KR100512915B1 - A Method and a Computer Program To Simulate Laboratory Gene Cloning Procedure Under Virtual Conditions for Generating Gene Clone Database. - Google Patents

Abstract

The present invention provides a program and an implementation method for supporting gene cloning in a molecular biology laboratory, through which the entire sequence of newly created genes and gene carriers (vectors) can be stored as accurate records on a computer. It is about. This process is characterized by maximizing the user's convenience by going through the same process as the work performed experimentally and by organizing a series of tasks so that the user can leave the result of the work as an accurate record on the computer. would.

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 relates to a method for genetic analysis, a method of enabling a computer to simulate genetic analysis and cloning performed by experimenters in the field of molecular biology, and to store the result in a database form through a process similar to the actual experiment. In existing programs, the sequence of steps required for cloning was arranged from the programmer's point of view, so real users would have to read through hundreds of pages of manuals to perform the task. Genes that manipulate and express genes also have limitations that cannot accurately express the actual form.

delete

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 need to facilitate the research activities by enabling the retrieval of the status of recombinant gene retention between experimenters (largerly between laboratories and institutes) and by improving mutual exchange such as gene exchange.

In the case of university laboratories and corporate research institutes in Korea, researchers are replaced on average within five years. In this process, the predecessor's work is organized in the form of a report, a takeover, or a dissertation and passed on to the successor, but the reality is that the cloning product does not remain a record of the entire sequence of the plasmid containing the specific genome. As a result, successors can't reuse the cloning results of their predecessors and perform new gene cloning, or do something completely different, leading to the loss of national resources as well as the laboratory or research center.

Therefore, there is a need to secure a sequence of cloning results as a database and to obtain sequence information. In addition, there is an increasing 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 inventors have attempted to develop a gene expression method that corresponds to a modular concept of programming, and expresses the characteristics of a gene to be manipulated, as experimenters need to perform an actual cloning operation. 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.

The Cloning Editor achieves this goal by breaking down the actual gene cloning process into a series of steps and then modularizing it into seven modules: In other words,

First, Primer Designer for designing and manufacturing primers to perform DNA polymerase chain reaction (PCR, Polymerize Chain Reaction, "PCR");

Secondly, PCR to examine PCR for annealing between the primer and the sequence to produce a PCR product;

Third, a DNA cutter (DNA Cutter) for producing a DNA product produced when the sequence is cut with a restriction enzyme;

Fourth, a ligation unit which calculates an overhang of two sequences and combines the two sequences into one to produce one sequence;

Fifth, cut and ligation (Cut & Ligation) to input all the cloning process at once by inputting the vector sequence object, insert sequence object, restriction enzyme required for cloning;

Sixth, a sequence editor for editing a sequence according to a sequence object expression used in the cloning editor;

Seventh, a sequence object database (Sequence Database) that can store all the data generated in each process and the sequence object objectized 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 of. This process is the same as that performed in the laboratory.

1 illustrates a method of constructing a PCR and a ligation unit in a cloning editor. A new plasmid is prepared by ligating a vector sequence object and a sequence object that is a PCR result. 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 DNA cutter and a ligation unit in a cloning editor is described. When a sequence to be ligated is not prepared, a vector sequence (hereinafter referred to as vector) and an insert sequence (hereinafter referred to as insert) are created. The process 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 a suitable restriction enzyme in the DNA cutter to make an overhang capable of ligating the vector and the insert, and then performs the ligation. In addition, the process of FIG. 2 may be performed at once by cut and ligation.

Referring to FIG. 3, the process of creating the insert in FIG. 2 is described in detail, and may be omitted if the insert is already prepared. Primers are required to amplify specific parts of the sequence in the insert using PCR. Create primers that can be PCR in the primer designer. The primers and template sequence objects are then subjected to PCR to generate ready-to-cut inserts 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 a vector and an insert, a PCR process is needed to amplify a specific range of sequences in the insert, and primers are created using the primer designer to create the primers needed for this PCR. Serve This process can be omitted if you have a primer already made or do not need it. When a blunt sequence is generated by PCR, a truncated insert for ligation is generated via a DNA cutter. When a vector has an overhang or a blunt cut vector from the DNA cutter, a ligation process produces 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 such 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 has known overhang states of 5 'and 3', combine them with two commas (..) 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.

Restriction enzymes are also input from primer designers and DNA cutters. 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 recognition of the mixed IUPAC code is processed using a regular expression search method in a program having a set concept.

Extension of the recognition sequence is, for example, restriction enzyme 'Bsp1286I' has a recognition sequence of "GDGCHC", which is expanded according to Table 3, "{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 is a program that automatically creates primers needed for PCR. Basically, primers for PCR of a user-designated region can be created by giving a Tm value or a length in which sequence objects and primers are annealed. Several methods are provided for automatically determining regions for PCR.

Methods for automatically determining the PCR region include CDS search in the feature table of the sequence object, coding sequence search of the sequence object, longest ORF, longest ORF include without start or stop codon, and full length. There is a way. The method of searching the coding sequence of a sequence object is to find the ORF in a given sequence in a program and set its range. The longest ORF uses the start codon and end codon in the 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 the codon is at the end of the sequence object, at the beginning of the sequence. That part is also an ORF-recognized method.

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 put into the added linker sequence, the PCR product can be cleaved with the restriction enzyme and used for ligation.

To check if the primers designed in the primer designer are suitable for use in PCR, the internal sequence into which the generated primers are annealed has a predetermined set of restriction enzymes and a recognition sequence that is cut by a specific restriction enzyme specified by the user. You can check whether the designed primer is annealed to the internal sequence to see if it is inappropriate for the primer. 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 using primer designers or prepared primers to obtain PCR products. The PCR program examines the binding of 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 larger than the Tm value specified by the user. Sequence objects resulting from PCR in a PCR program produce blunt sequence objects with no overhangs. The sequence 'A' can also be added at the 3 'position of the PCR product made for T-vector cloning.

In the DNA cutter, it takes sequence objects such as insert sequences or vector sequences from PCR products, cuts the sequence object with a given restriction enzyme, adds an overhang according to the restriction enzyme, and then moves on to ligation. At this time, if the sequence object input to the DNA 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 unit determines whether the ligation is possible by judging an overhang as shown in FIG. 9 when there is an ENDS item of the vector and the insert. To 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.

According to the present invention, the experimenter can operate the program consisting of each module and obtain the result as a complete sequence on the computer as if the actual cloning of the gene 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.

1 shows a cloning editor according to the invention.

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

3 is a diagram of PCR of the cloning editor of FIG.

4 is a diagram illustrating a sequence editor among the cloning editors of FIG. 1.

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 a ligation possibility.

Claims (36)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete Editing and storing the sequence by the sequence editor to objectize the gene sequence as independent data communicated on the program and to express a blunt or an overhang, respectively; Calling and ligating the objectized sequence to identify and ligate the ligation site; And Genetic cloning simulation method comprising the step of storing the ligated result in a database, A gene cloning simulation method comprising, in the ligation step or a step before, a DNA cutter includes a step of identifying a restriction enzyme recognition site sequence in an objectized sequence and inputting a restriction enzyme to cut the sequence. 28. The method of claim 27, wherein a standard position marker (SPM) is displayed and edited on the sequence object to mark the start of the sequence object. delete delete delete 28. The method of claim 27, wherein the blunt or overhang is represented by "(overhang of the beginning of the sequence object) .. (overhang of the end of the sequence object)". 28. The method of claim 27, wherein when there is one sequence object input in the ligation step, both ends of the input sequence object are treated as self-ligation. 28. The method of claim 27, wherein the restriction enzyme name to be input is case-insensitive and the Roman numerals are sequentially recognized as being identical to the corresponding Arabic numerals. 29. The method of claim 27, wherein the restriction enzyme recognizes the extended sequence according to the following table. [table] A process by which a sequence editor edits and stores a sequence to objectize a gene sequence as independent data mutually transferred on a program and to express a blunt or an overhang, respectively; And a process of calling and ligating the objectized sequence to identify and ligate the ligation site; And a program storing a program for implementing a process in which the ligated result is stored in a database.