JP6418800B2 - Method for determining the position of introduction of foreign DNA into the genome - Google Patents

Description

  The present invention relates to a method for determining the insertion position of DNA inserted into genomic DNA.

  A method for determining the position of DNA introduced into a recombinant animal, a recombinant plant or the like has been conventionally performed by the TAIL (Thermal asymmetric interlaced) -PCR method (Non-patent Document 1). The TAIL-PCR method is a method of isolating an unknown region adjacent to a specific primer by performing nested PCR using a genomic cDNA as a template and a specific primer and an arbitrary primer. According to the TAIL-PCR method, the genomic DNA sequence adjacent to the inserted DNA fragment is amplified by PCR amplification using a primer specific to the DNA inserted into the genomic DNA and an arbitrary primer having a random sequence. Can be confirmed.

  If the DNA to be inserted (for example, an exogenous gene) is inserted into genomic DNA while maintaining the structure as originally designed, the insertion position can be determined by the TAIL-PCR method. it can. However, in many cases, the inserted DNA is not necessarily inserted while maintaining the structure as designed. For example, if the inserted gene is introduced by a method such as the Agrobacterium method or gene-gun used to create a recombinant plant, either end of the inserted gene is greatly reduced. There are many cases that are missing. When inserted into the genome in a state different from the designed state, it was often difficult to accurately determine the insertion position by the TAIL-PCR method.

When creating a transgenic animal or plant, it is strictly necessary to determine where the inserted DNA is inserted in the genome and whether it will have an adverse effect on the genetic structure and function of the host animal or plant. Need to check.
Therefore, a further improved method for accurately confirming the position and structure of DNA inserted into the genome has been desired.

Liu et al., Genomics, 25, 674-681 1995

  In view of the above circumstances, the present invention provides a method for determining the insertion position of exogenous DNA inserted into genomic DNA.

  In order to determine the position on the genome of the DNA introduced into the genomic DNA of the plant, the present inventors have introduced the genome of the plant into which the DNA has been introduced (that is, a plant genome containing exogenous DNA at any position). The entire sequence data is obtained, and the sequence of the sequence group is compared with the entire genome sequence of the plant already known to determine the position on the genome of the DNA fragment not retaining the designed structure. In particular, the present invention has been completed.

That is, this invention is the following (1)-(4).
(1) A method for determining an insertion position of an exogenous DNA on genomic DNA into which the exogenous DNA has been inserted, including the following steps (a) to (c).
(A) determining a sequence from both ends of each fragment of the fragmented total genomic DNA;
(B) extracting a sequence unique to the foreign DNA from the sequence determined in step (a),
(C) extracting a sequence at the other end paired with each sequence included in the sequence group extracted in step (b);
(D) The sequence group extracted in step (c) was mapped to a foreign DNA sequence that was different from the “sequence unique to the foreign DNA” in (b) above, and was not mapped. Extracting the sequence;
(E) Performing a homology search for the sequence group extracted in step (d) with respect to the genomic DNA sequence, and specifying a position on the genomic DNA sequence having homology with the sequence of the extracted sequence group (2) The method according to (1) above, wherein the foreign DNA-specific sequence is a vector-derived sequence.
(3) The method according to (1) or (2) above, further comprising confirming the insertion position of the identified exogenous DNA by a PCR method.
(4) A method for determining the structure of the inserted foreign DNA based on the results obtained by the PCR method described in (3) above.

  According to the present invention, it is possible to accurately determine the position of exogenous DNA introduced into the genome. As a result, it is possible to confirm the safety of foods and vaccine preparations obtained from transgenic crops and animals. Furthermore, it becomes possible to identify a plant or animal into which a specific foreign DNA fragment has been introduced from a group of plants or animals into which the DNA fragment has not been introduced.

The result of confirming the introduction of CTB gene is shown. It is the result of analyzing the integration state of the donor nucleic acid by PCR (A) and Southern blot (B). It is a schematic diagram which shows the position of seven arrangement | sequences of LB-1 to LB-3 and RB-1 to RB-4. It is the figure which showed the procedure which confirms the position of inserted DNA. The result of having analyzed about the rice 3 chromosome introduction | transduction site | part of the donor nucleic acid in a recombinant rice (# 51A) is shown. The result of having analyzed about the rice 12 chromosome introduction | transduction site | part of the donor nucleic acid in a recombinant rice (# 51A) is shown. Recombinant rice (# 51A) The results of reconfirming the insertion of donor nucleic acid into chromosome 3 (A) and chromosome 12 (B) by PCR are shown. Recombinant rice (# 51A) The difference between the sequence introduced into chromosome 3 and the design structure is schematically shown. Recombinant rice (# 51A) schematically shows the difference between the sequence introduced into chromosome 12 and the structural structure. The confirmation result of the insertion position by TAIL-PCR method is shown. A is the result of confirming the PCR product by agarose gel electrophoresis. The three lanes of LB side TAILR3 show the results of amplification of the TAILR3 primer and Random1, Random2 and Random3 as a pair, and the lane of RB side TAILR3 has the TAILF3 primer and Random1, Random2 and Random3 primers as a pair. Amplified results are shown. The band indicated by the arrowhead is an amplified fragment containing an adjacent region between LB and chromosome 3, and an amplified fragment containing an adjacent region between RB and chromosome 3. Note that the band containing the chromosome 12 region was not amplified.

One embodiment of the present invention is a method for determining the insertion position of an exogenous DNA on genomic DNA into which the exogenous DNA has been inserted, including the following steps (a) to (c).
(A) determining a sequence from both ends of each fragment of the fragmented total genomic DNA;
(B) extracting a sequence unique to the foreign DNA from the sequence determined in step (a),
(C) extracting a sequence at the other end paired with each sequence included in the sequence group extracted in step (b);
(D) The sequence group extracted in step (c) was mapped to a foreign DNA sequence that was different from the “sequence unique to the foreign DNA” in (b) above, and was not mapped. Extracting the sequence;
(E) A step of performing homology search for the sequence group extracted in step (d) with respect to the genomic DNA sequence, and specifying a position on the genomic DNA sequence having homology with the sequence of the extracted sequence group

The present invention relates to a method for accurately confirming the position of foreign DNA inserted into genomic DNA of an arbitrary organism on genomic DNA. Here, the arbitrary organism is not particularly limited, but it is particularly preferable and applicable to, for example, higher eukaryotes such as animals and plants.
The exogenous DNA is not particularly limited, and DNA derived from an organism different from the host organism (that is, the organism into which the exogenous DNA has been inserted) or artificially prepared DNA is desirable. Fragmentation of genomic DNA into which exogenous DNA has been inserted can be performed based on techniques well known to those skilled in the art. Examples of the implementation method include a method of fragmenting with an appropriate restriction enzyme, a method of fragmenting using a physical method (for example, a method of ultrasonicating with an ultrasonic crusher), and the like. it can. The length of the fragmented DNA is, for example, about 100 to 2,000 bp, or 200 to 1,000 bp, preferably about 300 to 500 bp.
The method for determining the sequence from both ends of each fragmented DNA fragment may be any method, and can be easily selected by those skilled in the art. For example, adapter DNA containing a sequence to which a primer for sequencing is bound may be bound to both ends of each fragment, and sequencing may be performed using the adapter portion. Thus, by clarifying the sequences at both ends of each DNA fragment, the position of each fragment on the genome can be accurately identified. In addition, when determining the sequence by mixing genomes from multiple individuals, by including a sequence unique to each individual in the adapter portion, it is possible to determine which individual the sequenced fragment is derived from. It becomes possible to identify.
Sequencing from both ends of each fragment is performed by, for example, clustering each DNA fragment two-dimensionally on a sample plate called a flow cell (see also examples described later). The sequence of each fragment contained in may be determined from both ends. For example, each DNA fragment is clustered by binding oligo DNA complementary to the adapter sequence, immobilizing each DNA fragment on the sample plate via the DNA, and then amplifying each DNA fragment on the sample plate. Thus, a cluster composed of DNAs having the same sequence can be formed. In this case, as the DNA fragment amplification method, for example, solid phase amplification method (or bridge amplification method: see Illumina Corporation manual, etc .; http://www.illuminakk.co.jp/document/pdf/ techspotlight_sequencing-J.pdf) or the like. In addition, each DNA fragment is identified together with the adapter information using the position information of the cluster formed from the same DNA fragment (determined by the distance between the X and Y axes set on the sample plate). can do.
In order to determine the sequence from both ends of each DNA fragment, it is necessary to determine the sequence of a large amount of DNA fragments. For example, it is desirable to carry out by a technique such as next-generation sequencing technology. For example, sequencing by next-generation sequencing technology can be performed by requesting an analysis company such as Illumina, Inc.

  Next, from the sequence data obtained as a result of sequencing (sequence data determined from both ends of each DNA fragment), a sequence including a sequence unique to the foreign DNA is extracted. Here, the “sequence unique to exogenous DNA” is a sequence that does not exist in the genome of the host organism and is included in the exogenous DNA, and preferably is inserted into the exogenous DNA. A sequence near the both ends of DNA (for example, a sequence existing in a region of about 0 to 1,000 bases and about 0 to 500 bases from either end), and a sequence existing in the vicinity of the adjacent region of genomic DNA is preferable. As such a sequence, for example, when a foreign gene or the like is inserted, the sequence derived from the foreign DNA itself (for example, the cholera toxin gene itself when the cholera toxin gene is inserted) Or a sequence derived from a vector used to insert a foreign gene or the like (for example, LB-1 to LB-3, RB-1 to RB-4 shown in Examples). May be. A plurality of sequences may be used as the “sequence unique to exogenous DNA” (for example, LB-1 to LB-3, RB-1 to RB-4 shown in Examples).

  After extracting sequences peculiar to exogenous DNA, it is related to the other end paired with these sequences (the other end paired with the end of each DNA fragment that contains a sequence peculiar to exogenous DNA) Extract sequence information. Next, a sequence including a sequence unique to genomic DNA is extracted from the obtained sequence (sequence information at the other end). In order to extract a sequence peculiar to genomic DNA, for example, from the obtained sequence (the sequence of the other end paired with the end including the sequence peculiar to the exogenous DNA), the sequence peculiar to the exogenous DNA Map the sequence of the foreign DNA different from the “sequence” as a reference sequence (paste or align to a sequence portion highly homologous to the sequence of the foreign DNA different from the “sequence unique to the foreign DNA”) . The sequence group that has not been mapped can be determined to contain a sequence peculiar to genomic DNA, or it can be determined that the sequence group includes a sequence peculiar to genomic DNA. The mapping can be performed using an alignment tool such as BWA (Burrows-Wheeler Aligner).

A sequence group including sequences unique to genomic DNA extracted as described above is subjected to homology search against the genome sequence database of the host organism. As a homology search method, for example, BLAST search can be easily performed (for example, NCBI BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi, etc.). High homology with the extracted sequence group (high homology means, for example, that the compared sequences have 90-100% or more, preferably 95% or more, more preferably 98% or more identity) ) By confirming the position of the genomic DNA sequence, the position where the foreign DNA is inserted can be identified. That is, the sequence portion containing the sequence unique to the genomic DNA extracted in [0014] is a part of the genomic DNA fragment containing the sequence unique to the foreign DNA at the other end. Considering that the length of the genomic fragment is about several hundred bp, it is determined that the vicinity of the sequence unique to the genomic DNA extracted in [0014] is the position on the genome where the foreign DNA is inserted. be able to.
As for the insertion position on the genome of the foreign DNA identified as described above, create a primer pair that sandwiches the estimated insertion position, amplify the part, and sequence the amplified DNA Then, the insertion position may be confirmed.

After the position on the genomic DNA in which the foreign DNA is inserted is identified by the method of the present invention, the structure of the inserted foreign DNA is confirmed, or the genomic DNA in which the foreign DNA is inserted is detected. It is possible to confirm whether there is a structural change near the position. Exogenous DNA inserted into the genomic DNA of the host organism may have structural changes such as deletion or transfer without maintaining the DNA structure before insertion. In such a case, since the function of the inserted foreign DNA in the host organism may be affected, it is very important to confirm the structure of the inserted DNA. In addition, depending on where the foreign DNA is inserted in the genome, it is possible to predict the influence on the gene of the host organism (for example, when it is inserted into the exon part, The function of the gene may be lost or destroyed).
The vicinity of the introduction site into the genomic DNA is sequenced, and based on the result, the change in the structure of the inserted foreign DNA and the structure of the genomic DNA into which the foreign DNA has been inserted can be confirmed. The genome structure of the host organism before the foreign DNA is inserted can be easily confirmed by using an available database.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the examples. In this example, using rice as a host, DNA expressing cholera toxin B (CTB: Cholera toxin B) chain was introduced as a donor nucleic acid (CTB-10Li45GB3A region) onto the rice chromosome, and the introduced position was This is confirmed by the method of the invention.

1. 1. Production of transgenic rice expressing cholera toxin B chain 1-1. Construction of RNAi cassette-carrying plasmid pZAAMP-10Li45GB3A
The RNAi cassette-carrying plasmid pZAAMP-10Li45GB3A was prepared through the following 8 steps.
(1) First, pZAAMP (pZ2028AAMP), which incorporates the recognition sequences of restriction enzymes AsiSI, AscI, MluI, and PacI into the gene integration region (MCS) of the binary vector pPZP202, and the restriction enzyme AscI and MluI recognition sequences into the MCS of pUC19. Two plasmids of pUC198AM were prepared (Kuroda et al., Biosci. Biotechnol. Biochem. 74, 2348-2351 2010).
(2) 10 kDa prolamin promoter (P10L), 10 kDa prolamin terminator (T10L), rice aspartic protease gene third intron region (RAPint), 13 kDa prolamin gene 45 bp fragment (45), glutelin B gene 228 bp fragment using rice genomic DNA as a template (GB3) was PCR amplified. Of these, T10L was subcloned into MUC of pUC198AM to prepare pUC198AM-T10L. RAPint was subcloned into pUC19 MCS to produce pUC19-RAPint. The remaining three were subcloned into the pGEM-T-Easy plasmid by the TA cloning method to prepare pGEM-T-Easy-P10L, pGEM-T-Easy-45, and pGEM-T-Easy-GB3.

(3) EcoRI and SpeI recognition sequences present in P10L were modified and deleted by site-directed mutagenesis.
(4) pGEM-T-Easy-P10L subjected to the above modification was double-digested with HindIII and XbaI to excise the P10L fragment and incorporated into MUC of pUC198AM-T10L to prepare pUC198AM-P10LT10L.
(5) Double digestion of pGEM-T-Easy-GB3 with SpeI and XbaI to excise the GB3 fragment and insert it into the XbaI site at the end of the 45 bp fragment of pGEM-T-Easy-45 to create pGEM-T-Easy -45GB3 was produced.
(6) pGEM-T-Easy-45GB3 was double-digested with SpeI and XbaI to cut out a 45GB3 fragment and incorporated into the SpeI site present in pUC19-RAPint to prepare pUC19-RAPint45GB3.
(7) Following the above, pUC19-RAPint45GB3x2 was prepared by incorporating the 45GB3 fragment into the XbaI site present in pUC19-RAPint45GB3.
(8) Double digestion of pUC19-RAPint45GB3x2 with XbaI and SacI, excise the fragment from RAPint45GB3x2 to pUC19-derived SacI, and incorporate pUC198AM-10Li45GB3A between the XbaI and SacI sites present in pUC198AM-P10LT10L Produced.
pUC198AM-10Li45GB3A was double-digested with AscI and MluI, the 10Li45GB3A part was excised together with MCS, and inserted between the AscI and MluI sites present in pZAAMP to complete pZAAMP-10Li45GB3A.

1-2. Construction of plasmid pUC198AM-13110NTL-CTB carrying cholera toxin B (CTB) chain expression cassette 1-2-1. Construction of plasmid pUC198AM-13110NTL
pUC198AM-13110NTL was prepared through the following five steps.
(1) First, plasmid pUC198AM was prepared by incorporating restriction enzyme AscI and MluI recognition sequences into MUC of pUC19 (Kuroda et al., Biosci. Biotechnol. Biochem. 74, 2348-2351 2010). In addition, pGEM-T-Easy plasmid (Promega) or pUC19 was used for subcloning of the PCR fragment.
(2) Using the rice genomic DNA as a template, a total of three types of fragments of 13 kDa prolamin promoter (P131), 10 kDa prolamin signal sequence region (10N), and 13 kDa prolamin terminator (T131L) were PCR amplified. All amplified fragments were subcloned into the pGEM-T-Easy plasmid by the TA cloning method to prepare pGEM-T-Easy-P131, pGEM-T-Easy-10N, and pGEM-T-Easy-T131L.

(3) Double digestion of pGEM-T-Easy-10N with SpeI and XbaI, excise the 10N fragment, and construct pGEM-T-Easy-P13110N by incorporating it into the XbaI site present in pGEM-T-Easy-P131 did.
(4) pGEM-T-Easy-P13110N was double digested with HindIII and XbaI to excise the P13110N fragment and subcloned into MUC of pUC198AM to prepare pUC198AM-13110N.
(5) pUC198AM-13110NTL was completed by double digesting pGEM-T-Easy-T131L with SacI and EcoRI to cut out the T131L fragment and incorporating it between the SacI and EcoRI sites present in pUC198AM-13110N.

1-2-2. Construction of plasmid pUC198AM-13110NTL-CTB carrying CTB expression cassette
pUC198AM-13110NTL-CTB was prepared through the following procedure.
Targeted by double digestion with XbaI and SacI of an artificially synthesized gene synthesized with a base sequence with the XbaI recognition sequence added to the 5 'end and the SacI recognition sequence added to the 3' end of the CTB gene (SEQ ID NO: 1) The CTB gene fragment was excised and inserted between the XbaI and SacI sites present in the plasmid pUC198AM-13110NTL constructed in 1-2-1 above to complete pUC198AM-13110NTL-CTB.

1-3. Preparation of donor nucleic acid introduction plasmid pZAAMP-CTB-10Li45GB3A A donor nucleic acid introduction plasmid pZAAMP-CTB-10Li45GB3A was prepared by the following procedure.
CTB expression cassette-carrying plasmid pUC198AM-13110NTL-CTB is double-digested with AscI and MluI, and the target CTB expression cassette fragment is excised and incorporated into the AscI site present in RNAi cassette-carrying plasmid pZAAMP-10Li45GB3A. -10Li45GB3A was completed.

1-4. Preparation of plasmid pZH2B for introduction of HPT gene 1-4-1. HPT gene transfer plasmid pZH2B
pZH2B was prepared through the following 6 steps.
(1) Total of cauliflower mosaic virus 35S promoter (P35S), hygromycin phosphotransferase N-terminal region (HPTN), C-terminal region (HPTC), nopaline synthase terminator (Tnos) using pLAN101 plasmid or pBI221 plasmid as a template Four types of fragments were PCR amplified. Restriction enzyme sites MluI and SalI are added to both ends of the primer used for amplification of the cauliflower mosaic virus 35S promoter. The primers used to amplify the hygromycin phosphotransferase N-terminal region have restriction enzyme sites XhoI and BspHI, and the primers used to amplify the C-terminal region have restriction enzyme sites NcoI and XbaI added to both ends. . Restriction enzyme sites SpeI and AscI are added to both ends of the primer used for amplification of the nopaline synthase terminator.
All amplified fragments were subcloned into the pGEM-T-Easy plasmid by the TA cloning method to obtain pGEM-T-Easy-P35S, pGEM-T-Easy-HPTN, pGEM-T-Easy-HPTC, pGEM-T-Easy- Tnos was made.

(2) By using site-directed mutagenesis, EcoRI and PstI recognition sequences present in HPTN were modified and deleted to prepare pGEM-T-Easy-mHPTN.
(3) pGEM-T-Easy-P35S is digested with SalI, and the fragment from pGEM-T-Easy-derived SalI site to P35S is excised and incorporated into the XhoI site present in pGEM-T-Easy-mHPTN. -T-Easy-35SmHPTN was prepared. During this process, the excised pGEM-T-Easy-derived SalI site, the SalI site at the end of the P35S fragment, and the XhoI site at the tip of the mHPTN fragment disappear together to produce a 35SmHPTN fragment.
(4) pGEM-T-Easy-Tnos is digested with SpeI, and the fragment from Tnos to the SpeI site derived from pGEM-T-Easy is excised and incorporated into the XbaI site present in pGEM-T-Easy-HPTC. T-Easy-HPTCTnos was prepared. In this process, the excised pGEM-T-Easy-derived SpeI site, the SpeI site at the tip of the Tnos fragment, and the XbaI site at the end of the mHPTC fragment are extinguished to generate an HPTCTnos fragment.
(5) Double digestion of pGEM-T-Easy-35SmHPTN with NcoI and BspHI to excise the fragment from pGEM-T-Easy-derived NcoI site to 35SmHPTN, and to the NcoI site present in pGEM-T-Easy-HPTCTnos Incorporating, pGEM-T-Easy-P35SmHPTTnos was produced. In this process, the cut out BspHI site at the end of the 35SmHPTN fragment and the NcoI site at the end of the HPTCTnos fragment are extinguished to generate a P35SmHPTTnos fragment. In addition, the N-terminal region and C-terminal region of mHPT are ligated to generate a full-length mHPT gene, but the 118th alanine is mutated to valine depending on the base sequences of the BspHI site and NcoI site used for ligation. To do. This portion was presumed to be unrelated to the enzyme activity that degrades hygromycin, and in fact, a hygromycin-resistant trait was conferred on rice.
(6) pZH2B was completed by double digesting pGEM-T-Easy-P35SmHPTTnos with MluI and AscI to excise the P35SmHPTTnos fragment and incorporating it into the AscI site present in pZ2028. In this process, the MluI site at the tip of the excised 35SmHPTTnos fragment and the AscI site of pZ2028 disappear by ligation, while the AscI site at the end of the 35SmHPTTnos fragment remains intact. As a result, the 35SmHPTTnos fragment cannot be excised in pZH2B, and all MCS from pZ2028 is preserved from AscI to PacI.

1-5. Gene introduction into rice Cholera toxin B chain expression recombinant rice (# 51A) was prepared through the following seven steps.
(1) The donor nucleic acid introduction plasmid pZAAMP-CTB-10Li45GB3A and the HPT gene introduction plasmid pZH2B, respectively, 20 ng by applying an electric pulse of 200 Ω / 25 μF / 2.5 kv using a 0.2 cm cuvette, Rhizobium radiobacter EHA101 It was introduced into the strain (electroporation method).
(2) 100 μg / ml spectinomycin and 50 μg / ml kanamycin-containing LB agar medium. Two types of recombinant EHA101 strain (1) are smeared and cultured independently, and cultured at 25 ° C for 2-3 days. Colonies were formed.
(3) Next, Agrobacterium-mediated transformation into rice was performed according to the method of Toki (The Plant Journal. 47,969-76 2006). Nipponbare rice seeds were sterilized with 70% ethanol and sodium hypochlorite solution (effective chlorine concentration 2.5%), placed on N6D medium containing auxin (2,4-dichlorophenoxyacetic acid), and at about 28 ° C in a bright place. Callus was induced and dedifferentiated from the scutellum by culturing for 6 weeks.

(4) The Agrobacterium solution into which pZAAMP-CTB-10Li45GB3A was introduced was suspended in an AAM medium so that the final concentration was OD = 0.05, and the Agrobacterium solution into which pZH2B was introduced was final concentration OD = 0.01. Add 10 μg / ml acetosyringone to the two Agrobacterium mixed bacteria, inoculate the callus for 2 minutes, place it in 2N6AS medium containing 10 μg / ml acetosyringone, and then in the dark at 25 ° C Infection treatment was performed by culturing for 2 days.
(5) Subsequently, the callus was washed about 10 times with 30 ml of sterilized distilled water and then left in a 500 μg / ml carbenicillin solution to sterilize Agrobacterium, and 200 μg / ml carbenicillin / 50 μg / It was placed on N6D medium (containing auxin and cytokinin) containing ml hygromycin and cultured at 28 ° C in a bright place for 2 weeks. Then, placed on MS medium (containing auxin and cytokinin) containing 100 μg / ml carbenicillin and 50 μg / ml hygromycin, and cultured for 4-5 weeks at 28 ° C in the light to select and regenerate the callus. Was trained. Furthermore, it was placed in 1 / 2MS medium (containing selected drug 50 μg / ml hygromycin) without hormones (auxin and cytokinin), and further roots were extended by culturing for 1-2 weeks at 28 ° C in a bright place. .
(6) Redifferentiated plants were selected, and insertion of CTB gene and HPT gene into rice genome was confirmed by PCR.
(7) Transplanted primary seedlings (T0 individuals) in which both CTB and HPT genes have been inserted were transplanted into pots filled with culture soil and grown in a growth chamber (light period 27 ° C, 12 hours, dark period) It was cultivated for about 3 months at 22 ° C for 12 hours.

  Through the above steps, rice (# 51A strain) in which the CTB gene and the HPT gene were introduced into the chromosome was produced. Next, as for the progeny (1st generation to 6th generation) that self-bred, an individual into which the CTB gene was inserted and the HPT gene was not inserted was selected by PCR. As a result, in this recombinant rice (# 51A), introduction of the HPT gene was recognized only in the redifferentiated individuals (T0 individuals), and HPT in the progeny (1st generation to 6th generation) after genetic isolation by self-breeding It has been confirmed that the gene has not been introduced.

2. 2. Analysis of state of integration of donor nucleic acid into chromosomal DNA 2-1. Presence of CTB gene
Confirmation of CTB gene introduction was confirmed by PCR and Southern blot analysis for the entire CTB gene.
The recombinant rice (# 51A) 6th generation young rice leaves were finely chopped, and genomic DNA was extracted using Nucleon PhytoPure (GE Healthcare).
2-1-1. PCR
PCR was carried out using primer sets CTB-F and CTB-R using 25 ng of the obtained recombinant rice (# 51A) genomic DNA as a template. PCR was performed using GeneAmp PCR System 9700 (Applied Biosystems). Amplification was performed at 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds for 35 cycles, and electrophoresed on a 2.0% Agarose gel at 100 V for 25 minutes. The presence or absence of a PCR product was confirmed.
CTB gene detection primer
CTB-F: 5'-ACACCTCAACAGATTACTGA-3 '(SEQ ID NO: 2)
CTB-R: 5'-TCAATTTGCCATACTAATTGCG-3 (SEQ ID NO: 3)
Amplified fragment length: 312bp

2-1-2. Southern blot analysis Genomic Southern hybridization was performed using 20 μg of the obtained recombinant rice (# 51A) genomic DNA. Rice genomic DNA was digested overnight at 37 ° C. with restriction enzymes EcoRI and SacI. The treated genomic DNA was purified by the phenol / chloroform method, electrophoresed on 0.7% Agarose gel for 100 V for 3.5 hours, and allowed to stand overnight using 10 × SSC to transfer the genomic DNA to the nitrocellulose membrane. . After completion of the transcription, the membrane was irradiated with UV using a UV Cross Linker (Tomy Seiko), and the genomic DNA digested with the restriction enzyme was immobilized on the membrane. Next, the detection probe was AP-labeled using AlkPhos Direct Labeling Module (GE Healthcare) and hybridized overnight at 55 ° C. The next day, detection was performed by chemiluminescence using a CDP-Star detection reagent (GE Healthcare). ImageQuant LAS 4000mini (GE Healthcare) system was used to capture an image with an exposure time of 1 hour.
Probe preparation primer for CTB gene detection
CTB-F: 5'-ACACCTCAACAGATTACTGA-3 '(SEQ ID NO: 2)
CTB-R: 5'-TCAATTTGCCATACTAATTGCG-3 (SEQ ID NO: 3)
Probe length: 312bp
The results of PCR and Southern blot analysis are shown in FIG.

As can be seen from FIG. 1A, the CTB gene was inserted into the genome of this recombinant rice (# 51A), so PCR was performed using the 6th generation genomic DNA as a template, and the gene ( A band visible at the position of the arrow in FIG. 1 (312 bp) was amplified. On the other hand, Nipponbare, a non-recombinant rice plant, did not have a CTB gene, so that gene was not detected. From this result, it was confirmed that the CTB gene was introduced into the rice genome in the sixth generation of recombinant rice (# 51A) corresponding to the seed bank.
In addition, as shown in Fig. 1B, this recombinant rice (# 51A) has CTB gene inserted into its genome. Therefore, when Southern blot analysis using 6th generation genomic DNA was performed, Three bands visible at the position of the arrow were detected by the probe. On the other hand, Nipponbare, a non-recombinant rice plant, did not have a CTB gene, so no band was detected by the CTB gene detection probe. From this result, it was confirmed that 3 copies of the CTB gene were introduced into the rice genome in the sixth generation of recombinant rice (# 51A) corresponding to the seed bank.

3. Confirmation of chromosome introduction site 3-1. Acquisition of Sequence Data Genomic DNA was extracted using Nucleon PhytoPure (GE Healthcare) by finely chopping 2 g of 6th generation rice young leaves of this recombinant rice (# 51A). The extracted genomic DNA (about 1.0 μg) was fragmented with an ultrasonic crusher (Covaris). Both ends of the cleaved DNA were smoothed and phosphorylated, and the 3 'dA overhang and the index adapter were connected. A DNA fragment of 250-500 bp (excluding the index adapter sequence) was selected by agarose gel electrophoresis, and PCR amplification (about 10 cycles) was performed to prepare a sequence library.
In order to perform sequencing with Illumina's next-generation sequencer (HiSeq2000 Sequencer; Illumina), the cBot clustering system (Illumina) is used to index DNA fragments with index adapters on a slide glass called a flow cell. The adapter sequence was immobilized via a complementary DNA fragment to form a cluster which is a group of DNA fragments having the same sequence. DNA sequencing was performed using the Illumina HisEsq2000 system (Illumina). The pair-read (two sequence information read from the index adapter sequences at both ends) obtained as a result of sequencing was obtained using CASAVA software (v1.13.48; Illumina).

3-2. Identification of introduction site
In order to clarify the insertion position of the DNA fragment, first, a DNA fragment containing both the genomic sequence and the sequence unique to the inserted DNA was identified. Seven sequences of LB-1 to LB-3 and RB-1 to RB-4 located in the vicinity of the following LB and RB regions existing in the T-DNA vector designed for gene transfer are inserted DNA. Used as a unique sequence for the fragment (FIG. 2).
Near LB
LB-1 (90 bases)
ACAAATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTGCTCTAGCATTCGCCATTCA (SEQ ID NO: 4)
LB-2 (90 bases)
GGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAA (SEQ ID NO: 5)
LB-3 (89 bases)
GTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTGGCGATCGCTTTGGCGCGCCAAGCTTTT (SEQ ID NO: 6)

Near RB
RB-1 (80 bases)
GAATTCACGCGTTTTAATTAACCAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATT (SEQ ID NO: 7)
RB-2 (80 bases)
CCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTT (SEQ ID NO: 8)
RB-3 (80 bases)
GCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCG (SEQ ID NO: 9)
RB-4 (67 bases)
GTTTGCGTATTGGAGCTTGAGCTTGGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAACTATCAGTG (SEQ ID NO: 10)

The sequence information (read) of a base sequence of about 100 bp was obtained from both ends of all the fragments obtained by fragmenting the genome containing the inserted DNA fragment by sequencing. Reads containing the above 7 types (LB-1 to RB-4) of the fragment sequences in either end sequence were extracted from the sequence data. Further, based on the index adapter sequence information of all the extracted leads, the lead at the opposite end to which the index adapter paired with the index adapter is coupled was extracted (FIG. 3).
Mapping the extracted sequence information (read) group using the inserted sequences other than the above LB-1 to LB-3 and RB-1 to RB-4 as a reference (paste a sequence highly homologous to the reference to that part) Then, the sequence group which is not mapped was obtained. A sequence group that has not been mapped is likely to contain a non-reference or genomic sequence. When this unmapped sequence group is subjected to a rice genome BLAST search (Oryza sativa (rice) Nucleotide BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=OGP__4530__9512) Of these, it was found that these sequences were highly homologous at specific positions on chromosomes 3 and 12 (around 24,697, 53 bp of rice chromosome AP008209 and 7,164,931 bp of rice chromosome NC_008405). .

PCR using this recombinant rice genome as a template, primers designed to sandwich the presumed place of introduction of chromosomes 3 and 12 and the primer set shown in the table below designed in the highly specific part of the inserted sequence Then, the amplified product was subjected to DNA gel extraction and then cloned into TOPO TA Cloning Vector (Invitrogen). The positions of the following primers are shown in FIG.
Subsequently, the sequence was confirmed, and the introduction site into the genome was confirmed. Whether or not the presumed place of introduction was included in the obtained sequence was determined by searching with NCBI BLAST BLAST Assembled RefSeq Genomes (Oryza sativa) or Basic BLAST (nucleotide blast).
The insertion position into chromosome 3 AP008209 and chromosome 12 NC_008405 was confirmed by searching with NCBI BLAST BLAST Assembled RefSeq Genomes (Oryza sativa).

It was confirmed by searching with NCBI BLAST whether or not a gene having a function in the vicinity of the insertion position was destroyed as the donor nucleic acid was inserted.
FIG. 4 shows the results of analyzing the chromosome 3 introduction site of the donor nucleic acid in this recombinant rice (# 51A). As a result of confirming the sequence of introduction into the genome, it was revealed that in this recombinant rice (# 51A), the introduced donor nucleic acid was inserted into 24,697,531 of rice chromosome 3 AP008209. As the donor nucleic acid is inserted, the genomic DNA 18bp (24,697,513-24,697,530: GATGTCAAGCTCCTCTAT) (SEQ ID NO: 11) of rice chromosome 3 AP008209 is deleted. According to the latest genome information registered on September 19, 2012, the 3 ′ end of the gene Os03g0627500 (24,685,816-24,694,363) registered upstream of the insertion site is 3,168 bp away from the insertion site 24,697,531, As you can see, it is not inserted between exons of Os03g0627500. Note that Os03g0627500 is registered as a hypothetical protein and is not a translation region for which an apparent function has been identified. Further, since the insertion site is located downstream of Os03g0627500, it is not involved in the promoter region and translation start point of Os03g0627500. In addition, the 5 'end of the gene Os03g0628800 (24,749,451-24,757,823) registered downstream of the insertion site is 51,920 bp away from the insertion site 24,697,531, and the vicinity of the insertion site is unlikely to be the promoter region of Os03g0628800. Thus, the insertion site is not inserted between exons of Os03g0628800. Note that Os03g0628800 is registered as a hypothetical protein and is not a translation region for which an apparent function has been identified. Moreover, since the translation start point of Os03g0628800 is 24,749,575-24,749,577 of rice chromosome AP008209, the insertion site is not involved in the translation start point of Os03g0628800.
From the above, when the genes registered in the vicinity of the insertion site were examined, the functional gene was not destroyed by the insertion of the donor nucleic acid.

FIG. 5 shows the results of analysis of the rice chromosomal introduction site of the donor nucleic acid in this recombinant rice (# 51A).
As a result of confirming the sequence of introduction into the genome, it was revealed that in this recombinant rice (# 51A), the introduced donor nucleic acid was inserted into 7,164,931 of rice chromosome 12 NC_008405. As the donor nucleic acid was inserted, genomic DNA 150bp (7,164,932-7,165,081: CCCACTTAGCTAGCTTGCAATTGAGATCTTGCAGAGAATTGTGAAGATCTAGAGCTATAGCCATGGCTCTGCAAGTGCAAGGCCTTGTGGACTGGAGGTGAAGACCGGTTGATCCTGTGAGGCATGCTGGT
The gene information of Os12g231000 (7,164,860-7,170,071) registered in the insertion site was updated in 2008 by The Rice Annotation Database Project and registered in Genbank on June 8, 2010 (NM_001072977.1 ). As of October 29, 2013, the literature that elucidated the function and regulation of this gene in rice is not relevant. From the gene sequence homology search, the protein encoded by Os12g231000 contains a domain corresponding to the Peptide transporter (PTR) 2 family. In Arabidopsis thaliana, the expression and function of the protein AtPTR2 belonging to the same family has been studied, and gene expression is observed in a wide range of plant tissues, and it is known to function to transport many types of dipeptides into and out of the cell membrane.
As can be seen from FIG. 5, the donor nucleic acid does not divide the translated region of the Os12g231000 gene in chromosome 12, but is inserted into the promoter region. When the insertion site was confirmed by FASTA analysis, the introduced sequence was located in the upstream region of the promoter region, and was assumed to be a site that hardly affects transcription complex formation.

3-3. Confirmation by PCR In order to reconfirm the introduction of the donor nucleic acid into chromosomes 3 and 12 of this recombinant rice (# 51A), a primer specific to the introduction site of each chromosome was designed. Using the obtained recombinant rice (# 51A) genomic DNA (25 ng) as a template, PCR was performed using primer set ch3 51A-F1 · ch3 51A-R1 and primer set ch12 51A-F1 · ch12 51A-R1. PCR was performed using GeneAmp PCR System 9700 (Applied Biosystems), amplification was carried out at 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute for 35 cycles, and electrophoresed on a 2.0% Agarose gel at 100 V for 25 minutes. The presence or absence of a PCR product was confirmed.
Primer for confirming the introduction of chromosome 3
ch3 51A-F1: 5'-GGCCAGCTGCACAACCCTCA-3 '(SEQ ID NO: 13)
ch3 51A-R1: 5'-TCCAGGCACTTCCCTGCTGT-3 '(SEQ ID NO: 14)
Amplified fragment length: 945 bp
Ch3 51A-F1 and ch3 51A-R1 are primers designed as complementary strands of rice genomic DNA sequences.
Primer for confirming chromosome 12 introduction
ch12 51A-F1: 5'- GGAGTCAAGACTGCGACACA-3 '(SEQ ID NO: 15)
ch12 51A-R1: 5'-CATTGTGCCATGCAGGTAGC-3 '(SEQ ID NO: 16)
Amplified fragment length: 900bp
Ch12 51A-F1 and ch12 51A-R1 are primers designed as complementary strands of rice genomic DNA sequences.

In addition, the following primers were also used.
131out2: 5'-CTTTCTTTTGAACTGAACGGG-3 '(SEQ ID NO: 17)
10TLseq5B: 5'-AGTGGGTAGTGGTTACTAGT-3 '(SEQ ID NO: 18)
RAP-R: 5'-TGTCAATGCATCCAAGTAAAC-3 '(SEQ ID NO: 19)
131out2 is a primer designed as a complementary strand of a sequence of 1118 to 1138 bp on the 13 kDa prolamin promoter (1256 bp) of the CTB expression cassette.
10TLseq5B is a primer designed with a sequence of 532 to 552 bp on the 10 kDa prolamin terminator (643 bp) of the RNAi cassette.
RAP-R is a primer designed as a complementary strand of the 898 to 918 bp sequence on the RAP intron (995 bp) of the RNAi cassette.

The results of PCR are shown in FIG. 6A.
As can be seen from FIG. 6A, in the PCR using the combination of primers ch3 51A-F1 and ch3 51A-R1 for confirming the introduction of chromosome 3, an expected 945 bp band was amplified when Nipponbare of non-recombinant rice was used as a template. In contrast, the target band is not amplified in this recombinant rice (# 51A). This was thought to be due to the increase in amplification length from 945 bp due to the insertion of donor nucleic acid into rice chromosome 3 AP008209 · 24,697,531 bp.
On the other hand, in the PCR combining the chromosome 3 confirmation primer ch3 51A-F1 · 131out2 or 10TLseq5B · ch3 51A-R1, the non-recombinant rice Nipponbare does not have a donor nucleic acid and thus is not amplified. In contrast, in this recombinant rice (# 51A), bands corresponding to sizes of 560 bp and 1201 bp calculated from the introduction site of chromosome 3 of the donor nucleic acid confirmed by TAIL-PCR in any combination of primers. I was able to detect it.
Therefore, it was confirmed that the donor nucleic acid was introduced into the rice chromosome 3 AP008209 · 24,697,531 in the sixth generation of this recombinant rice (# 51A).

In addition, as can be seen from FIG. 6B, in the PCR using the combination of primers ch12 51A-F1 and ch12 51A-R1 for confirming the introduction of chromosome 12, when using Nipponbare of non-recombinant rice as a template, an expected 900 bp band is obtained. In contrast, the target band was not amplified in this recombinant rice (# 51A). This was thought to be due to the increase in the amplification length from 900 bp due to the insertion of the donor nucleic acid into the chromosome 12 of rice, NC_008405, 7,164,931 bp.
On the other hand, in the combination PCR with the chromosome 12 confirmation primer ch12 51A-F1 · 131out2 or RAP-R · ch12 51A-R1, the non-recombinant rice Nipponbare does not have a donor nucleic acid and thus is not amplified. In contrast, in this recombinant rice (# 51A), in any primer combination, bands corresponding to sizes of 339 bp and 757 bp calculated from the chromosome 12 introduction site of the donor nucleic acid confirmed by TAIL-PCR were used. I was able to detect it.
Therefore, it was confirmed that the donor nucleic acid was introduced into the chromosome 12 NC_008405 · 7,164,931 in the sixth generation of this recombinant rice (# 51A).

4). Confirmation of chromosomal transfer sequence (confirmation of PCR product sequence)
The full length nucleotide sequence of the donor nucleic acid inserted into rice chromosome 3 and rice chromosome 12 was evaluated by confirming the sequence of the PCR product performed under the following conditions.
4-1. PCR
The recombinant rice (# 51A) 6th generation (corresponding to a seed bank) rice young leaves 2 g was finely minced, and genomic DNA was extracted using Nucleon PhytoPure (GE Healthcare).
Confirmation of the sequence of rice chromosome 3 Using the obtained recombinant rice (# 51A) genomic DNA (500 ng) as a template, TaKaRa LA Taq HS (Takara Bio) and primer set ch3 51A-F1, 51A LongPCR PCR was performed with a combination of -R and 51A LongPCR-F · ch3 51A-R1. Amplification was performed 35 cycles of 98 ° C. for 10 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 7 minutes.
Confirmation of the sequence of rice chromosome 12 Using the obtained recombinant rice (# 51A) genomic DNA (500 ng) as a template and using TaKaRa LA Taq HS (Takara Bio), primer sets ch12 51A-F1, CTB-R and CTB- PCR was performed with a combination of F · ch12 51A-R1. Amplification was performed 35 cycles of 98 ° C for 10 seconds, 60 ° C for 30 seconds, 72 ° C for 4 minutes 30 seconds.
The obtained PCR product was electrophoresed on 0.7% Aarose gel at 100 V for 25 minutes, the amplified band was excised from the gel, extracted with DNA gel, and then cloned into TOPO TA Cloning Vector (Invitrogen).

4-2. Sequence analysis The plasmid was purified, and the nucleotide sequence was confirmed using T3 primer and T7 primer designed on TOPO TA Cloning Vector, and 15 kinds of sequencing primers designed inside.
The primers used in the sequence are shown in the table below.
4-3.3 Difference between the sequence introduced into chromosome 3 and the design structure The actual structure (donor nucleic acid) and the design structure differ in the three structures surrounded by the dotted lines shown in FIG. It was. In addition, on chromosome 3, two copies of the donor nucleic acid were inserted in tandem.
(1) Of LB 25 bp, 9 bp (GGTGGCAGG) on the 5 ′ side is deleted.
(2) RB 25bp and its upstream region 43bp, LB 25bp and its downstream region 13bp, 106bp in total are deleted.
(3) Among 25 bp of RB, 3 ′ 20 bp (CAGGATATATTGGCGGGTAA) (SEQ ID NO: 48) is deleted.

4-4. Difference between the sequence introduced into chromosome 12 and the designed structure In the actual structure (donor nucleic acid) and the designed structure, the two structures surrounded by the dotted line shown in FIG. It was.
(1) LB 25bp, downstream region 96bp, 121bp in total is deleted.
(2) RNAi cassette 13kDa prolamin 45bp partial sequence, glutelin B1 228bp partial sequence, part of RAP intron 5 'side, 1018bp including 10kDa prolamin terminator, RB 25bp and upstream region 326bp, 1344bp in total were deleted ing.

5. Confirmation of Insertion Site by TAIL-PCR The insertion position of foreign DNA in the genome has been confirmed conventionally by the TIAL-PCR method (Non-patent Document 1). Thus, the insertion positions of chromosomes 3 and 12 revealed by the method of the present invention were confirmed using the TAIL-PCR method (Liu et al., Genomics 25: 674-681 1995).
The following primers were used in the PCR reaction.
Random 1: 5'-NGTCGASWGANAWGAA-3 '(SEQ ID NO: 39)
Random 2: 5'-GTNCGASWCANAWGTT-3 '(SEQ ID NO: 40)
Random 3: 5'-WGTGNAGWANCANAGA-3 '(SEQ ID NO: 41)
TAILF 1: 5'-TGTTATCCGCTCACAATTCCAC-3 '(SEQ ID NO: 42)
TAILF 2: 5'-CCAGCTGCATTAATGAATCGG-3 '(SEQ ID NO: 43)
TAILF 3: 5'-AGAGGCGGTTTGCGTATTGGAG-3 '(SEQ ID NO: 44)
TAILR 1: 5'-CTGGGAAAACCCTGGCGTTA-3 '(SEQ ID NO: 45)
TAILR 2: 5'-GCCAGCTGGCGTTAATAGCGA-3 '(SEQ ID NO: 46)
TAILR 3: 5'-GAATGCTAGAGCAATTCGGCG-3 '(SEQ ID NO: 47)
TAILF 1-3 and TAILR 1-3 were used to amplify the RB and LB adjacent regions of T-DNA, respectively (see FIG. 9B). Random 1-3 was a degenerate primer used as a primer that anneals to the genome sequence. TAIL-PCR was performed in 20 μl of reaction solution using TaKaRa LA Taq Hot Start Version (TaKaRa Bio) and Veriti Thermal Cycler (Applied Biosystems). TAIL-PCR reaction products were purified using Wizard SV Gel and PCR Clean-Up System (Promega), cloned into pCR4 TOPT plasmid (Invitrogen Life Technologies), and sequence confirmed. It was.
As a result, the insertion position in chromosome 3 could also be confirmed by the TAIL-PCR method (FIG. 9). In FIG. 9A, the band indicated by the arrowhead is an amplified fragment containing an adjacent region of LB and chromosome 3 and an amplified fragment containing an adjacent region of RB and chromosome 3.
On the other hand, since the fragment corresponding to the chromosome 12 region was not amplified, the presence of the inserted DNA in the chromosome 12 could not be confirmed by the TAIL-PCR method. This is probably because TAIL-PCR did not run because part of the 3 'side of the transgene to chromosome 12 was introduced with a deletion of 1,000 bp or more. However, if this fact is unknown, it is impossible to predict the introduction site by TAIL-PCR. From this, it was shown that according to the method of the present invention, the insertion position of the foreign DNA that cannot be confirmed by the conventional TAIL-PCR method can be accurately identified.

  The present invention relates to a method for accurately confirming the position and structure of DNA inserted into the genome of an arbitrary organism. Since it is important for the management of genetically modified crops and animals to accurately grasp the position of the inserted gene in the organism or plant into which the gene is introduced, the present invention is applied in the field of animal and plant breeding. Provide useful technology.

Claims (4)

A method for determining an insertion position of an exogenous DNA on a genomic DNA into which the exogenous DNA has been inserted, comprising the following steps (a) to ( e ), wherein both ends of the exogenous DNA are derived from a vector: The method of claim 1, wherein
(A) determining a sequence from both ends of each fragment of the fragmented total genomic DNA;
(B) a step of extracting a vector-derived sequence existing near both ends of the exogenous DNA from the sequence determined in step (a),
(C) extracting a sequence at the other end paired with each sequence included in the sequence group extracted in step (b);
(D) The sequence group extracted in step (c) is a foreign DNA sequence and is mapped to a sequence different from the “ vector-derived sequence ” in (b) above, and an unmapped sequence is extracted. The process of
(E) A step of performing homology search for the sequence group extracted in step (d) with respect to the genomic DNA sequence, and specifying a position on the genomic DNA sequence having homology with the sequence of the extracted sequence group The method according to claim 1, wherein the vector-derived sequence in the step (b) is present at a position of 0 to 1,000 bases from either end of the exogenous DNA.   The method according to claim 1 or 2, further comprising confirming the insertion position of the identified exogenous DNA by a PCR method.   A method for determining the structure of inserted foreign DNA based on the results obtained by the PCR method according to claim 3.