JPH10191981A - Analysis of restriction enzyme length polymorphism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for simply and efficiently analyzing a restriction enzyme length polymorphism of a DNA and selecting a polymorphic marker. SOLUTION: At least two kinds of adapters having a base sequence capable of ligating to each one of DNA fragments obtained by cleaving a DNA with at least two restriction enzymes are ligated to the DNA fragments. An oligonucleotide set is then selected for each pair from at least two pairs of oligonucleotide sets in which the number of two oligonucleotides having a structure of a nucleotide, forming no base pair with the adapters and protruding to the side of the 3'-terminal when carrying out the annealing with each adapter is equal among those belonging to the same oligonucleotide set and different among those belonging to the different oligonucleotide sets in the decreasing order of the protruding nucleotides to provide primers. A fluorescent labeled primer is used as at least one of the primers of the oligonucleotide set having the largest number of the protruding nucleotides to successively conduct a polymerase chain reaction. The resultant reactional product is then analyzed by using an electrophoretic apparatus.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for analyzing restriction enzyme fragment length polymorphisms, and more particularly, to a method for analyzing restriction enzyme fragment length polymorphisms useful for selecting polymorphic markers used for plant breeding.

[0002]

2. Description of the Related Art As a method for examining the presence or absence or genotype of a certain gene, restriction fragment length polymorphism (Restriction fr.
An analysis technique based on agment length polymorphisms (RFLP) is known. RFLP is based on the fact that the size of a restriction enzyme fragment differs when a restriction enzyme site is present at a site having a different gene structure between individuals of an organism. When a DNA region showing a polymorphism is identified, an individual from which a certain gene is derived can be identified by using a probe targeting the region as a marker, and plant breeding using this gene can be performed by: This is an important technology that can greatly shorten the conventional breeding period.

However, in order to detect a polymorphism and select a polymorphic marker, a method using a probe having an arbitrary sequence or a probe having a random sequence is used (Theor. Appl. Genet, 80,
437-448, 1990), but in plants such as tomato, wheat, cotton, and soybean, there is less polymorphism among cultivars than in other plants, so that RFLP does not provide sufficient polymorphism markers.

As a means for detecting a gene, a polymerase chain reaction (PCR: Polymer
se Chain Reaction, Science, vol.239, p.487-491 (19
88)) method is being used. This is because heat denaturation of double-stranded DNA to single-stranded DNA, annealing of the oligonucleotide corresponding to the sequence at both ends of the site intended for amplification with the heat-denatured DNA, and polymerase using the oligonucleotide as a primer This is a method of amplifying the DNA sequence exponentially by repeating an amplification cycle consisting of a reaction. The difference in the length of the amplified gene fragment makes it possible to identify the gene.

As a polymorphism analysis method using PCR, an RAPD analysis method using an arbitrary primer is known.
By RFLP and RAPD, a high-resolution molecular map (high-resolution) is obtained in tomato.
lmolecular map) has been completed.

[0006] In addition to RFLP and RAPD analysis techniques, several more effective polymorphism detection techniques have been developed. One of those methods is AFLP (Amplifie
d fragment length polymorphisms: Nucleic Acids Rse
arch, 1995, Vol.23, No.21, 4407-4414) analysis is known. The greatest advantage of this approach is that it can
This is where it becomes possible to compare polymorphisms in regions. However, in order to resolve and separate these regions by electrophoresis, radiolabeled PCR primers are required, and much labor and labor are often required for the work,
Attention was also required in terms of safety. On the other hand, DN
As a base sequence determination device for A, a fluorescence-labeled primer used for the dideoxy method and an automated device using an electrophoresis device equipped with a fluorescence detection means are known. AFLP analysis using a fluorescence-labeled primer is known. No successful case is known.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for simply and efficiently analyzing restriction fragment length polymorphisms of DNA and selecting a polymorphism marker.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that at least two types of restriction enzymes are used for cutting DNA and a plurality of types of primers are used. It was found that AFLP analysis was possible even when fluorescent-labeled primers were used, and that the reaction was efficiently performed by increasing the concentration of DNA polymerase used in the PCR reaction. The invention has been completed.

[0009] That is, the present invention provides a method for preparing DNA of at least 2
At least two types of adapters each having a base sequence connectable to one end of the DNA fragment are ligated to a DNA fragment obtained by digestion with various kinds of restriction enzymes, and a base sequence complementary to each of the adapters is ligated. An oligonucleotide set comprising two types of oligonucleotides having a structure in which a nucleotide that does not form a base pair with an adapter protrudes to the 3 ′ end when annealed to each adapter, the number of the protruding nucleotides But,
Oligonucleotides belonging to the same oligonucleotide set are equal, and at least two pairs of oligonucleotide sets different between oligonucleotides belonging to different oligonucleotide sets are selected from one set of oligonucleotide sets in ascending order of the protruding nucleotides. As a primer, and using a fluorescently labeled at least one primer of the oligonucleotide set with the largest number of protruding nucleotides,
Using the DNA fragment to which the adapter is connected as a template, a polymerase chain reaction is sequentially performed, and the reaction product is analyzed by an electrophoresis apparatus equipped with a fluorescence detecting means.
This is a method for analyzing restriction fragment length polymorphisms of the above.

[0010] The present invention also relates to the above method, wherein each oligonucleotide belonging to a different oligonucleotide set and having a sequence complementary to the same adapter has the same sequence at a portion where the length overlaps. Provide a way. The present invention further provides the method as described above,
A first oligonucleotide set consisting of two kinds of oligonucleotides in which the number of projecting nucleotides is 1 and a second oligonucleotide set consisting of two kinds of oligonucleotides in which the number of projecting nucleotides is 2 or 3; After performing a polymerase chain reaction using the first oligonucleotide set as a primer and a DNA fragment to which an adapter is ligated as a template, the second oligonucleotide set as a primer and the reaction product as a template A method for performing a polymerase chain reaction is provided.

Further, the present invention provides a method as described above, wherein a DNA polymerase in an amount of 0.5 units or more is used in the final polymerase chain reaction. Hereinafter, the present invention will be described in detail.

[0012]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides AFLP analysis,
In the AFLP analysis using at least two kinds of restriction enzymes and using at least two pairs of primers, a primer labeled with fluorescence is used. In a preferred embodiment of the present invention, the PCR reaction is preferably at least 0.3 unit, particularly preferably 0.5 unit.
A unit amount of DNA polymerase is used. Usually 0.
It reaches a peak at about 5 units, but more DNA polymerases can be used. Here, one unit is:
Using activated salmon sperm DNA as template / primer,
11. The activity of incorporating 10 nmol of total nucleotides into an acid-insoluble precipitate in 30 minutes at 74 ° C.

The method of the present invention is the same as the AFLP method (Amplified fragment length polymorphiy) except for the amount of primers and DNA polymerase used.
sms: Nucleic Acids Rsearch, 1995, Vol.23, No.21, 4
407-4414).

The principle of the AFLP reaction will be described below.
First, genomic DNA is completely digested with a specific restriction enzyme (FIG. 1). Next, an adapter was ligated to the cut fragment (FIG. 1).
), And cleave the fragment using primers complementary thereto.
Amplify with CR (FIG. 1). At this time, 3 'of the primer
On the side, nucleotides that do not form a base pair with the adapter (in this figure, A for the 5 ′ primer and C for the 3 ′ primer) are made to protrude. What is formed, that is, the base at the 5 'end of the DNA fragment excluding the adapter is A
And only the fragment whose base at the 3 ′ end is C is amplified (FIG. 1). Hereinafter, this first PCR is referred to as “preliminary amplification”. In the method of the present invention, at least two types of restriction enzymes having different recognition sequences, preferably a restriction enzyme that recognizes 4 bases and a restriction enzyme that recognizes 6 bases, are used. As a result, a fragment having a length that allows efficient PCR is obtained.
Using only one restriction enzyme and adapter, PCR
During the reaction, both ends of the DNA fragment are associated with each other to inhibit the reaction, and the reproducibility is often insufficient. Specific examples of the restriction enzymes used include EcoRI, MseI, and BamHI.
And TaqI. The adapter is a double-stranded DNA fragment containing a partially single-stranded structure having a restriction enzyme-cleaved end and a cohesive end, and usually has a length of about 20 bases.

In the above steps, there are two chances of detecting a polymorphism, that is, selection of a restriction enzyme to be used and selection of a primer. That is, the former is based on the same principle as RFLP, and the latter is based on RAP
It is the same as the principle of D analysis. Furthermore, in the present invention, using the amplification product as a template, more nucleotides protrude than the primer (3 nucleotides in FIG. 1), and the first nucleotide protruding is the same as the primer (FIG. 1).
When a PCR reaction is performed using a primer set of AAC for the 5′-side primer and CAA for the 3′-side primer, the base at the 5 ′ end of the DNA fragment excluding the adapter is AAC and the base at the 3 ′ end is CAA. Is amplified (FIG. 1, hereinafter). Hereinafter, this second PCR is referred to as “selective amplification”. When the selective amplification of the restriction enzyme-cleaved DNA fragment linked to the adapter is performed directly without performing the pre-amplification reaction, the detection of the reaction product becomes unclear,
Often poor reproducibility. Usually, one selective amplification gives satisfactory results, but two or more selective amplifications may be performed. In that case, the number of protruding nucleotides of the primer used for the selective amplification is made larger than that of the primer used for the previous round of the selective amplification. In addition, each oligonucleotide belonging to a different oligonucleotide set and having a sequence complementary to the same adapter can effectively perform selective amplification if the overlapping portion is the same sequence. .

The method of the present invention uses, in the AFLP reaction, one or both of the primers for the final selective amplification, which are fluorescently labeled. As the fluorescent dye used for the fluorescent labeling, those usually used for a probe for hybridization or a primer for a base sequencer can be used. Specifically, for example, FITC (fluoroscein isoform) can be used. Thiocyanate), Cy
5 (indodicarbocyanine dye
dye)) and the like. The labeling of the primer with these dyes is possible by using a commercially available DNA synthesizer and synthesizing oligonucleotides using nucleotides to which the corresponding dyes are bound. The length of the primer is usually about 16 to 20 bases. The analysis of the AFLP product is performed using an electrophoresis apparatus equipped with a fluorescence detecting means. In particular, a high-resolution device, for example, an electrophoresis device for determining a base sequence, can separate DNA fragments even if only one base length is different.
preferable. In addition, an automated device capable of detecting a fluorescently labeled DNA band is commercially available and can be suitably used in the present invention. Examples of such a device include: L. F. DNA Sequencer (manufactured by Pharmacia; ALF is a trademark of Pharmacia). Except for the detection of fluorescence, the gel used for electrophoresis and the conditions for electrophoresis are not particularly different from ordinary AFLP or base sequence determination.

From the results of the analysis in the examples described below, it was found that the method of the present invention enables comparison of 100 or more regions by one AFLP reaction (FIG. 2). Assuming that two types of varieties are compared, for example, if the number of lanes of the electrophoresis gel is 40, analysis can be performed with a maximum of 20 types of primers, and a total of 10 × 20 = 2000 regions can be searched at once. It is possible. Assuming that all random regions are amplified in one AFLP analysis, the probability of detecting a target region is expressed by the equation P = 1− (1-t / G) nx (GB Martin et al. ., Proc. N
atl. Acad. Sci. USA., 88, 2336 (1991)). Here, the genome size (G) of tomato is 9.5 × 10 ⁸ = bp (GB Martin et al., Science., 262, 1432 (199
3)), the size of the target region (t), and the average number of bands (x) detected in one AFLP analysis (x) is assumed to be 100, and the number of analysis required for various probabilities and region sizes ( Table 1 shows the results of the determination of the type of primer.

[0018]

[Table 1]

However, the above formula does not assume that the two types of target regions to be compared have the same pattern, so that it is expected that the number of times of analysis actually required will be increased. Table 1 shows that, for example, in order to detect one marker within a target area size of 10 ⁶ bp (1000 kb), 69.9% probability is required to detect 6 markers.
6 times (66 types of primers), 22% with 90% probability
This means that AFLP analysis should be performed twice (22 types of primers).

On the other hand, tomato genome size 9.5 × 1
0 ⁸ , from the genetic scale of the tomato genome of 1300 cM (centimorgan), 1 cM of tomato corresponds to about 730 kb. Therefore, the size of the target area is set to 1000 k.
It is considered that a strongly linked marker within 1 cM can be obtained by the number of analyzes assuming b.

[0021] The method of the present invention facilitates the acquisition of genetic markers. In particular, isolation of markers linked to jointless traits, and furthermore, conventional polymorphism analysis methods such as dominant sterility traits and high pigment mutations, etc. It is expected that traits that are expected to be very difficult can be isolated.

[0022]

【Example】

<1> Preparation of tomato total DNA Three tomato varieties, lycopersicon esculentum
Kagome 77 (Licopersicon esculentum Kagome77) and Lycopersicon esculentum KG204, a jointless variety of Kagome 77, were grown in a field.

[0023] Leaves are collected from the grown tomato, and commercially available A
FLP Kit (AFLP ^TM Analysis System I AFLP Starte
r Primer Kit (manufactured by Gibco BRL, purchased from Life Technology) was used to prepare total DNA by the method recommended in the manual attached to the kit.

That is, 0.3 to 0.5 g of tomato leaves are ground under liquid nitrogen, and 5 μl of a lysis buffer [lysis buffer: 1% CTAB (cetyltrimethylammonium bromide), 5% PVP (polyvinylpyrrolidone) ), 1.4M NaCl, 20m
M EDTA, 10 mM Tris-HCl (pH 8.
0), 350 mM 2-mercaptoethanol] and incubated at 65 ° C. for 10 minutes. 5ml for this
Add phenol / chloroform and stir, add 300
After centrifugation at 0 rpm for 5 minutes, the upper layer was separated. This is 5
Add 3 ml of chloroform and stir to 3000 rpm
After centrifugation at for 5 minutes, the upper layer was separated, and 2.5 volumes of ethanol was added. The precipitate was collected by centrifugation, washed with 70% ethanol, and dissolved in 50 μl of TE to obtain a total DNA sample.

<2> AFLP Reaction The DNA obtained as described above and the DNA of the cultivar lycopersicon esculentam ailsa craig 2 (Ailsa Claig 2) contained in the AFLP kit
Was used as a template to perform an AFLP reaction.

The AFLP reaction was carried out using the AFLP kit according to the method described in the manual attached to the kit. However, the concentration of Taq polymerase at the time of selective amplification was modified 5-fold. Also,
Among the primers used for the AFLP reaction, FITC (fluorescein isothiocyanate) -labeled primers were requested to be manufactured by Pharmacia.

(1) Restriction enzyme digestion of DNA sample 5 μl of 5 × reaction buffer was placed in a 1.5 ml centrifuge tube.
250 μg of DNA sample (18 μl or less), restriction enzyme (Eco
RI / MseI) 2 μl, add 25 μl with water (AFLP-grade water)
and mixed gently, centrifuged briefly and collected at the bottom of the tube and allowed to stand at 37 ° C. for 2 hours. After incubating at 70 ° C. for 15 minutes to inactivate the enzyme, the reaction solution was collected by light centrifugation at the bottom of the tube and allowed to stand on ice.

(2) Ligation of Adapter To a DNA sample (25 μl) digested with a restriction enzyme, 24 μl of an adapter ligation solution (T
4 1 μl of DNA ligase was added, mixed gently at room temperature, centrifuged lightly, and allowed to stand at 20 ° C. ± 2 ° C. for 2 hours.

The above reaction solution was aliquoted in 10 μl portions into 1.5 ml centrifuge tubes, and 90 μl of TE buffer was added and mixed well to dilute the reaction solution 10-fold. The remainder used for the next reaction was stored at -20 ° C.

(3) Preamplification reaction
eaction) In a 0.5 ml centrifuge tube, add the 10-fold diluted D
5 μl of NA sample, pre-amplification reaction mixture (pre-amp primer
mix) 40μl, 10x PCR buffer (AFLP use) 5μ
1, 1 μl of Taq DNA polymerase (lu / μl)
And gently mixed and centrifuged, and then 2-3 drops of mineral oil were added. The preamplification reaction mixture contains E
A primer (SEQ ID NO: 1) containing a sequence complementary to the sequence of the coRI adapter (30 ng) and a primer containing a sequence complementary to the sequence of the MseI adapter (30 ng) (30 ng) are included. When these primers anneal to each adapter, one nucleotide that does not form a base pair with the adapter protrudes to the 3 'end. The protruding nucleotides are A (deoxyadenosine) for EcoRI and C (deoxycytidine) for MseI.

Subsequently, a dissociation reaction at 94 ° C. for 30 seconds, 56
After repeating 20 cycles of an association reaction at 60 ° C for 60 seconds and an extension reaction at 72 ° C for 60 seconds, the reaction was stopped at 4 ° C. Reaction products were stored at -20 ° C.

(4) Selective amplification for polymorphism analysis (Sele
ctive AFLP Amplification) One of a primer containing a sequence complementary to the EcoRI adapter (EcoRI primer ~, SEQ ID NOs: 3 to 5) and a primer containing a sequence complementary to the MseI adapter (MseI primer ~, SEQ ID NOs: 6 to 13) Selected one by one. EcoRI primer is FITC
The labeled product was outsourced to Pharmacia. M
As the seI primer, the one attached to the kit was used. When these primers anneal to each adapter, three nucleotides that do not form a base pair with the adapter protrude to the 3 'end. The protruding nucleotides are AAC, ACA, ACC for EcoRI and CAA, CAC, CAG, CAT, for MseI.
CTA, CTC, CTG, and CTT.

The selected EcoRI primer (10 ng)
/ Nl) 5 μl, MseI primer (6.7 ng, d
45 μl (containing NTP) was placed in a 1.5 ml centrifuge tube and mixed. This is referred to as Mix1. Also, 1.5m
1 μl of AFLP-grade water
l, 10 x PCR buffer (AFLP use) 20 l, Taq
1 μl of DNA polymerase (5 u / μl) was added.
This is referred to as Mix2. 0.5 μl of the pre-reaction product,
4.5 μl of AFLP-grade water, Mix1 (pri
mer / dNTPs) (5 μl) and Mix2 (10 μl) were added to a 0.5 ml tube, mixed gently and centrifuged. Then, 2-3 drops of mineral oil were added, and Reactions 1 to 11 were continued under the following conditions. To perform PCR. In addition, by this mixing, Taq per reaction was obtained.
The polymerase amount is 0.5 units. For 5 μl of the reaction solution, 7.5 μl of Stop Reaction Buffer (Stop Reaction Buffer)
Buffer: blue dextran dissolved in formamide deionized to 5 mg / ml), treated at 95 ° C for 2 minutes, and cooled with ice to obtain a sample for electrophoresis.

[0034]

[Table 2] Table 2 ───────────────────────────── Dissociation reaction Association reaction Extension reaction ────────温度 Temperature Time Temperature Time Temperature Time Cycle (° C) (second) (° C) (second) (° C) (second) (times) ──────────── ───────────────── 1 94 60 65 60 72 90 1 2 94 60 64 60 72 90 1 3 94 60 63 60 72 90 1 4 94 60 62 60 72 90 1 5 94 60 61 60 72 90 1 6 94 60 60 60 72 90 1 7 94 60 59 60 72 90 1 8 94 60 58 60 72 90 1 9 94 60 57 60 72 90 1 10 94 60 56 60 72 90 1 11 94 30 56 30 72 60 23 ─────────────────────────────

<3> Analysis of AFLP Reaction Product by Electrophoresis The above-mentioned analysis of the AFLP reaction product by electrophoresis is an automatic electrophoresis apparatus (ALF DNA Sequencer, Pharmacia, Inc.) capable of analyzing the base sequence by detecting a fluorescent label. ) According to the instruction manual of the apparatus. This device can be controlled by electrophoresis software (ALF manager) attached to the device. Electrophoresis is performed using a commercially available preparation (ready mix).
gel, manufactured by Pharmacia: 6% polyacrylamide), a gel having a thickness of 0.5 mm and a length of 28 cm, and a 0.6 × TBE buffer solution, 1500 V, 38 mA
Was performed under the following conditions. The analysis of the electrophoresis was performed using analysis software (Fragment manager).

FIG. 2 shows the results. This figure is a waveform display of a part of the fluorogram by fluorescence detection,
The top row shows chain length markers that were simultaneously electrophoresed. 100
The DNA fragment is amplified from bp to 500 bp,
Among them, a clear polymorphism was detected when the MseI primer (SEQ ID NO: 6) was used.

[0037]

[Comparative Example 1] Except that a pre-amplification reaction was not performed and a restriction enzyme-cleaved DNA fragment to which an adapter was ligated was selectively amplified.
An AFLP reaction was performed in the same manner as in Example 1. As a result, the number of amplified fragments increased, the electrophoresis pattern was smeared, and the resolution was reduced.

[0038]

Comparative Example 2 In a selective amplification reaction, 0.1 unit of T
An AFLP reaction was performed in the same manner as in Example 1 except that aq DNA polymerase was used. As a result, no amplification product was obtained.

[0039]

Example 2 Linkage analysis between a polymorphism pattern and a jointless trait was performed by the method of the present invention. In addition to the varieties used in Example 1, Kagome 932, Kagome 941, Kagome 952, 95-1 (all jointless varieties), KG
From 138, chromosomal DNA was prepared in the same manner as in Example 1, and AFLP analysis was performed. In addition, EcoRI primers and MseI primers were used as primers for selective amplification.

FIG. 3 shows the results of the electrophoresis. as a result,
Except for 95-1, a region where the jointless trait and the polymorphism pattern coincided was found (in FIG. 3, a portion surrounded by a square). Furthermore, subsequent analysis revealed that the trait and polymorphism pattern were consistent in 14 of the 16 cultivated species in total, suggesting that this polymorphism was linked to the jointless trait (Table 2).

[0041]

[Table 3] Phenotype No: No joint, Yes: With joint Polymorphism pattern a: Same pattern as Kakazume 77, b: Same pattern as KG204 Presence or absence of linkage ○: With linkage, ×: Without linkage

[0042]

According to the method of the present invention, restriction fragment length polymorphism of DNA can be simply and efficiently analyzed to select a polymorphism marker. As a result, the present method can be used to isolate traits that are expected to be extremely difficult with conventional polymorphism analysis methods, such as isolation of markers linked to jointless traits, dominant sterility traits, and high pigment mutations. It is expected.

[0043]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GACTGCGTAC CAATTCA 17

SEQ ID NO: 2 Sequence length: 17 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GATGAGTCCT GAGTAAC 17

SEQ ID NO: 3 Sequence length: 19 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GACTGCGTAC CAATTCAAC 19

SEQ ID NO: 4 Sequence length: 19 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GACTGCGTACCAATTCACA

SEQ ID NO: 5 Sequence length: 19 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GACTGCGTACCAATTCACC 19

SEQ ID NO: 6 Sequence length: 19 Sequence type: number of nucleic acid chains: single stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GATGAGTCCT GAGTAACAA 19

SEQ ID NO: 7 Sequence length: 19 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GATGAGTCCT GAGTAACAC 19

SEQ ID NO: 8 Sequence length: 19 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GATGAGTCCT GAGTAACAG 19

Sequence number: 9 Sequence length: 19 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GATGAGTCCT GAGTAACAT 19

SEQ ID NO: 10 Sequence length: 19 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GATGAGTCCT GAGTAACTA 19

SEQ ID NO: 11 Sequence length: 19 Sequence type: number of nucleic acid chains: single stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GATGAGTCCT GAGTAACTC 19

SEQ ID NO: 12 Sequence length: 19 Sequence type: number of nucleic acid chains: single stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GATGAGTCCT GAGTAACTG 19

SEQ ID NO: 13 Sequence length: 19 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GATGAGTCCT GAGTAACTT 19

[Brief description of the drawings]

FIG. 1 is a diagram showing the principle of an AFLP reaction.

FIG. 2 is a view showing the results of electrophoretic analysis of the AFLP reaction product of Example 1.

FIG. 3 is a diagram showing the results of electrophoretic analysis of the AFLP reaction product of Example 2.

Claims (4)

[Claims] 1. A DNA fragment obtained by cleaving DNA with at least two kinds of restriction enzymes, at least two nucleotides having a base sequence which can be ligated to each of the ends of the DNA fragment.
Two types of adapters each having a structure in which nucleotides that do not form a base pair with an adapter protrude to the 3 ′ end when each adapter contains a nucleotide sequence complementary to each of the adapters and is annealed to each adapter. At least two pairs of oligonucleotides, wherein the number of protruding nucleotides is the same between oligonucleotides belonging to the same set of oligonucleotides and different between oligonucleotides belonging to different sets of oligonucleotides. From the nucleotide set, a pair of oligonucleotide sets are selected one by one in ascending order of the protruding nucleotides as primers, and at least one primer of the oligonucleotide set having the largest number of protruding nucleotides is fluorescently labeled. Using the obtained DNA fragment, the polymerase chain reaction is sequentially performed using the DNA fragment linked to the adapter as a template, and the reaction product is analyzed by an electrophoresis apparatus equipped with a fluorescence detection means. Analysis method. 2. The method according to claim 1, wherein each oligonucleotide belonging to a different set of oligonucleotides and having a sequence complementary to the same adapter has the same sequence at the overlapping portion. 3. The oligonucleotide set according to claim 1, wherein the oligonucleotide set comprises two types of oligonucleotides each having one protruding nucleotide, and two types of oligonucleotides each having two or three protruding nucleotides. A second oligonucleotide set consisting of nucleotides, wherein the first oligonucleotide set is used as a primer, a polymerase chain reaction is performed using a DNA fragment to which an adapter is connected as a template, and then the second oligonucleotide set is used as a primer, 3. The method according to claim 2, wherein a polymerase chain reaction is performed using the reaction product as a template. 4. In the final round of polymerase chain reaction, 0.
2. The method according to claim 1, wherein the DNA polymerase is used in an amount of 3 units or more.
The described method.