JP2009268360A - Primer for producing fused dna fragment and method for producing fused dna fragment using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a primer available for producing a DNA fragment in which a plurality of DNA fragments are fused by using PCR method, and a method for effectively producing a fused DNA fragment thereby. <P>SOLUTION: In a primer available for overlap extension PCR method, a pair of primers comprises a specific sequence in which, the 5' end of primer allowing to overlap has 50% or more of guanine (G) plus cytosine (C) content, is 9-45 bp in length, and allows target DNA fragments to complementarily bind together. A method for producing a fused DNA fragment uses the same. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a primer capable of effectively producing a DNA fragment (fused DNA fragment) obtained by fusing a plurality of DNA fragments, and a method for producing a fused DNA fragment using the primer, and more specifically, PCR (Polymerase chain reaction). And a primer capable of producing one fusion DNA fragment from a plurality of target DNA fragments and a method for producing a fusion DNA fragment using the same.

  In the field of molecular genetics, production of a recombinant DNA fragment is a technique widely used for various purposes such as mass production of a target protein, production of a tool for protein detection, and modification of a functional protein. Among these, in particular, when a detection tool is manufactured or a functional protein is modified, a technique for fusing DNA fragments encoding a plurality of domains is required. Examples of these include techniques for producing a DNA fragment encoding a fluorescent fusion protein by fusing a DNA fragment encoding a target gene and a DNA fragment encoding GFP (Green Fluorescent Protein). For normal production of recombinant DNA fragments, a core DNA fragment is produced by PCR (Polymerase chain reaction), etc., the E. coli plasmid is cleaved with a restriction enzyme, and the core DNA fragment is fused therein using ligase. In general, a method of transforming E. coli with the plasmid is used as a vector, and the operation is complicated and takes time.

The overlap extension method (Overlap extension) is known as a method for producing a fusion DNA fragment via PCR. The principle of this technique is that when a target DNA is amplified in a PCR reaction, a new sequence is added to the PCR product by adding another sequence to the 5 ′ end side of the target DNA-specific primer. By designing the sequences to be complementary between multiple target DNAs, the ends of multiple target DNAs are fused together during annealing, and then a single PCR product is synthesized by an extension reaction using DNA polymerase. Is. Various techniques for applying this principle to insert mutations into genes have been developed so far (Non-Patent Documents 1-4, Patent Documents 1 and 2). However, the overlap extension method is not generally used for the production of fusion DNA fragments, and mainly in vitro recombination with restriction enzymes and DNA ligases has been developed and used. Free fusion of DNA fragments is a basic technology for gene manipulation, and its high operability and certainty are required. The overlap extension method is a very useful method in theory. Until now, there have been many problems such as severe optimization of PCR conditions, and in principle, the advantage of being able to freely design DNA sequences has not been fully utilized. It has been desired to develop a technique that can easily and reliably perform overlap extension, does not require optimization of PCR conditions, and can fuse a plurality of DNA fragments at a time.
US Patent 2004/0053267 US Patent 2006/0257876 Kuwawa H. et al. 2002. Biotech. Lett. 24: 1307-1312. Charlier N. et al. 2003. J. et al. Virol. Methods 108: 67-74. Yang L. et al. 2004. Eukaryot. Cell 3 (5): 1359-1362. Heckman K. & Pease L. 2007. Nature Protocols 2 (4): 924-932. Brachmann et al. 1998. Yeast 14: 115-132.

  In view of the above-mentioned present situation, the present invention provides a primer that can be used for the production of a DNA fragment obtained by fusing a plurality of DNA fragments using the PCR method, and an effective method for producing a fused DNA fragment using the primer. With the goal.

  In order to solve the above-mentioned problems, the present inventors have studied a technique for efficiently performing an overlap extension. In the process, two target DNA fragments having a guanine (G) and cytosine (C) content of 50% or more and a length of 9-45 bp on the 5 ′ end side of a pair of primers to be overlapped The present inventors have completed the present invention by finding the fact that by using a specific complementary sequence capable of binding the, the overlap extension can be performed with a much higher efficiency than before.

That is, the first aspect of the present invention provides a set of primers characterized in that it comprises the following two regions (a) and (b) and is used to bind two types of target DNA fragments: To do.
(A) 3 'terminal region having homology with the partial sequence at the end of the target DNA fragment (b) containing 50% or more of guanine (G) and cytosine (C), with a total length of 9-45 bp, 5 'terminal region (hereinafter referred to as "fusion sequence") capable of complementary binding of target DNA fragments

The second aspect of the present invention provides the primer according to the first aspect, wherein the fusion sequence has a GC content of 80% or more.
The third aspect of the present invention provides the primer according to the second aspect, wherein the fusion sequence does not have a continuous (GC) m (m is an integer of 5 or more and 20 or less).
In the fourth aspect of the present invention, a fusion DNA fragment containing n target DNA fragments (n is an integer of 2 or more and 10 or less) is produced by PCR using the following primers (1) and (2). Provide a method.
(1) Primer for amplifying the full length of the fusion DNA fragment (2) Primer according to any one of the first to third aspects

  By using the primer provided by the present invention, it is possible to produce one DNA fragment from a plurality of, preferably 2-4, DNA fragments. This method is a method for producing a DNA fragment that enables efficient and simple production of a gene or the like encoding a new protein fused with an arbitrary functional domain.

The best mode for carrying out the present invention will be described below. The present invention is a set of primers used to bind two types of target DNA fragments, specifically, a set of primers comprising the following two regions.
(A) 3 'terminal region having homology with the partial sequence at the end of the target DNA fragment (b) containing 50% or more of guanine (G) and cytosine (C), with a total length of 9-45 bp, 5 'terminal region (hereinafter referred to as "fusion sequence") capable of complementary binding of target DNA fragments
FIG. 1 shows the basic concept of the present invention. As shown in FIG. 1A, the primer provided by the present invention is a set of primers composed of the two regions (a) and (b) above, and the binding mode of the target DNA fragment using this is shown in FIG. 1B. ing. The target DNA fragments 1 and 2 are complementarily bound through the fusion sequence of the primer of the present invention.
Moreover, the schematic diagram of the manufacturing method of the fusion DNA fragment using the primer which this invention provides is shown in FIG. This embodiment is a primer capable of producing one DNA fragment (fusion DNA fragment) from n target DNA fragments (n is a positive integer of 2 or more and 10 or less) by PCR (Polymerase chain reaction), (1): a pair of primers having sequences complementary to the partial sequences of the 5 ′ end and 3 ′ end of the fusion DNA fragment, and primer (2): 5 ′ of the target DNA fragment located inside the fusion sequence It has a base sequence complementary to the terminal sequence or the partial sequence at the 3 ′ end, and further has a GC content of 50% or more and a length of 9-45 bp on its 5 ′ end side, A fusion DNA fragment characterized by using the primer according to the first to third aspects, preferably n-1 sets of the primers, having a base sequence (fusion sequence) capable of binding. It is a manufacturing method of a piece. Here, the fusion sequences of each set of the primers (2) are complementary to each other. The present invention is a so-called overlap extension called a nucleic acid amplification method using PCR, which can be applied not only to two or three target sequences but also to four or more target sequences. is there.

In FIG. 2, the case where n = 2 is shown in the upper stage. Primer (1) is a sequence complementary to the 5 ′ and 3 ′ ends of the fusion DNA fragment, while primer (2) is a sequence located inside the fusion DNA fragment. The 3 ′ end side of the primer (2) has a sequence complementary to the end of each target DNA fragment, while the 5 ′ end side is a fusion sequence, and the primers (2) -5 ′ and (2) -3. The 'fusion sequences are complementary to each other. PCR with this combination, that is, PCR using the primer (2) (1st PCR) is first performed to obtain a product in which a fusion sequence is added to the target DNA fragment, and the product obtained by this PCR is used as a template for the primer (1) By performing PCR (2nd PCR) using a DNA, a fusion DNA fragment in which a fusion sequence is sandwiched between the target DNA fragment 1 and the target DNA fragment 2 can be produced.
The lower part of FIG. 2 shows a case where n ≧ 3. As a representative example, n = 3 is shown, but the principle is common except for 3. Primer (1) is a sequence complementary to the 5 ′ and 3 ′ ends of the fusion DNA fragment, while two sets of primers (2a) and (2b) are sequences located inside the fusion DNA fragment. The design of the primers is the same as in the case of n = 2, but the important point is that the fusion sequences of each set of primers can be complementaryly bound only to the partner. By performing PCR using this combination, in principle, one kind of PCR product is amplified in the order of the target DNA fragments 1, 2, and 3 from the 5 ′ end side.

The most important of the primers provided by the present invention is the design of the fusion sequence. As shown in the examples below, the fusion sequences need not simply be complementary to each other. The conditions are required to have a base sequence having a guanine (G) and cytosine (C) content of 50% or more and a length of 9-45 bp, preferably 12-24 bp.
In complementary binding of DNA, the binding of GC is more specific than the binding of AT, so it is considered that the specificity is higher, and primers that have been conventionally used for overlap extension However, the condition that the GC content of the fusion sequence of the primer provided by the present invention is 50% or more, preferably 80% or more, and more preferably 100% can be one measure. As shown in Example 1 below, when the GC content of the fusion sequence is 20-25%, good results cannot be obtained, and 50% or more is preferable.
In addition, it is important that the fusion sequence is a base sequence that does not contain (GC) m (m is a positive integer of 5-20). As shown in Example 4, with a fusion sequence such as (CG) _15, no PCR product was obtained even though the GC content was 100%. From RNA and other studies, it is known that single-stranded nucleic acids with a certain length have complementary loops when their ends have complementary base sequences to form a loop structure. It is conceivable that the PCR amplification efficiency is remarkably reduced when this occurs during annealing with primers. Therefore, it is important that the fusion sequence does not have (GC) m (where m is a positive integer of 5-20) or that 30% of the 5 ′ end and 3 ′ end are not complementary each other. become.

Preferable examples of the fusion sequence satisfying the above conditions include a sequence in which any one of G and C is connected, preferably 9-30. Furthermore, a sequence in which one or more, preferably 1-5, of A or T are added to one or both ends thereof is also effective as long as the above conditions are satisfied. A sequence in which A or T is inside rather than at the end is also effective as long as the above conditions are satisfied.
As a preferable example of the fusion sequence, as shown in Example 4 below, a sequence in which a base of G or C and a base of A or T are alternately repeated, for example, (GA) m, (GT) m, (CA) m, Examples thereof include sequences represented by (CT) m, (AG) m, (TG) m, (AC) m, and (TC) m (m is a positive integer of 5-20). m is preferably a positive integer in the range of 6-15.
As a fusion sequence, a sequence having a more complicated structure can also be applied as a preferred example of the present invention as long as the above conditions are satisfied. For example, 3G3C3G3C3G, 3C3G3C3G3C, 5C5G5C, 5G5C5G, etc.

Since the fusion sequence enters the interior of the fusion DNA fragment, attention must be paid to the number of bases in the fusion sequence when a protein is synthesized from the fusion DNA fragment. In the case where the fusion DNA fragment is produced for the purpose of gene disruption (Example 5), it is not necessary to pay particular attention to the number of bases. However, in the case of producing an improved gene in which a plurality of domains are combined, etc. Therefore, it is necessary to devise such as designing the fusion sequence to be a multiple of 3 so that no frame shift occurs during translation.
In addition, the longer the fusion sequence, the higher the specificity. However, from the viewpoint of the possibility that the primer takes a secondary structure and the efficiency of PCR itself, it is preferably 9 bp to 45 bp, more preferably 15-24 bp. The length of is good. Examples of the present invention are shown below, but the present invention is not limited to the examples.

  (Yeast, its culture, and DNA extraction) As a yeast strain, BY-series yeast (Non-patent Document 5) was used. The yeast culture method was cultivated using YPD agar medium (1% yeast extract, 2% polypeptone, 2% glucose, 2% agar), and DNA extraction from yeast was performed using Zymolase (Seikagaku Corporation) powder. 3 mg was dissolved in 0.9 ml of SET buffer (1.2 M Sorbitol, 20 mM Tris-HCl, 10 mM EDTA, pH 7.5), and 0.1 ml of β-mercaptoethanol was added to form a DNA extraction solution. It was dissolved in a DNA extraction solution and then in a 10% SDS solution, followed by phenol-chloroform extraction / ethanol precipitation to extract and purify DNA. The extracted DNA was dissolved in TE buffer, and RNA was previously removed by RNase treatment.

(Manufacture of target DNA fragment) A large amount of plasmid pST106 prepared in advance was cleaved with EcoRI and HindIII, electrophoresed on 0.7% agarose gel to cut out a 1.8 kbp band, centrifuged with a filter, and URA3TDH3p fragment Got.
The 1.2 kbp and 1.0 kbp PpHIS3 fragments are from RAK3600, the 2.1 kbp TDH3pTAA fragment is from the p316TDHTAA plasmid, the 1.2 kbp URA3 fragment is from the URA3TDH3p fragment, the 2.4 kbp TDH3pTAAPGK fragment is from the p316TDHTAApkK plasmid, URA3-5 'and 1.3 kbp URA3-3' fragments from BY4704, 0.8 kbp ADE2-5 'and 2.0 kbp ADE2-3' fragments from BY4741, 1.2 kbp URA3 fragment From BY4704, each was amplified by PCR. All PCR reactions (10 μl) were performed according to usage using KOD Plus Polymerase (Toyobo). The reaction conditions for PCR are as shown in Table 1 below. In the subsequent PCR reaction, the annealing temperature is appropriately changed, and this is clearly indicated when changing.

(Overlap extension 1) Overlap extension PCR was carried out by fusing a plurality of target DNA fragments produced above as a template. PCR was carried out in a 10 μl system, and the reaction conditions were basically the same as in Table 1 above, and the optimum conditions were searched by appropriately changing the annealing temperature between 50-68 ° C.
First, the effect of the GC content of the fusion sequence on overlap extension PCR was verified. In order to fuse TDH3pTAA and PpHIS3 among the above target DNA sequences, TDH3-572 primer (SEQ ID NO: 1) and URA3-200c primer (SEQ ID NO: 2) as primer (1), and primer (2) As a primer having a GC content of 5/20 (25%) and a length of 20 bases, that is, a combination of PGK1ter1-20c-TAAc primer (SEQ ID NO: 3) and PGKter1-20-PpHIS3 (SEQ ID NO: 4), or primer (2) A combination of primers having a GC content of the fusion sequence of 7/30 (about 23%) and a length of 30 bases, ie, a PGKter1-30c-TAAc primer (SEQ ID NO: 5) and a PGKter1-30-PpHIS3 primer (SEQ ID NO: 6); Or as primer (2) 3 of a primer having a GC content of 8/40 (20%) and a length of 40 bases, ie, a combination of a PGKter1-40c-TAAc primer (SEQ ID NO: 7) and a PGKter1-40-PpHIS3 primer (SEQ ID NO: 8). PCR was performed using various types of primer sets. The temperature conditions for annealing were four conditions of 50 ° C., 55 ° C., 60 ° C., and 68 ° C. The outline of PCR is shown in FIG.

  In addition to the above fusion sequences, studies were also conducted to increase the GC content. In PCR using the same target sequence and the same conditions as described above, PCR was performed using primers of 15G-TAAc (SEQ ID NO: 9) and 15C-PGKter1 (SEQ ID NO: 10) as the primer (2). The GC content of the fusion sequence was 100% and the annealing temperature was 68 ° C. An outline of the PCR reaction is shown in FIG.

  The results of PCR are shown in FIG. The figure shows an electrophoretogram of the PCR product, lane M shows the molecular weight marker (kb ladder), arrows show the direction of migration, lanes 20, 30, and 40 show the length of the fusion sequence, respectively. Shows the annealing temperature conditions. From this result, when a fusion sequence with a GC content as low as 20-25% was used, the fusion DNA fragment (3.3 kbp) could be obtained in any of 20, 30, 40 under annealing temperature conditions of 60 ° C. or lower. It was shown that it was not amplified. At 60 ° C, a band appeared when the fusion sequence length was 30, 40, but it was not clear, and even at 68 ° C, the amplification efficiency was not good.

  On the other hand, the rightmost lane in FIG. 5 shows the results of PCR using a primer having 15 C fusion sequences. Unlike each lane on the left, a clear single band was confirmed at 3-4 kbp, and this size was consistent with the theoretical size when two target DNA fragments were fused. Despite the fact that the fusion sequence is shorter than the fusion sequence with a low GC content, the overlap extension product is amplified, and using a fusion sequence with a high GC content (GC rich) is a success for the overlap extension. It was shown to be important.

  Since good results were obtained with a fusion primer having a fusion sequence of 15G, the length of the fusion sequence consisting only of G (or C) was examined. Using TDH3pTAAPGK and URA3 sequences as target sequences as templates, TDH3-572 primer (SEQ ID NO: 11) and KanMX75-TDHu1 primer (SEQ ID NO: 12) as primers (1), and the number of consecutive G / Cs as primers (2) A combination of nine 9G-PGKterC primers (SEQ ID NO: 13) and 9C-ASC primer (SEQ ID NO: 14), or 12G-PGKterC primer (SEQ ID NO: 15) and 12C-ASC primer with 12 G / Cs ( Three types of PCR were performed: a combination of SEQ ID NO: 16) or a combination of 15G-PGKterC primer (SEQ ID NO: 17) and 15C-ASC primer (SEQ ID NO: 18) having 15 G / Cs. Three annealing temperatures of 50 ° C., 55 ° C., and 65 ° C. were examined. An outline of the PCR reaction is shown in FIG.

The results of the PCR are shown in FIG. In FIG. 7, the underlined “Example 2” shows the PCR results. The drawings are common to the second, third, and fourth embodiments. In this figure, the PCR product is electrophoresed, lane M represents the molecular weight marker, and the horizontal number represents the molecular weight (kbp). A, B, and C show the results at annealing temperatures of 50 ° C, 55 ° C, and 65 ° C, respectively. The product amplified by PCR appeared as a single band at the position of 3 kbp-4 kbp, which was consistent with the size predicted from the target sequence (3.5 kbp). Lane 9C is a PCR product having 9 G / Cs in the fusion sequence. No amplification was observed at 50 ° C. and 55 ° C., and a product was observed at 65 ° C.
Lane 12C is a PCR product with 12 G / Cs in the fusion sequence. Although the amount of product is small at 50 ° C, amplification is observed at 55 ° C and 65 ° C. It was shown that it was obtained.
Lane 15C is a PCR product having 15 G / Cs in the fusion sequence, and a clear PCR product was confirmed from 50 ° C to 65 ° C. The amount of PCR product (represented by the intensity of fluorescence) was highest at 15C under all temperature conditions. From these results, when the fusion sequence in primer (2) is represented by Gm or Cm (m is a 9-45 positive integer), the number is important for the efficiency of PCR, and m is 9 or more and 30 or less. It has been shown that a primer that is preferably 12 or more and 21 or less, and more preferably 15 or more and 18 or less is suitable.

(Overlap extension 3) Since the fusion sequences represented by C ₉ , C ₁₂ and C ₁₅ showed good results, the efficiency of PCR amplification when T was added to the fusion sequence C ₁₅ was examined next. . As in Example 2, the target sequences were TDH3pTAAPGK and URA3 sequences as templates, TDH3-572 primer (SEQ ID NO: 11) as primer (1) and KanMX75-TDHu1 primer (SEQ ID NO: 12) as primers (2) 15CCT-ASC primer (SEQ ID NO: 19) and 15AGG-PGKterC primer (SEQ ID NO: 19) designed to add (T) between C of (5C-T-4C-T-3C-T-2C-T-CT) 20), or a combination of T-15C-T-ASC primer (SEQ ID NO: 21) and A-15G-A-PGKterC primer (SEQ ID NO: 22) with T added to both ends of 15 consecutive Cs, or 3T-15C-3T-AS with 15 Ts added to both ends of C Primer (SEQ ID NO: 23) and 3A-15G-3A-PGKterC combination of primers (SEQ ID NO: 24) or 3 T, then is added during 15 C _(5C3T) 3 -ASC primer (SEQ ID NO: 25) And (3A5G) ₃ -PGKterC primer (SEQ ID NO: 26) combination, 4 types of PCR were performed. Three annealing temperatures of 50 ° C., 55 ° C., and 65 ° C. were examined.

In FIG. 7, the underlined “Example 3” shows the PCR results. Lane 15C-T in the figure is a PCR product with a fusion sequence of (5C-T-4C-T-3C-T-2C-T-CT). Although amplification is slightly weak at 50 ° C, it is 55 ° C and 65 ° C. The amount of product increased as the annealing temperature increased, and it was shown that the amount of product was as high as 15C at 65 ° C.
Lane T-15C-T is a PCR product with a fusion sequence in which T is added to both ends of 15 consecutive Cs. Like 15C-T, the product amount tends to increase as the annealing temperature increases. At 65 ° C., a product similar to 15C was obtained.
Lanes 3T-15C-3T are PCR products with a fusion sequence in which 3 Ts are added to both ends of 15 consecutive Cs. At this time, the GC content of the fusion sequence was about 70%. The amplification efficiency was better than that of the above two primers at 50 ° C. and 55 ° C., and a product similar to 15C was obtained at 65 ° C.
Lane (5C3T) ₃ is a PCR product of a fusion sequence in which 3 Ts are added after 5 C, and the GC content of the fusion sequence was about 60%. Although amplification efficiency was somewhat poor at 50 ° C., a product similar to 15C was obtained at 55 ° C. and 65 ° C., indicating that this fusion sequence is effective.

From the above results, 5C-T-4C-T-3C-T-2C-T-CT, T-15C-T, 3T-15C-3T, and (5C3T) ₃ sequences are suitable as fusion sequences. It has been shown.

(Overlap extension 4) Even when the GC content of the fusion sequence was about 60%, the overlap extension showed good results. Next, the case where different base species were repeated in the fusion sequence was examined. As in Example 2, the target sequence was TDH3pTAAPGK and the URA3 sequence as a template, TDH3-572 primer (SEQ ID NO: 11) and KanMX75-TDHu1 primer (SEQ ID NO: 12) as primer (1), CG as primer (2) and CG Was repeated 15 times (CG) _15- ASC primer (SEQ ID NO: 27) and (GC) _15- PGKterC primer (SEQ ID NO: 28) combination, or primer (2), CT was repeated 15 times (CT) Combination of _15- ASC primer (SEQ ID NO: 29) and (AG) _15- PGKterC primer (SEQ ID NO: 30), or CT repeated 10 times as primer (2) (CT) _10- ASC primer (SEQ ID NO: 31) and _(AG) 10 -PGKterC Reimer (SEQ ID NO: 32) the combination of or a combination of the primers CA is Repeat 10 times as (2) _(CA) 10 -ASC primer (SEQ ID NO: 33) and _(TG) 10 -PGKterC primer (SEQ ID NO: 34), the Each was examined. As primer (2), 15A-ASC primer (SEQ ID NO: 35) and 15T-PGKterC primer (SEQ ID NO: 36) in which A was repeated 15 times were examined in the same manner. Three annealing temperatures of 50 ° C., 55 ° C., and 65 ° C. were examined.

In FIG. 7, the underlined “Example 4” shows the result of the PCR. Lane (CG) _{15 in the} figure shows the result of the fusion sequence in which CG was repeated 15 times. Although this fusion sequence had a GC content of 100%, no PCR product was observed under all temperature conditions. This was thought to be due to the fact that the ends of the fusion sequences were complementarily joined during PCR annealing to form a loop-like structure, and the efficiency of the PCR reaction was extremely reduced. This result showed that it is important for the overlap extension that the fusion sequence is not represented by (GC) m or (CG) m (m is a positive integer of 5-20).
Lane (CT) ₁₅ shows the result of the fusion sequence in which CT was repeated 15 times. Unlike (CG) ₁₅ , the PCR results with this fusion sequence were good, especially at 55 ° C. and 65 ° C., indicating high amplification efficiency. In (CT) ₁₅ , since the ends of the fusion sequence are not complementary, it can be said that the result supports the above prediction.
Lane (CT) ₁₀ shows the result of the fusion sequence in which CT was repeated 10 times. Although amplification efficiency was slightly lower at 50 ° C. than (CT) ₁₅ , good results were obtained at 55 ° C. and 65 ° C., indicating that this fusion sequence was suitable.
Lane (CA) ₁₀ shows the result of the fusion sequence in which CA was repeated 10 times. Good amplification was seen under all temperature conditions, indicating that this fusion sequence is suitable.
Lane 15A shows the result with a fusion sequence of 15 consecutive As. Unlike the 15C results, no PCR product was observed with this fusion sequence under any temperature conditions. This reiterated the importance of GC content in the fusion sequence.

  From the above results, it was shown that (CT) m and (CA) m (m is a positive integer of 5-20, preferably 10-15) are suitable as the repeated pattern in the fusion sequence.

(Overlap Extension Using Three Target DNA Fragments-Gene Disruption) Since the overlap extension using two target DNA fragments showed good results, the target DNA fragment was examined for three overlap extensions. The method of fusing three target DNA fragments can be used for “gene disruption” in which a specific sequence is inserted between the 5 ′ terminal region and the 3 ′ terminal region of the target gene to destroy the function of the gene. Therefore, the fusion DNA fragment was produced and verified using a yeast system.

As a target gene for gene disruption, an ADE2 (Phosphoribolaminomidazole carboxyylase) gene was used, and an overlap extension was attempted in which a PpHIS3 fragment was inserted 379 bases downstream from the start codon of the ADE2 gene.
The PpHIS3 gene fragment was amplified from the genomic DNA of the yeast RAK3600 strain, and the ADE2-5 fragment and the ADE2-3 fragment were each amplified by PCR from the yeast BY4741 strain in order to disrupt the ADE2 gene. Using these three target sequences as templates, ADE2-797 primer (SEQ ID NO: 37) and ADE2 + 2392c primer (SEQ ID NO: 38) as primer (1), 15C-PpHIS3-1 primer (SEQ ID NO: 39) as primer (2) and Overlap extension was performed using a combination of 15G-ADE2-1c primer (SEQ ID NO: 40) and a combination of 5C5G5C-ADE2 + 379 primer (SEQ ID NO: 41) and 5G5C5G-PpHIS3c primer (SEQ ID NO: 42). The outline of PCR is shown in FIG. The PCR product was confirmed by 0.7% agarose gel electrophoresis, and the result is shown in FIG. The figure is an electrophoretic image of a PCR product, lane M represents a molecular weight marker (kbp ladder), and lane 2 represents an overlap extension PCR product. As shown in the figure, a single band was observed in the vicinity of 4 kbp in lane 2, which agreed well with the predicted value of the overlap extension (3.8 kbp).
Using 50 ng of the obtained PCR product, yeast BY4741 strain was transformed by the lithium acetate method. 120 μl of 60% PEG 3350, 10 μl of carrier DNA (10 mg / ml), 5 μl of 4M lithium acetate, 65 μl of BY4741 strain culture solution and 50 ng of PCR product were mixed, and heat shock at 42 ° C. was added to the yeast cells. PCR product was introduced. The transformed cells were seeded in a histidine-deficient medium, and the appearing colonies were observed. Since the ADE gene-disrupted strain showed a red color on this medium, the appeared red colonies were counted as ADE2-disrupted strains. The ratio of the gene-disrupted strain to the whole was 28.9% (red colony 488 / red colony + white colony 1688).

  (Overlap extension using 4 target sequences) Since the overlap extension using 3 target sequences showed good results, fusion of 4 target sequences was examined. ADE2-5 (0.8 kbp), PpHIS3 (1.0 kbp), ADE2-3 (2.0 kbp), URA3 (1.2 kbp) were used as target sequences, and ADE2-797 primer (SEQ ID NO: 43) was used as primer (1). ) And URA3-200c primer (SEQ ID NO: 44) as a combination of 5G5C5G-PpHIS3 primer (SEQ ID NO: 45) and 5C5G5C-ADE2-1c primer (SEQ ID NO: 46) as primer (2), and 3C3G3C3G3C-ADE2 + 379 primer (SEQ ID NO: 47) and 3G3C3G3C3G-PpHIS3c primer (SEQ ID NO: 48), and 15C-URA3-223 primer (SEQ ID NO: 49) and 15G-ADE2-1c primer (SEQ ID NO: 50). The used, PCR was performed at an annealing temperature of 68 ° C.. FIG. 10 shows an outline of the PCR of Example 6.

  The results of PCR are shown in FIG. The figure shows the results of agarose gel electrophoresis of the PCR product, lane M represents the molecular weight marker (kbp ladder), and lane 1 represents the PCR product. As shown in the figure, a band was observed around 5.0 kbp, which was consistent with the molecular weight estimated from the four target DNA fragments. It was shown that the overlap extension of the present invention can be effectively used even when the number of target sequences is four.

An outline of the technology of the present invention will be described. The schematic diagram of the manufacturing method of the fusion DNA fragment using the primer which this invention provides is shown. The outline | summary of the overlap extension of Example 1 (first half) is shown. The outline | summary of the overlap extension of Example 1 (latter half) is shown. The electrophoresis image of the PCR product of Example 1 is shown. Example 2 An outline of the overlap extension of Example 4 is shown. Example 2 shows an electrophoresis image of the PCR product of Example 4. The outline | summary of the overlap extension of Example 5 is shown. The electrophoresis image of the PCR product of Example 5 is shown. The outline | summary of the overlap extension of Example 6 is shown. The electrophoresis image of the PCR product of Example 6 is shown.

Claims (4)

A set of primers comprising the following two regions (a) and (b), which are used to bind two types of target DNA fragments.
(A) 3 'terminal region having homology with the partial sequence at the end of the target DNA fragment (b) containing 50% or more of guanine (G) and cytosine (C), with a total length of 9-45 bp, 5 'terminal region (hereinafter referred to as "fusion sequence") capable of complementary binding of target DNA fragments   The primer according to claim 1, wherein the GC content of the fusion sequence is 80% or more.   The primer according to claim 2, wherein the fusion sequence does not have continuous (GC) m (m is an integer of 5 or more and 20 or less). A method for producing a fusion DNA fragment containing n target DNA fragments (n is an integer of 2 or more and 10 or less) by PCR using the following primers (1) and (2).
(1) Primer for amplifying the full length of the fusion DNA fragment (2) Primer according to any one of claims 1 to 3