KR101067899B1 - Method for preparation of deletion cassette using synthesis of gene comprising artificial sequences linkers - Google Patents

Abstract

The present invention relates to a method for producing a defective cassette using a gene synthesis method including an artificial sequence linker, which is a method required for preparing a gene targeting model. More specifically, (a) designing oligomers of gene fragments comprising artificial sequence linkers at both ends to prepare a deletion cassette; (b) conjugating the fragments by phosphorylating the oligomer and then using a ligase chain reaction (LCR); (c) performing amplification PCR using the conjugated LCR product as a template using primer pairs present in an artificial sequence linker; And (d) removing the artificial sequence linker present at both ends of the PCR amplified deletion cassette, and a deletion cassette prepared by the method. By using the above method, systematic gene synthesis is possible, which can dramatically increase the efficiency of gene synthesis.

Artificial sequence linkers, gene synthesis, gene targeting, deletion cassettes

Description

METHODS FOR PREPARATION OF DELETION CASSETTE USING SYNTHESIS OF GENE COMPRISING ARTIFICIAL SEQUENCES LINKERS}

The present invention relates to a method for producing a defective cassette using a gene synthesis method including an artificial sequence linker, which is a method required for preparing a gene targeting model. More specifically, (a) designing oligomers of gene fragments comprising artificial sequence linkers at both ends to prepare a deletion cassette; (b) conjugating the fragments by phosphorylating the oligomer and then using a ligase chain reaction (LCR); (c) performing amplification PCR using the conjugated LCR product as a template using primer pairs present in an artificial sequence linker; And (d) removing the artificial sequence linker present at both ends of the PCR amplified deletion cassette, and a deletion cassette prepared by the method.

Gene targeting is a genetic technique that knocks out or introduces a specific gene in an individual by homologous recombination, and has developed in various ways since 1989 when the first genetically targeted mouse was born. As a result, it is now possible to inject target genes into mature adults as well as developmental growth periods, and to inject mutant genes that are expressed only at certain times. Three people, Martin J. Evans, Oliver smithies, and Mario R. Capecchi, were awarded the 2007 Nobel Prize for their work.

For gene targeting, a DNA fragment structure called a deletion cassette may be used. The deletion cassette is a tool having a constant genetic structure for inducing targeted mutagenesis by directly targeting and deleting a target gene using a homologous DNA sequence, and the DNA to be replaced with the target gene to be deleted. The fragment is introduced into the cell to induce homologous recombination at the target gene site so that the target gene is deleted and the externally introduced DNA fragment enters the chromosome.

In order to prepare the deletion cassette, a technique for selectively amplifying a specific gene sequence is required, and the most widely used technique is a polymerase chain reaction. Polymerase chain reaction (PCR) is a molecular biological technique for copying and amplifying DNA (US Patent No. 4,683,195, US Patent No. 4,683,202, US Patent No. 4,800,159, and European Patent No. 0200362). , 0201184 and 0229701 and Methods in Enzymology, 1987, 155 : 335-350, Murakawa et al., DNA 1988, 7 : 287-295), which can selectively amplify only specific DNA fragments of interest in trace amounts of DNA solution. To be. PCR generally amplifies the desired DNA through three to three cycles of denaturation, annealing, and extension. Such PCR is a multiplex PCR. , Nested PCR, quantitative PCR, real-time PCR, reverse transcription PCR, RT-PCR, touchdown PCR, and the like.

As described above, after the deletion cassette is prepared by using PCR to prepare the gene targeting model, a transformation process of introducing the deletion cassette into a specific individual is performed. Transformation is the transformation of an organism by the acceptance of external DNA, resulting in recombination of homologous regions in the deletion cassette and the target gene of the individual, thereby replacing specific gene regions in the individual and the selection marker portion in the deletion cassette. As a result, the trait of the individual is changed. In general, selectable markers are labeled on the DNA to be introduced to distinguish cells which have been successfully transformed. As a selection marker, adenine, uracil, leucine, and the like, which are related to nutrition of amino acids, may be used. However, there is a disadvantage in that non-specific strains are selected at the time of selection, and in practice, a marker that is involved in antibiotic resistance is generally used. Representative examples include ampicillin, tetratracycline, or kanamycin, and after transformation, cultivating the bacteria in a medium containing antibiotics facilitates only strains that successfully transformed. Can be obtained.

The genome hit model organism can be used as a tool for not only knowing the function of a specific gene and information about the protein expressed by the gene, but also as a tool for detecting the action point at which a specific drug acts. Furthermore, it can be used as an effective tool to predict the point of action for drug development (Lum et al., Cell. 116: 121-137). The preparation of the deletion cassette is essential for preparing the gene targeting model.

However, in preparing the deletion cassette for gene targeting as described above, synthesizing a gene including both a homologous recombination site and a selection marker gene site is uniform due to the high A / T ratio of the promoter and the terminator of the gene in the deletion cassette. Difficulties in designing primer pairs with Tm values present difficulties in the PCR amplification step. This becomes a factor that lowers the PCR amplification rate, and as a result, the success rate of defective cassette production is only 50% or less.

Accordingly, the present inventors have developed a method for producing a defective cassette using a gene synthesis method including an artificial sequence linker, which can dramatically increase the efficiency of gene synthesis method as a result of diligent efforts to produce a systematic and automated effective deletion cassette. The present invention has been completed.

An object of the present invention is to (a) design an oligomer of a gene fragment comprising an artificial sequence linker at both ends to prepare a deletion cassette; (b) conjugating the fragments by phosphorylating the oligomer and then using a ligase chain reaction (LCR); (c) performing amplification PCR using the conjugated LCR product as a template using primer pairs present in an artificial sequence linker; And (d) to provide a method for producing a deletion cassette (deletion cassette) comprising the step of removing the artificial sequence linker at both ends of the PCR amplified deletion cassette.

Another object of the present invention is to provide a deletion cassette prepared by the above method.

Thus, in one aspect, the present invention provides a method for preparing a deletion cassette comprising the steps of: (a) designing an oligomer of a gene fragment comprising artificial sequence linkers at both ends to prepare a deletion cassette; (b) conjugating the fragments by phosphorylating the oligomer and then using a ligase chain reaction (LCR); (c) performing amplification PCR using the conjugated LCR product as a template using primer pairs present in an artificial sequence linker; And (d) removing the artificial sequence linker present at both ends of the PCR amplified deletion cassette.

As used herein, the term "ligase chain reaction (LCR)" is one of the main components of the gene synthesis method, the desired small amount of 2 through the overlap (ligation) after overlapping the oligomer of a specific length This is the reaction required to obtain strand DNA fragments. The oligomeric conjugation reaction can be carried out by linking with conventional conjugation reactions based on the nucleic acid conjugation principle. Therefore, enzymes commonly used in the art may be used as a ligase for inducing linkage of nucleic acids without limitation. Preferably, in order to increase the conjugation efficiency, a heat resistant conjugation enzyme (Tfi) may be used. In the case of using the Tfi enzyme, the conjugation reaction is repeatedly performed like the PCR reaction, thereby increasing conjugation efficiency. This is called LCR. The basic principle of the gene synthesis method is a technique obtained by amplifying the final gene by PCR method using a double-stranded DNA fragment conjugated through the LCR reaction as a template.

Gene lengths that can be successful in one gene synthesis are typically less than 1000 bp, and for longer gene synthesis, a method of concatenating LCR with DNA fragments less than 1000 bp obtained by the above method can be used. The gene hit success rate of the deletion cassette is determined by homologous recombination efficiency, and homologous recombination efficiency in the same strain depends on the length of the chromosomal homology site (Michael et al., Gene. 158: 113-17; Palaniyandi et al., Nucl Acid Res. 3: 2799-2800), in order to increase the success rate of gene targeting, the length of the chromosomal homology region in the deletion cassette should be increased. The deletion cassette used in the preferred embodiment of the present invention also has a homologous recombination site of about 250 bp at both ends, and a selectable marker has a length of about 1.43 kb. The defect cassette was designed to be divided into three fragments. Each of the three oligomers prepared using the Block2Oligo program and the linking oligomers connecting these three fragments of the DNA fragments prepared by iBlocks2Assembly were joined to one DNA fragment using LCR to complete one missing cassette. .

The three fragments of the fragments used in the present invention are fragmented into appropriate sizes necessary for convenience in order to prepare a deletion cassette, and the sequences of 5 'and 3' recombination sites or selection markers of a target gene of interest are used. To have. In addition, each gene fragment is composed of a plurality of oligomers, any oligomer used in the art can be used without limitation, the length of the oligomer is also unlimited, but preferably within about 10 bp to 60 bp, more preferably 12 bp to 35 bp. In a preferred embodiment according to the present invention, the defect cassette was designed and joined in three fragments, and the number and length of oligomers constituting each fragment were conveniently designed and manufactured to suit a desired purpose. The length of the oligomers was average 27 mer in length for gene-specific artificial sequence linkers, 5 'and 3' homologous recombination sites and oligomers of barcode sites, except that the 5 'top strand of DNA at both ends of the deletion cassette was used. Only the sock end of the deletion cassette was prepared at the blunt end using an oligomer of 12-16 bp in length. Common oligomers of Kan ^r selective marker fragments used 30 or 31 mer. In a preferred embodiment of the present invention, Block2Oligo, a program developed by itself, was used to determine the oligomeric nucleotide sequences constituting the DNA fragment.

Preferably, the linking oligomer may be used to connect the three fragments of the gene fragments constituting the deletion cassette according to the production method of the present invention.

By performing LCR as described above, three fragments of DNA fragments can be linked to obtain a deletion cassette. However, since the amount of the two-stranded deletion cassette DNA is only a very small amount, the deletion cassette DNA is used as a template. Amplification techniques are essential. Therefore, PCR technology is still required for sufficient gene synthesis.

However, as mentioned in the background, the conventional gene synthesis method has difficulty in final PCR amplification of trace cassette DNA fragments present in trace amounts after LCR reaction due to the high A / T ratio of promoter and terminator of the gene. Existed. In other words, it is difficult to design primer pairs for PCR amplification having an equivalent Tm value, resulting in a very low PCR amplification rate, thus reducing the success rate of the deletion cassette to 50% or less. The mean value of the double-stranded DNA becomes the single-stranded DNA, which is dependent on the length and base composition of the DNA, and the higher the Tm value, the stronger the binding. The Tm value refers to the degree of binding between the primer and the target DNA. The Tm value is calculated based on the experimental value (Tm = 81.5 + 16.6 * (log10 ([Na +] + [K +])) + 0.41 * (% GC)-675 / N).

However, when the artificial sequence linker is conjugated to both ends of the LCR resultant as in the manufacturing method of the present invention, it is possible to easily design a primer pair having an equal Tm value, thereby significantly increasing the yield of the PCR amplification step. This, in turn, results in a significant increase in the success rate of the deletion cassette. Thus, the present invention is the first time that a large amount of LCR output was obtained by performing gene synthesis using an artificial sequence linker.

As used herein, the term "artificial sequence linker" is a linker consisting of a sequence which does not exist in a subject to be transformed with a deletion cassette, and any artificial sequence used in the art as a linker without limitation. Can be used. The artificial sequence linker should consist of sequences that are not present in the gene hit strain, and preferably the linker may comprise a PCR primer binding site, or a PCR primer binding site and a restriction enzyme site. . The PCR primer binding site of the artificial sequence linker of the present invention, which is used to improve the low PCR success rate of gene synthesis, enables the design of primer pairs having a uniform Tm value, which has an artificial sequence having a uniform Tm value. Linkers can be placed at both ends of the primer pairs to increase the yield of PCR. As a preferred embodiment, the linker sequence may comprise two pairs of primer binding sites and restriction enzyme sites, but the linker sequences of the present invention are not limited by the above examples.

Polymerase chain reaction (PCR) is a molecular biological technology that replicates and amplifies DNA as described in the background art. Examples of the method according to the method include multiplex-PCR, nested PCR, and quantitative PCR. PCR, real-time PCR, reverse transcription PCR (RT-PCR), touchdown PCR, and the like may be used, but the present invention is not limited by the above examples. In the present invention, the purpose of PCR is to obtain a high yield after the reaction, rather than to measure the quantification of the PCR product according to the number of amplifications, and a general method may be used. In a preferred embodiment of the present invention was carried out 35 times for 2 minutes at 94 ℃ 30 seconds, 60 ℃ 30 seconds and 72 ℃ 2 minutes over a second, the deletion cassette gene synthesized by LCR by reacting for an additional 5 minutes at 72 ℃ It was confirmed that can be obtained with a significantly high yield. In addition, the amplification PCR using a primer having a uniform Tm value, any type of systematic PCR, such as 96 well, 384 well and 1536 well is possible.

Another object of the PCR in the present invention may be to cut out the unnecessary linker when introducing the strain of the deletion cassette. In order to remove unnecessary artificial sequence linker, a method using nested PCR may be performed in addition to the above-mentioned restriction enzyme. Nested PCR is a PCR method that removes both ends of a template using an inner primer pair of an existing template DNA fragment. For such PCR, the artificial sequence linker may include three pairs of primer binding sites.

As used herein, the term "deletion cassette" is a component used for gene deletion, and may include without limitation the structure of a deletion cassette commonly used in the art. The structure of a conventional deletion cassette includes a selection marker, a homologous recombination site at both ends of the selection marker. When the deletion cassette is transformed and introduced into a heterozygous strain, the selection marker gene in the deletion cassette is replaced with the target gene on the chromosome by homologous gene recombination of the homologous recombination site on the chromosome. The marker gene in the deletion cassette is then inserted into the chromosome and the target gene is hit and deleted.

Selection markers in the deletion cassettes used in the present invention include, but are not limited to, auxotrophic markers, neomycin resistance gene (neo ^R ), kanamycin resistance gene (kan ^R ), hygromycin (hyg), histi Dinol dehydrogenase (hisD), guanine phosphoromosyl transferase (gpt), tetracycline (tet ^R ), hypoxanthine phosphoribosyl transferase (hprt), ura3, ble and sacB, preferably It is the kanMX which applied kanamycin resistance gene. The deletion cassette comprising the selection marker can be substituted into the strain chromosomal DNA through genetic recombination to a chromosomal DNA site with homology. Preferably, ura4, leu1, and his3 may be used as nutritional markers, but in order to use such markers, a strain having one or more auxotrophic mutations must be used. In addition, gene conversion of nutritionally demanding mutations by DNA introduction may occur, and the nutritional demand mutations themselves, along with other mutations, may be osmosensitivity or nitrogen-deficient differentiation, spores. Unpredictable phenotypes such as sporulation and pseudohyphae can be exhibited, and many nutritional mutations may also inhibit growth in normal media.

Therefore, to overcome this limitation of nutritional markers, Philipsen's team developed the kanMX module, a dominant drug resistance marker. It has also been used in the deletion project of Saccharomyces cerevisiae , which lacks the gene of yeast (Giaever et al., Nature. 418: 387-391, 2002). The kanMX module is manufactured to be resistant to the antibiotic G418 and to be used as a selectable marker. The marker of G418 has advantages over nutritional requirement markers due to the high success rate of gene deletion even when using PCR-DNA fragments. Currently, dominant drug resistance markers of hygromycin B, nourseothricin, and bialaphos / phosphinothricin have been developed.

Thus, as a preferred embodiment, 810 bp KanMX4, the selection marker used in the present invention, originates from the transposon Tn903 gene of Escherichia coli , which is an amino-glycoside phosphotransferase activity. It is reported to inhibit the activity of kanamycin or its derivative G418 (Lang-Hinrichs et al., Current Genetics. 18: 511-6, Oka et al., J. Mol. Biol. 147: 217-26). . The kanMX module, one of the dominant drug resistance markers, uses an open reading frame (ORF) known as kan ^r of the E. coli transposon Tn903 to the yeast type Ashbya gossypii . It was inserted into the transcriptional promoter and terminator of the TEF gene, and was prepared to be used as a selection marker by showing resistance to the antibiotic G418.

In a preferred embodiment of the present invention, the structure of the kanMX4 module is another type of yeast ahsiba notice blood gene the TEF promoter (promoter) 381 bp of (Ashbya gossypii) to express the kanamycin resistance gene of the 810 bp Tn903 in yeast ATG is placed in front of the start codon of ATG, and 242 bp of the TEF terminator of the Assybian gossip is located after the stop codon of the gene. When using Saccharomyces cerevisiae or a cleavage yeast promoter or terminator, it is preferable to use a heterologous yeast promoter because overlapping homologous sites in the chromosome may occur and false homologous recombination may occur. After the above process, the length of KanMX4 module becomes 1,433 bp. In the case of gene deletion, the frequency of insertion failure increases when the PCR product is used or when the homology region of the target gene where recombination occurs in the deletion cassette is short. G418 markers such as KanMX4 are heterologous selection markers that exhibit dominant resistance, and have a high success rate of gene deletion even when using PCR-DNA fragments.

The deletion cassette of the present invention can be used without limitation the deletion cassette that targets the gene by the above principle, and preferably may further include a barcode array sequence for the micro array in the deletion cassette. The barcode array sequence for the microarray is a kind of identification code specifically assigned to each strain. Preferably, a gene-specific barcode composed of 20 mer oligos has a 5 'up barcode at each end of a selection marker of a deletion cassette. A total of two can be assigned, named UP-tag and 3 'sub-barcode (downtag, DN-tag). The deletion cassette model comprising the barcode sequence is disclosed in the inventor's prior application (Korean Patent Application No. 2008-0037420).

Although the length of the homologous recombination sites in the deletion cassette of the present invention is not limited, as mentioned above, the length of the homologous recombination sites is determined by different homologous recombination efficiencies for each strain, but is dependent on strain characteristics, but homologous recombination in the same strain is performed. Efficiency depends on the length of the chromosomal homology region (Michael et al., Gene. 158: 113-17; Palaniyandi et al., Nucl Acid Res. 3: 2799-2800), corresponding to the chromosomal homology region of the gene It is desirable to optimize the length of the DNA fragment to be as long as possible. The length of such homologous recombination sites may preferably be at least 45 bp, more preferably at least 80 bp, even more preferably between 80 and 450 bp. In a preferred embodiment of the present invention, the deletion cassette was prepared by optimizing the length of homologous recombination to 250 bp in the case of fission yeast.

The present invention provides a method for attaching a gene fragment using LCR, performing PCR using an artificial sequence linker to sequences at both ends of a deletion cassette to be prepared, and removing the artificial sequence, thereby providing a method for producing a deletion cassette. . Preferably the artificial sequence linker at both ends of the deletion cassette may comprise two or more primer binding sites mentioned above, and more preferably may further comprise restriction enzyme cleavage sites. Removal of the artificial sequence can be removed by performing nested PCR using a primer other than the primer binding site, and if a restriction enzyme cleavage site is included, it may be easily removed using a restriction enzyme. Restriction enzymes are endonucleases that recognize DNA specific nucleotide sequences and cleave double chains, preferably type II restriction enzymes can be used. Type II restriction enzymes recognize and cleave four, five or six sequences, such as Eco RI (GAATTC cleavage, sticky end), Bam HI (GGATTC cleavage, sticky end), Bgl II (AGATCT cleavage, sticky end), Hind III (AAGCTT cleavage, sticky end), Pvu II (CAGCTG cleavage, blunt end), Sac I (GAGCTC cleavage, sticky end) and Sam I (CCCGGG cleavage, blunt end). Restriction enzyme sequences are not limited by the above examples. Preferably the restriction enzyme may be a Bsa Ⅰ, in the preferred embodiment of the present invention by inserting the _{5'-GGTCTC (N) 1 -3} Bsa restriction sites consists of 'and _{3'-CCAGAG (N) 2 -5} ' The deletion cassette was prepared by exposing the chromosomal homology sites at both ends of the deletion cassette during restriction enzyme treatment.

As another aspect, the present invention relates to a deletion cassette produced by the above method.

In a preferred embodiment of the present invention, the kanamycin resistance gene (Kan ^R ) as a selection marker, in inserting the barcode sequence and homologous recombination site at both ends, the deletion cassette using the gene synthesis method comprising the artificial sequence linker Prepared. The deletion cassette first comprises 1) a 5 'linker, a 5' homologous recombination site and a 5 'barcode site, 2) a kanamycin selection marker site, and 3) a 3' linker, a 3 'homologous recombination site and a 3' barcode site. It was designed as a piece, and the oligomer sequence of each piece was determined using Block2Oligo, a program developed by itself, and a linking oligomer was designed to connect the 3 pieces using iBlocks2Assembly, which is also a program developed by itself. The conjugated gene was amplified by nested PCR as described above, and the amplified result was introduced into Schizosaccharomyces pombe , a cleavage yeast, to confirm that successful gene hits occurred.

In the case of using a method for preparing a deletion cassette using a gene synthesis method including an artificial sequence linker of the present invention, one phosphorylation reaction and a polymerization conjugation instead of the four PCR and four DNA fragment purification processes used for the preparation of the deletion cassette were performed. Deletion cassettes can be prepared by the reaction and the two PCR reactions, which eliminates the cumbersome conventional purification process, as well as the deletion cassette having high strain production success rate and production efficiency and low point mutation rate. By using the highly efficient heterozygous gene hit strain can be produced.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1 Structure of Deletion Cassettes and Design of Oligomers

Using the gene synthesis method, a deletion cassette having the structure of FIG. 1 was prepared. The length of the deletion cassette is about 2160 bp including 1) kanamycin resistance gene region 1.43 kb, 2) 70 bp barcode region of the sock end and 250 bp gene homology region, and 3) 46 bp linker of the sock end.

In order to manufacture the gene deletion deficiency cassette of FIG. 1 by a gene synthesis method, a large number of expensive oligomers are required, and thus a strategy for minimizing the cost of oligomer synthesis was required. Therefore, the following two points were considered in design. First, the length of the chromosomal homology region, which is the most important factor for determining the efficiency of strain preparation, was set to 250 bp in consideration of efficiency and cost. The 5 'chromosomal homology region is 250 bp in the direction of the gene's promoter relative to the protein coding start codon ATG, and the 3' chromosomal homology region is the poly-A based on the protein coding end codon TGA, TAG or TAA. 250 bp was taken from the chromosome and used. The base sequences of the 5 ′ and 3 ′ chromosomal homology regions of 250 bp length are different for each gene and are omitted because those skilled in the art can easily estimate the base sequences with knowledge of general genome information. Second, oligomers corresponding to the 1.43 kb long Kan ^r module, which are common to all missing cassettes, were commonly used to reduce costs. Although all deletion cassettes have a common Kan ^r module of 1.43 kb length and 92 bp corresponding to each of the 46 bp artificial sequence 5 'and 3' linkers, the common use of the artificial sequence linker is realistic due to the difficulty of the oligomer design. It was impossible.

As shown in FIG. 2, the deletion cassette was identified as 1) a gene specific 5 ′ chromosomal homology region and 5 ′ barcode region comprising a 5 ′ linker, 2) a common kanamycin resistance gene (Kan ^r ) region, 3) 3 ′ The design was divided into three fragments of the gene-specific 3 'barcode region including the linker and the 3' chromosome homology region. At this time, in order to prepare the missing cassette by connecting the three pieces of the above, the overlapping portion of the "linking oligomer" having the base sequence necessary for the connection of each piece was disposed. The gene-specific fragments 1 and 3 were designed with 27 oligomers of 27 mer length each, and the kanamycin resistance gene (Kan ^r ) fragment corresponding to the commonly used fragment 2 was 30 or 30 using a known sequence. 93 were designed with 31 mer oligomers. However, oligomers corresponding to the 5 'strand of DNA at both ends of the deletion cassette were arranged to have blunt ends at both ends of the deletion cassette by placing a relatively short 12 to 16 bp oligomer. The oligomer corresponding to each part is omitted because it can be easily designed by those skilled in the art having ordinary knowledge about the genome.

The oligomers for the deletion cassette preparation were designed to overlap without gaps between each other to prepare two strands of missing cassette DNA fragments having nicks with overlapping oligomers alone. At this time, all Tm values of the oligomers were designed to be 60 ± 3 ° C. uniformly, and the calculation of Tm values of the oligomers was used by SantaLucia's well-known formula, which is as follows (SantaLucia PNAS. 95: 1460-5).

To determine the gene specific oligomer sequences corresponding to three fragments of the deletion cassette, a self-developed program called Block2Oligo was used. In addition, the iBlocks2Assembly program, which was developed in-house, was also used for the design of oligomers connecting each of the three designed pieces. The oligomer design principle of each program is to properly dissociate the G / C ratio within a given length so that the Tm value of each oligomer is constant.

Example 2. Phosphorylation of oligomers and ligase chain reaction (LCR)

In this step, the 5 'end of the overlapping oligomer is phosphorylated with a kinase (kinase, T4 polynucleotide kinase), followed by ligation of all gaps between the overlapping oligomers to prepare a defective cassette DNA fragment consisting of two strands. This step can proceed in 96-well format (96-well format) or 384 well (384-well format), but in the 96-well format for ease of operation. The first process is a process of phosphorylating oligomers, as shown in Table 1 below, each solution was mixed and reacted at 37 ° C. for 5 hours, and then 3 μl of kinase was added for 5 hours. The second process is a conjugation process that adds Tfi heat-resistant ligase to phosphorylated oligomer and then performs LCR reaction. As shown in Table 2 below, after the solution was mixed, the reaction of condensation enzyme chain reaction was performed by repeating the reaction of condensation of DNA for 5 minutes at 95 ° C. and conjugation at 50 to 60 ° C. for 5 minutes.

 Phosphorylation solution mixing and reaction conditions solution Volume or quantity Oligomer 29 (20 pmole) Phosphorylation Buffer 5 μl Phosphatase (T ₄ polynucleotide kinase) 5 μl ATP [10 mM] 8 μl Total volume 47 μl Reaction conditions: After reaction at 37 ° C. for 5 hours, additionally 3 μl of enzyme was added for 5 hours.

LCR reaction solution mixing and reaction conditions solution Volume (μl) Phosphorylated oligomers (each oligo 8.7 pmole) 25 μl Junction Buffer 3 μl Tfi Conjugation Enzyme (Bioneer) 3 μl Total volume 31 μl Reaction conditions: 40 cycles of DNA modification at 95 ° C for 5 minutes and conjugation of 40 times at 50 ~ 60 ° C (typically 58.6 ° C) for 5 minutes

Example 3. Amplification by PCR of LCR Defected Cassette

Since the DNA amount of the defective cassette which has undergone the LCR reaction is extremely small, the method for amplifying the defective cassette by PCR is necessary for successful transformation.

In order to perform amplification PCR without a linker by the conventional method, it is necessary to perform a difficult task of selecting amplification PCR primer binding sites at both ends of the defective cassette. However, in the case of using a linker, it is possible to easily PCR amplification by overcoming these problems. The nucleotide sequence used as the linker is an artificial nucleotide sequence not present in the genome of the cleavage yeast, and the BsaI restriction for removing the linker sequence and the sequences of L1, L2, L3 and L4, which are 20 mer primers for the second round PCR amplification. The enzyme site should be included, and the Tm value of each primer should be about 60 ° C. The nucleotide sequence of the artificial sequence linker and L1, L2, L3 and L4 is shown in Table 3 below. Primary PCR was performed using primers L1 and L4, and secondary PCR was performed using L2 and L3 (FIG. 3). To this end, each cycle was repeated 35 times using each primer set at the normal conditions (30 ° C, 60 ° C, 30 seconds, 72 ° C, and 35 minutes, followed by an additional 5 minutes at 72 ° C). Primary and secondary PCR procedures were performed. The mixing and reaction conditions of the solution for PCR amplification are shown in Table 4 below.

Base sequence of linker for amplifying a deletion cassette name Tm
(℃) Linker sequences Sequence orientation order
number 5'-L 5'-TGCAATGCGTTGGGCAACAATTACGCTTGTGCAAACGCCAGGTCTC-3 ' Forward One 3'-L 5'-GAGACCTTTTAATGCGCGCCCTTGCATCATAACGCGTTTGCGGCAT-3 ' Anti-sense 2 L1 60.6 5'-TGCAATGCGTTGGGCAACAA-3 ' Forward 3 L2 60.1 5'-TTACGCTTGTGCAAACGCCA-3 ' Anti-sense 4 L3 60.0 5'-TGCAAGGGCGCGCATTAAAA-3 ' Forward 5 L4 60.5 5’-ATGCCGCAAACGCGTTATGA-3 Anti-sense 6

Solution Mixing and Reaction Conditions for Defect Cassette Amplification PCR solution Volume (ul)  Primary PCR Template: LCR reaction solution (total 31 ul)  One Buffer  2 Pfu polymerase (Bioneer) [5U / ul]  0.5 Linker L1 and L4 [100 pmole / ul each] 1 (0.5 each) dNTP [10 mM] 0.5 water 15 Total volume 20 Reaction conditions: After repeated 35 times at 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 2 minutes, an additional 5 minutes at 72 ℃ 2nd PCR Template: Primary PCR reaction solution (20 ul total) One Buffer 2 Polymerase  0.5 Linker L2 and L3 [100 pmole / ul each] 1 (0.5 each) dNTP [10 mM] 0.5 water 15 Total volume 20 Reaction conditions: After repeated 35 times at 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 2 minutes, an additional 5 minutes at 72 ℃

Using two genes with systematic gene names SPCC548.04 and SPAC19B12.09 The optimal conjugation temperature for amplifying the deletion cassette was confirmed (FIG. 4), and the amplified deletion cassette of about 2.2 kb was obtained only at 58.6 ° C. LCR conditions as shown in the red dotted line box at the bottom of FIG. 4. If the conjugation reaction failed at 58.6 ° C, the conjugation reaction was performed at 54 ° C or lower. In addition, as a result of confirming the optimal dilution ratio of the primary PCR product in the second amplification PCR, when the experiment was performed by diluting the amount of the primary PCR product to 1/10, 1/20, 1/40, respectively, 1/10 The highest amount of secondary PCR product was obtained at, and the defect cassette was visually identified in the agarose gel as indicated by the white arrow in the lower figure of FIG. 4. At this time, as the dilution rate increases, the yield decreased. In conclusion, the LCR reaction was performed at 58.6 ℃, and the dilution ratio of the primary PCR product was determined to be 1/10.

Example 4 Removal of Linker Sites Present at Socks of Missing Cassettes

The linker for PCR amplification was essential to amplify the trace amount of the defective cassette after the LCR reaction. However, the linker should be removed before transformation into cleavage yeast because it will interfere with homologous recombination after transformation into the strain. Recognition between chromosome homology region and the linker nucleotide sequence is _{5'-GGTCTC (N) 1 -3} ' and _{3'-CCAGAG (N) 2 -5} ' ( complementary strand) insert the Bsa I restriction site to this It was. Chromosomal homology sites are exposed at both ends of the deletion cassette treated with Bsa I restriction enzymes, thereby increasing the efficiency of homologous recombination during transformation, thereby increasing the success rate of strain production.

The defective cassettes subjected to PCR amplification with LCR in the form of the 96-wells mentioned above were used for transformation by checking the concentration in agarose gel after treatment with Bsa I restriction enzymes (FIG. 6).

The contents mentioned in Examples 1 to 4 are collectively shown again in FIG. 6.

Example 5 Transformation of Defect Cassettes and Identification of Gene Hit Strains

Introducing the deletion cassette prepared through the procedure of Examples 1 to 4 into diploid split yeast SP286 strain (ade6-M210 / ade6-M216, ura4-D18 / ura4-D18, leu1-32 / leu1-32) cells For this purpose, the cells were transformed using a known lithium acetate method (Moreno et al., Methods Enzymol. 194: 795-823). The transformed strains were plated in a YES agar solid medium containing G418 antibiotics (200 mg / L) and incubated for 3 to 4 days at 30 ℃ until colonies were produced. Colony PCR was performed to confirm that the transformed colonies were the desired genes. A small amount of cells were taken from the edge of the transformed colonies grown in solid medium and suspended in tertiary distilled water, and some of them were taken and confirmed by PCR using oligomeric primer pairs for strain identification.

The 5 'region of the chromosome into which the deletion cassette was inserted was subjected to 5' colony PCR using CP5 and CPN1 or CPN10 oligomeric primer pairs, and the 3 'region was 3' using CP3 and CPC1 or CPC3 oligomer primer pairs. Confirmation was performed by colony PCR. CP5 and CP3 are located on the chromosomal region 100 to 200 bp outside the chromosomal homology region, CPN1 and CPC1 are located within 200 bp toward the KanMX4 gene, and CPN10 and CPC3 are located 300 bp inside the KanMX4 gene. Therefore, the size of a conventional colony PCR DNA fragment is 620-820 bp, which is equal to 250 bp of chromosomal homology, 70-bp barcode, 100-200 bp chromosome outside, and 200-300 bp KanMX4 gene length. It is about. Reaction conditions and method of colony PCR are described in Korean Patent Application No. 2008-0037420. In addition, the oligomeric sequences of CP5 and CP3 are specifically different for all genes, but are omitted because those skilled in the art can easily know. The nucleotide sequence of oligomer sites of CPN1, CPN10, CPC1 and CPC3 present in the kanamycin resistance gene is also specified in Korean Patent Application No. 2008-0037420, so it is omitted.

1 is a structural diagram of a deletion cassette to be produced by gene synthesis.

2 is a three-piece design diagram of a missing cassette.

Figure 3 is a PCR amplification of the LCR product using an artificial sequence linker.

4 is a diagram illustrating a method of determining an optimal Tm value and the amount of template for PCR amplification.

5 is a result of confirming the deletion cassette treated with BsaI restriction enzyme for transformation in agarose gel in 96-well form.

Figure 6 is a diagram showing a schematic diagram of the deletion cassette by the gene synthesis method using an artificial sequence linker.

<110> KOREA RESEARCH INSTITUTE BIOSCIENCE AND BIOTECHNOLOGY <120> METHOD FOR PREPARATION OF DELETION CASSETTE USING SYNTHESIS OF          GENE COMPRISING ARTIFICIAL SEQUENCES LINKERS <130> PA080412 / KR <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> forward primer for linker <400> 1 tgcaatgcgt tgggcaacaa ttacgcttgt gcaaacgcca ggtctc 46 <210> 2 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for linker <400> 2 gagacctttt aatgcgcgcc cttgcatcat aacgcgtttg cggcat 46 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for L1 <400> 3 tgcaatgcgt tgggcaacaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for L2 <400> 4 ttacgcttgt gcaaacgcca 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for L3 <400> 5 tgcaagggcg cgcattaaaa 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for L4 <400> 6 atgccgcaaa cgcgttatga 20  

Claims (9)

(a) designing oligomers of gene fragments comprising artificial sequence linkers at both ends to prepare a deletion cassette; (b) conjugating the fragments by phosphorylating the oligomer and then using a ligase chain reaction (LCR); (c) performing amplification PCR using the conjugated LCR product as a template using primer pairs present in an artificial sequence linker; And (d) removing the artificial sequence linker present at both ends of the PCR amplified deletion cassette. The method of claim 1, wherein step (c) is a step of performing a PCR by any one well system selected from the group consisting of 96 wells, 384 wells and 1536 wells. The method of claim 1, wherein the removing of the artificial sequence linker of step (d) is performed by performing restriction enzyme treatment or nested PCR. The method of claim 1, wherein the artificial sequence linker comprises a PCR primer binding site and a restriction enzyme cleavage site. The method of claim 4, wherein the primer binding site has a uniform Tm value of the binding site. The method of claim 4, wherein the base sequence of each primer binding site is a manufacturing method of the deletion cassette, characterized in that 17 to 25 mer. 5. The method of claim 4, wherein said restriction enzyme cleavage site comprises a sequence cleaved by a type II restriction enzyme. 5. A method according to claim 4, wherein the restriction enzyme cleavage site is cleaved by the restriction enzyme Bsa I. delete

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