder is mapped to the circular sequence. Gene Cloning Software A program that accepts restriction enzyme names during each step, automatically distinguishes the restriction enzyme name and displays the Roman numerals (I, II, III, IV, V, ... i, ii, iii, iv). , v ..., 1, 2, 3, 4, 5 ....) to facilitate user restriction enzyme input. Gene Cloning Software A method of treating mixed IUPAC codes as a set of inclusions during each process. 27. The method of claim 25, wherein the processing of mixed IUPAC code is handled by a regular expression representation in a software implementation via a set concept.