JPH05192147A - Method for specifying dna base sequence - Google Patents

Abstract

PURPOSE:To enable the specification of a DNA base sequence with good reproducibility, above all a genetic DNA derived from a bacterium, the easy classification and identification of the bacteria and wide utilization in a field of chemistry, medicines, foods, electronics (high-density integrated circuits), environment, etc., requiring rapid detection of polluting or useful bacteria. CONSTITUTION:The objective method for specifying a DNA base sequence is characterized by amplifying a DNA base sequence according to polymerase chain reaction (PCR), cleaving the resultant DNA base sequence with a specific restriction enzyme, then separating the cleaved fragments by a desired separating means, providing separation patterns of the cleaved DNA fragments and specifying the DNA base sequence using the separation patterns of the cleaved DNA fragments as an index. Furthermore, a microorganism is classified or identified by using the method.

Description

Detailed Description of the Invention

[0001]

This invention relates to PCR (Polymerase C
hain reaction) The present invention relates to a method for identifying a base sequence of an amplified relatively short DNA. In particular, the present invention relates to a method for easily classifying or identifying bacteria in a short time by amplifying a specific region of DNA encoding 16S ribosomal RNA of bacteria. And
This method can be widely used in chemical, medical, food, electronics (high-density integrated circuit), environment, and other industrial fields that require rapid detection of contaminating bacteria or useful bacteria.

[0002]

2. Description of the Related Art The PCR method is a method for amplifying a DNA in a specific region of interest from a small amount of DNA sample by 200,000 to 500,000 times in about 3 hours. In the PCR method,
The target double-stranded DNA is heat-denatured in a test tube to give single-stranded DNA, and then the oligomer DNA chemically synthesized to each single-stranded DNA is used as a primer and complementary by using a DNA polymerase that is a repair enzyme. Synthesize strand DNA.
By repeating this reaction, DNA can be synthesized logarithmically in a short time.

The method of determining the nucleotide sequence constituting DNA is called the DNA sequence method and is roughly classified into two methods. One is Maxa using base-specific chemical modification
m-Gilbert method (Maxam, AM, Gilbert, W . : Proc. Nat
L. Acad. Sci. USA , 74, 560-564 (1977)), and the other is the dideoxy method (Sanger, F., Nicklen, which uses a dideoxynucleotide to terminate the synthesis of DNA polymerase in a base-specific manner). S., Coulson, AR: Proc. Na
tl. Acad. Sci. USA , 74, 5463-5467 (1977)).
Other than this, that is, the base sequence of DNA is not directly determined, and means for specifying DNA is a restriction enzyme (an enzyme that can recognize a specific base sequence and cleave double-stranded DNA, For example, Eco R1 [Eco R
The restriction enzyme called [I] recognizes the base sequence of GAATTC and cuts the DNA between G and A), and examines the cut site to create a map called a restriction enzyme map. It is also possible to distinguish large DNA (eg plasmid) from other DNA. Conventionally, useful newly discovered plasmids and the like have been frequently used for DNA identification by the latter restriction map. Nowadays, among the DNA sequencing methods, the procedure is simple and kits are commercially available. Direct sequencing has become the mainstream method for DNA identification. The above is an outline of the latest technology in academic research.
Industrial use of the sequence method is hampered by the availability of radioactive isotope equipment, expensive equipment, and the need for highly specialized engineers.

Generally, bacteria have been detected by a selective culture method on a special medium. Classification and identification are performed using morphological observation, Gram's stain, oxygen requirement, nutritional requirement, and biochemical examination as indexes. However, these methods have drawbacks such as complexity, time and labor, and difficulty in determination. Therefore, it has been attempted to analyze DNA, which is a gene unique to each bacterium, but at present, an effective and rapid method has not been established. At the present time, all DNs are being applied as the chemical classification index of gene DNA.
A simple method that uses the ratio of guanine to adenine of A as GC%, or D using synthetic DNA specific to the bacterial species as a probe
A method of performing classification and identification by performing NA-DNA hybridization may be mentioned. The former is a qualitative auxiliary means in taxonomy, and the latter is a D-specific D species.
There is a drawback that a probe specific to the bacterium cannot be synthesized unless information on the NA base sequence is obtained.

16S ribosomal RNA, which is one of the constituent molecules of ribosomal RNA, is the most widely used molecule as a molecular chemical indicator of phylogenetic evolution. The reason for this is that all organisms contain a 16S ribosomal RNA molecule of almost the same size and the same function, and the total DNA encoded by them is short, so that nucleotide sequencing is relatively easy. Points etc. Attempts have been made to utilize these industrially, but as mentioned above, the process of DNA sequencing is an obstacle.

[0006]

As described above, complicated biochemical tests are required to identify bacteria, and methods using DNA as a chemical index have been attempted as an improved method for these. However, there were many problems such as the process of DNA sequencing, the extraction of DNA until the sequence was reached, and the selection of specific amplified bases, which made it unusable. The present invention has been made to solve such problems, and has a high reproducibility, high reliability, and safe method for identifying DNA,
In particular, it is an object of the present invention to provide a method for classifying and identifying bacteria by identifying gene DNA derived from bacteria.

[0007]

The present invention provides a DNA obtained by PCR amplification of a DNA base sequence with primers.
The method is characterized in that after the base sequence is cleaved with a specific restriction enzyme, the cleaved DNA fragment is separated by a desired separating means, and the separation pattern of the cleaved DNA fragment obtained is used as an index to identify the DNA base sequence. This is a method for identifying a DNA base sequence.

Electrophoresis, chromatography and the like are used as the means for separating the cut DNA fragments. The DNA base sequence may be any one as long as it is derived from a living organism, but a DNA base sequence derived from a microorganism is particularly preferable, and a specific example thereof is Lactobacillus (Lactobacillus).
Examples include bacteria belonging to the genus s ) or the genus Bacillus.

Further, the present invention is a DNA obtained by subjecting a microorganism to heat treatment with a surfactant to extract DNA, and PCR-amplifying with a primer using a DNA base as a template.
A microorganism is classified or identified by using the separation pattern of the cleaved DNA fragment obtained by cleaving the nucleotide sequence with a specific restriction enzyme and separating the cleaved DNA fragment by a desired separation means. This is a method for classifying or identifying microorganisms.

The DNA base sequence used as the template is as follows:
It is preferable to use a specific region of DNA encoding microbial 16S ribosomal RNA. The microorganism to which the method for classifying or identifying the microorganism of the present invention is applied,
Although not particularly limited thereto, preferably bacteria are exemplified, and examples thereof include Lactobacillus (Lactoba).
cillus) include the bacteria belonging to the genus or Bacillus (Bacillus) genus.

Furthermore, the present invention relates to a primer-DNA capable of PCR-amplifying a specific region of a DNA encoding a microbial 16S ribosomal RNA. And as this primer-DNA, SEQ ID NO: 1 is used.
And a nucleotide sequence of SEQ ID NO: 2 or a DNA capable of hybridizing complementarily to the nucleotide sequence of SEQ ID NO: 3 and / or SEQ ID NO: 4, respectively.

The present invention will be described in detail below. The most important feature of the present invention is to present the optimal base sequence of the gene DNA to be PCR-amplified so that it is suitable for the subsequent analysis, and the DNA extraction from bacteria is the first step, and finally the DNA bases unique to all bacteria are finally presented. The point is that the sequence can be specified. In order to achieve the above object, the present invention provides a method of extracting DNA from at least several tens of bacterial cells, and using this as a template to obtain a separation pattern of restriction enzyme cleavage fragments of DNA amplified by thermostable DNA polymerase. It is composed of

The first step of the present invention is the extraction of DNA from microorganisms. As a typical example, DNA extraction from bacteria is Ma
The purification of E. coli DNA by the rmur method (J. Marmur: J. Mol. Biol. , 3, 208 (1961)) is widely known. Specifically, 2-3 g of E. coli cells in the logarithmic growth phase is 25%. Lysis with sodium dodecylsulfate (SDS), and the lysate is added with 5M sodium perchlorate to a final concentration of 1M to dissociate nucleic acid and protein. To this, an equal amount of chloroform / isoamyl alcohol (24/1; V / V) was added, and the mixture was sealed and shaken to denature the protein. The nucleic acid fraction transferred to the aqueous layer by centrifuging the obtained solution at 5000-10000 rpm for 5 minutes was adjusted to 95% of double the volume.
Wrap around a glass rod by adding cold ethanol. Further, by adding a step of decomposing coexisting RNA with RNase, sufficiently purified DNA can be obtained so that it can be used as an enzyme reaction substrate of a DNA polymerase described later.

Other methods for purifying DNA from bacteria are enumerated. Saito and Miura's phenol treatment method (H. Sa
ito & K. Miura: Biochim.Biophys.Acta , 72,619 (1963))
, Zamenhof et al. (S. Zamenhof, G. Leidy, HEAlex
ander, PLFitzgerald & E.Chargaff: Arch.Biochem.Bi
ophys ., 40,50 (1952)), Thomas et al.'s method (KIBerns
& CAThomas, Jr .: J.MOl.Biol. , 11,476 (1965)) and the like are common. The outline of the procedure is as follows: Lysis with lysozyme / SDS, treatment with phenol saturated with Tris SDS buffer, deproteinization, and ethanol precipitation from a DNA solution.
NA is recovered and purified by RNase treatment. The DNA extraction in the present invention is also an improvement based on this method.

That is, since the purpose of the lysis operation is to destroy the cell membrane or cell wall of bacteria, heat treatment,
Freezing treatment, mechanical crushing, etc. are also processes that replace lysozyme / SDS. In the present invention, lysis treatment at 100 ° C or 120 ° C by boiling water or high-pressure heating, and rapid lysis in dry ice / ethanol or liquid nitrogen. It has been confirmed that the freezing treatment is also useful as a cell disruption method for the subsequent DNA purification operation. In the fraction after the bacterial cells were disrupted, high molecular weight DNA, R
NA or those partially mechanically or enzymatically decomposed, or a large amount of protein as a constituent of bacterial cells are mixed. Therefore, in the next step, deproteinization operation is necessary, but this operation applies the method of performing with phenol: chloroform mixed solution. That is, a mixed solution of phenol saturated with the buffer solution used and an equal amount of chloroform is mixed with the disrupted bacterial suspension, and the mixture is vigorously stirred with a vortex mixer. After that, 5 ℃, 10,000RP
Centrifuge at M rpm for 10 minutes. The separation layers are, in order from the top, the first layer is the phenol layer, the second layer is the insoluble fraction of the denatured protein, and the final third layer is the chloroform layer. Since the phenolic fraction in the first layer contains DNA, take it out with a Pasteur pipette without disturbing the liquid surface, add an equal volume of the phenol: chloroform mixture, and mix with the first vortex mixer. Repeat the stirring operation. This operation is performed at least three times, chloroform is added in an equal amount to the final phenol layer, and centrifugation is performed at room temperature at a rotation speed of 10,000 RPM for 10 minutes. The supernatant liquid was taken out with a Pasteur pipette, and 2 volumes of ethanol cooled to -25 ° C was slowly added thereto. If a large amount of DNA is contained, collect it by wrapping it around a glass rod, but if the amount is small, minus 25
Store in a freezer at ℃ for about 16 hours to perform sufficient DNA precipitation. After precipitation, the precipitate is collected by centrifugation at 10,000 RPM at 0 ° C. The precipitate is rinsed with 70% ethanol and dried under reduced pressure in a desiccator.
This precipitate was dissolved in Tris-EDTA buffer pH 7.5 and the absorbance at a wavelength of 260 nm (nanometer)
Calculate the concentration of DNA purified from bacteria and use it for PC
Used as a template for R.

The second step of the present invention is the PCR method (Polymera
This is amplification of DNA at a specific site encoding 16S ribosomal RNA by se chain reaction). The PCR method is a DNA amplification method utilizing a DNA polymerase reaction. DNA polymerase synthesizes complementary DNA using single-stranded DNA as a template (template), but requires a primer to initiate the reaction. Therefore, by using an appropriate primer, only the DNA region to be amplified can be synthesized. Since the DNA of an organism is usually double-stranded, by using two types of primers that sandwich the DNA region to be synthesized, the region can be duplicated in a single reaction. The principle of the PCR method is to repeat this DNA polymerase reaction, and in principle, the DNA can be amplified by N times the power of 2 by N cycles. PC
The sensitivity of the R method is surprising in that it can detect one molecule of DNA both in calculation and in the literature. In fact, one sperm D
It has been reported that NA (that is, one molecule of human DNA) can be analyzed (Li, H., Gyllensten, UB, Cui, X., Saik).
i, RK, Erlich, HAand Arnheim, N .: Nature , 335: 414-41
7,1989).

At present, there are about 160 kinds of bacterial 16S ribosomal RNA whose DNA base sequence has been determined or the DNA encoding the same, and these are registered as database. From this database, almost all 16S ribosomal RNA consists of about 1500 bases, and the DNA coding for this is 1.5 kilobase pairs (Kbp, base pairs). .. As a representative example, the nucleotide sequence of DNA encoding Escherichia coli 16S ribosomal RNA is shown in SEQ ID NO: 5. Of these about 1500 bases, complementary base sequences in which two primers anneal at two sites [519 to 536 (base sequence of SEQ ID NO: 3) and 1392 to 1406 (base sequence of SEQ ID NO: 4)] Was selected as. The criterion for this selection was that the nucleotide sequences were almost common to 160 kinds of bacteria obtained from the database.

As the universal primer used for PCR amplification, those shown in the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 were used. As the reaction conditions, each primer-5
00 nM, 4 kinds of deoxytriphosphate (dATP, d
GTP, dCTP, dTTP) 200 μM, 10 mM Tris-HCl buffer-pH 8.3, 50 mM KCl, 1.
In a solution containing 5 mM MgCl ₂ , 0.001% gelatin, 10-20 ng of bacterial DNA as a template was added, and 0.5 unit of Taq DNA polymerase was added for PCR amplification. The PCR amplification apparatus used was a DNA thermal cycler manufactured by Perkin-Elmer-Citas. The reaction conditions were 94 ° C denatured for 1 minute, annealing at 55 ° C for 2 minutes, and extension at 74 ° C for 3 minutes at 30 minutes.
It was carried out by cycle amplification.

Confirmation of the DNA after amplification is carried out by using 1.5% agarose.
Gel electrophoresis (Maniatis, T., Molecular Cloning La
boratory Mnual (1982) Cold Spring Harbor Laborator
(Y, NY) by becoming one band. Clostridium bifermenta is a bacterium confirmed to be capable of amplification other than Escherichia coli.
ns , Desulfotomaculum nigrificans , Enterobactercloa
cae , Bacillus licheniformis , Bacillus polymyx a, B
acillus cereus, Bacillus coagulan s, Klebsiella ox
ytoca , Lactobacillus casei , Lactobacillus confusu
s , Lactobacillus plantarum , Lactobacillus brevis ,
Zymomonasmobili s, Bacillus sphericu s, Bacillus ac
idocaldarius , Bacillus acidoterrestriu s, Bacillus
Cycloheptanicus , Listeria monocytogenes .

As the third step of the present invention, the details of the method of identifying the sequence of the amplified DNA, which is the most important step, will be described below. Since DNA is a polymer composed of four kinds of bases A, G, C, and T, in order to show the difference, the arrangement of these four kinds of bases A, G, C, and T,
That is, by examining the base sequence and the difference in the arrangement,
The method of identifying individual DNA is the most direct and reliable. However, in order to industrially use the DNA sequence method, there are obstacles such as facilities that can use radioactive isotopes, expensive equipment, and skilled advanced technicians.

Therefore, as a simple method, DN after PCR amplification is used.
A method was employed in which A was cleaved with a restriction enzyme that recognizes four bases, and the fragment was identified by a chromatographic separation method. This method will be specifically described below by taking Escherichia coli as an example. Escherichia coli ( E.col represented by the nucleotide sequence of SEQ ID NO: 5 )
519 of DNA encoding the 16S ribosomal RNA of i )
The 1406th to 1406th base sequences are PCR-amplified with the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 2 as a universal primer to synthesize a DNA as shown in SEQ ID NO: 6 consisting of 888 bases. To be done.

This 888-base DNA fragment was cleaved with ALU I, a restriction enzyme for 4-base recognition, that is, the AGCT portion in the DNA was found and cleaved between AG and CT as follows. Four DNA fragments are generated. That is, 132 bp (SEQ ID NO: 7), 211 bp
(SEQ ID NO: 8), 207 bp (SEQ ID NO: 9), 338b
A degradation product of p (SEQ ID NO: 10) is obtained. (Total base number is 888 bp).

The reason for using a restriction enzyme that recognizes four bases is 8
This is because it is theoretically calculated that a DNA having a length of 88 bases is cleaved to a length of about 4 to 5 pieces. The probability that four specific nucleotide sequences appear in a DNA of 888 nucleotides arranged at random is calculated (888 /
4 × 4 × 4 × 4 ≒) About 4 places, which is a degradation product D
There are 5 NA fragments. If an enzyme that recognizes 4 or more bases is used, there is a risk that it will not be cleaved at all. Conversely, if the enzyme recognizes a sequence of 4 bases or less, the number of cleaved fragments will be too large, making it difficult to separate and analyze the cleaved fragments later It is expected to become.

Similarly, when digested with another restriction enzyme, namely ACC II, 9 bp (SEQ ID NO: 11), 340 bp (SEQ ID NO: 12), 104 bp (SEQ ID NO: 13), 295 bp (SEQ ID NO: 14), 140 bp (SEQ ID NO: 15). 5 pieces of
58 bp (SEQ ID NO: 16), 531b when cleaved with HHA I
p (SEQ ID NO: 17), 136 bp (SEQ ID NO: 18), 163
Four fragments of bp (SEQ ID NO: 19) are obtained.

Furthermore, the cleavage fragments of 5 kinds of restriction enzymes are shown as follows: TAQ I has 305 bp (SEQ ID NO: 20),
53 bp (SEQ ID NO: 21), 86 bp (SEQ ID NO: 22), 3
5 of 60 bp (SEQ ID NO: 23) and 84 bp (SEQ ID NO: 24)
The fragment of the book is 96 bp for HAP II (SEQ ID NO: 25), 10
6 bp (SEQ ID NO: 26), 137 bp (SEQ ID NO: 27), 2
80 bp (SEQ ID NO: 28), 4 bp (SEQ ID NO: 29), 16
2 bp (SEQ ID NO: 30), 81 bp (SEQ ID NO: 31), 22
8 fragments of bp (SEQ ID NO: 32) are 373 in AFA I
bp (SEQ ID NO: 33), 502 bp (SEQ ID NO: 34), 13
3 fragments of bp (SEQ ID NO: 35) are 21 in HAE III
6 bp (SEQ ID NO: 36), 161 bp (SEQ ID NO: 37), 3
8 bp (SEQ ID NO: 38), 210 bp (SEQ ID NO: 39), 6
8 bp (SEQ ID NO: 40), 180 bp (SEQ ID NO: 41), 1
Seven fragments of 9 bp (SEQ ID NO: 42) are each obtained.

In order to separate the fragments cleaved by these restriction enzymes and pattern them, agarose gel electrophoresis method, high performance liquid chromatography method, capillary electrophoresis method and the like can be considered. The simplest and most effective method is the agarose gel electrophoresis method, and the following examples describe separation examples using this method.

[0027]

EXAMPLES The present invention will be described in detail below with reference to examples. However, the technical scope of the present invention is not limited by these examples.

[0028]

Example 1 [Preparation of chromosomal DNA from microorganism] Five kinds of bacteria
( Escherichia coli , B acillus subtilis , B acillus liche
niformi s, Bacillus confusu s, Bacillus stearothermoph
Each of ilis) Brain Heart Infusion Medium
(Beef brain extract 7.5 g, heart extract 8.0 g, peptone 10 g, glucose 2.0 g, sodium chloride 5.0
g, 2.5 g of potassium monohydrogen phosphate is dissolved in 1000 ml of distilled water and adjusted to pH 7.0) 37 in 100 ml
Cultivated at ℃ for 2-5 hours. The resulting culture solution is kept at 5 ° C for 10
After centrifuging at 000 rpm for 10 minutes to collect the cells as a pellet, 5 ml of 1 × TE containing 1 ml of 1% SDS
Suspended in. The suspension was heated at 100 ° C. for 5 minutes and extracted with 5 ml of chloroform: phenol (1: 1),
After the chloroform treatment, ethanol was added to the clarified solution to a final concentration of 70% to precipitate the DNA, and the temperature was kept at 5 ° C for 1 ° C.
0000RPM, centrifuged for 10 minutes, DNA 2-5μ
g was recovered. The obtained DNA was dissolved in 1 ml of TE, and the final concentration was determined by ethidium bromide staining after agarose gel electrophoresis. Figure 1 shows the UV absorption spectrum of chromosomal DNA extracted from E. coli.
A characteristic absorption of nucleic acids at 0 nm was confirmed.

[0029]

Example 2 [Synthesis of primer DNA] DNA encoding 16S ribosomal RNA of Escherichia coli sequenced by Kumano et al. (Kumano, M., Tomioka, N., Shimozaki, K. a.
nd Sugiura, M., Mol. Gen. Genet. 191, 46-50 (198
3)) Sequences complementary to the 519th to 536th and 1392nd to 1406th base sequences of (SEQ ID NO: 5), that is, the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 2 were selected as primers. .. The nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 were synthesized according to the manual using an oligo DNA synthesizer manufactured by Applied Biosystems. Then, about 50 μg of each of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 was obtained.

[0030]

Example 3 [PCR amplification of DNA] DNA (10-100 ng) extracted from Escherichia coli Escherichia coli and partially purified (1 × P) was used.
CR buffer (50 mM KCl / 10 mM Tris
-HCl, pH 8.3 / 1.5 mM MgCl2 / 0.
01% (W / V) Geratin), 1.25 mM d
NTPs, 250 ng of each primer and Taq DN
A polymerase (Perkin-Elmer Cet
us, Norwalk, ct. ) 0.5 unit is 10
It was dissolved in 0 μl and overlaid with mineral oil to prevent evaporation. This is Perkin-Elmer Cetu
PCR (Pol) using Thermocycler of s company
Ymerase Chain Reaction) reaction was performed under the following conditions. DNA was amplified at 30 ° C by performing denature at 94 ° C for 1 minute, annealing at 55 ° C for 2 minutes, and extension at 74 ° C for 3 minutes. As a result, 500 ng of amplified DNA was obtained.
The amplified DNA was confirmed by performing ethidium bromide staining by 2% agarose gel electrophoresis or 6% polyacrylamide gel electrophoresis.

[0031]

Example 4 [Determination of Nucleotide Sequence of Amplified DNA] (1) Subcloning to M13mp-based Phage DN of M13mp18 Phage (Toyobo Co., Ltd.)
A50 ng is cleaved with a restriction enzyme SmaI 1.5 unit, and 12 ng of the DNA amplified in Example 3 and 2.0 unit of T4 DNA ligase are reacted with each other to ligate both. 50 ng vector DNA, insert DNA
12 ng of 66 mM Tris.HCl (pH 7.
6), 1 mM ATP, 1 mM Spelmidin,
It was dissolved in a buffer solution of 10 mM MgCl ₂ and 15 mM DTT, and 2.0 units of T4 DNA ligase was added to the solution.
Ligation was carried out at 16 ° C for 16 hours. The resulting reaction solution was mixed with 100 µl of competent cells of Escherichia coli JM109 (manufactured by Toyobo Co., Ltd.) prepared by the calcium chloride method, and the mixture was instantaneously heat treated at 42 ° C for 2 minutes at 40 ° C for 40 minutes in ice for transfection. To complete.
Stored again on ice, 100 μl of the competent cells after this transfection and 0.6-0.7% 2 × TY
Agarose 3.0 mL, 100 mM IPTG 25 μl,
E. coli JM105 100 μl after overnight culture, 2% X-
Mix the Gal / dimethylformamide solution at 42 ° C and sprinkle on LB plates. After culturing at 37 ° C. overnight, semitransparent plaques are collected. Recombinant E. coli transformed with the DNA-inserted phage form translucent plaques. (2) Preparation of single-stranded DNA 20 μl of E. coli JM109 after overnight culture was added to 2 × TY medium (trypton 16 g, yeast extract 10).
g, NaCl 5 g dissolved in distilled water 1000 ml) 2 mL
And incubate with shaking at 37 ° C. for 5 to 6 hours. 120
00RPM, 5% centrifugation and 20% PEG60 in the supernatant
00 (polyethylene glycol), 2.5M NaCl
200 μl of the solution is added to precipitate single-stranded DNA. 12
After centrifugation at 000 RPM for 5 minutes, the phage pellet is suspended in 100 μl of 10 mM TE. To this suspension, 100 μl of phenol: chloroform (1: 1) was added, and the mixture was stirred and allowed to stand at room temperature and then centrifuged for 3 minutes. Then, 750 μl of ethanol was added to the aqueous layer to precipitate DNA, and bacterial DN was added.
18 ng of single-stranded DNA of A-inserted phage was obtained. (3) Dideoxy reaction G, A, T, C dN in four Eppendorf tubes
2 μl each of the TP / ddNTP mixed solution was dispensed, and the primer DNA was annealed to 16.
7 μM dCTP 1.0 μl, [α-32P] dCTP
(3000 Ci / mmol) 1.0 μl, Klenow
Add 1.0 μl of enzyme and incubate at 37 ° C. for 2 minutes.
Then, an acrylamide sequence gel was prepared, and after electrophoresis, autoradiography was performed. Then, the sharp band of the electrophoresis was confirmed, and the base sequence of the DNA was determined by examining the bands in each lane in order.

[0032]

[Example 5] [Cleavage of DNA fragment consisting of 888 bases by restriction enzyme] PCR-amplified DNA fragment 1-8 of 888 bases
μg is diluted with 70 μl of TE buffer and dispensed into each 7 eppendorf tubes. AluI,
AccII, HhaI, TaqI, HapI,
One unit of a restriction enzyme (Boehringer Mannheim / Yamanouchi Co., Ltd.) that recognizes four bases of AfaI and HaeIII was added, and the mixture was treated at 37 ° C. for 30 minutes in an optimum reaction buffer of each enzyme.

[0033]

Example 6 [Separation of DNA consisting of 888 bases after restriction enzyme treatment]
The DNA digested with the restriction enzyme was heated at 100 ° C. for 2 minutes to inactivate the enzyme, and then treated with phenol. This DNA
The decomposed product was subjected to electrophoresis on 4% NuSieve GTG agarose gel (manufactured by Takara Shuzo Co., Ltd.) to separate the cleaved fragments. The results are shown in 5 types of microorganisms ( Escherichia col
i, Bacillus subtili s, Bacillus licheniform is , Bacillu
s confusus , B acillus stearothermophilis )
The degradation products of A are shown in FIGS.

[0034]

Example 7 [Separation of DNA consisting of 888 bases after restriction enzyme treatment]
With respect to those derived from Escherichia coli, the DNA after digestion with the restriction enzyme was heated at 100 ° C. for 2 minutes to inactivate the enzyme and treated with phenol. This DNA degradation product 10μ
g was subjected to high performance liquid chromatography to separate the cleaved fragments. The results are shown in Fig. 7. In addition, high performance liquid chromatography uses TSKgel DEAE-NP as a column.
R using 20 mM Tris-HCl (pH
9.0) and 1.0 M NaCl were used.

[0035]

[Example 8] [Confirmed that the contaminating microorganism of food is Bacillus licheniformis ] The contaminated bacterium derived from almond was Brain Hear
DNA was prepared according to Example 1 by culturing in t Infusion Medium at 37 ° C. for 3 to 5 hours, and then PCR was carried out in the same manner as in Example 3 to obtain 500 μg of the amplified DNA.
Got This DNA is AluI, AccII, HhaI, TaqI, HapI
After treatment with 7 types of restriction enzymes I, AfaI and HaeIII, the DN
A was heated at 100 ° C. for 2 minutes to inactivate the enzyme and treated with phenol. The obtained DNA degradation product is 4% NuSi
The cleaved fragments were separated by subjecting eve GTG agarose gel (Takara Shuzo Co., Ltd.) to electrophoresis to obtain a separation pattern shown in FIG. This electrophoretic separation pattern is shown in FIGS.
As a result of comparison with the electrophoretic separation pattern of 5 kinds of bacteria,
The pattern of FIG. 8 completely coincided with the electrophoretic separation pattern of Bacillus licheniformis (FIG. 4). From this, it was confirmed that the bacterium contaminating almonds was Bacillus licheniformis .

[0036]

Example 9 [Confirmed that two kinds of contaminating microorganisms in food are different kinds of bacteria] Two kinds of contaminating bacteria A and B derived from fruit juice raw material
In Brain Heart Infusion Medium at 37 ℃,
Incubate for 3-5 hours to prepare DNA according to Example 1,
Then, this was carried out in the same manner as in Example 3 to carry out PCR amplification to obtain 500 μg of amplified DNA. This DNA is AluI, AccI
After treatment with 7 kinds of restriction enzymes of I, HhaI, TaqI, HapII, AfaI and HaeIII, the DNA was heated at 100 ° C. for 2 minutes to inactivate the enzyme and treated with phenol. The resulting DNA degradation product was subjected to electrophoresis on a 4% NuSieve GTG agarose gel (Takara Shuzo Co., Ltd.) to separate the cleaved fragments, and the separation patterns shown in FIGS. 9 and 10 were obtained. And when comparing the separation patterns of both, ACCII,
The cut bands of HAPII and AFAI did not match. From the above, it was confirmed that the two kinds of contaminating bacteria are different kinds of bacteria.

[0037]

INDUSTRIAL APPLICABILITY According to the present invention, a DNA base sequence can be specified with good reproducibility, and in particular, a gene DNA derived from a bacterium can be specified and the bacterium can be easily classified / identified. This method can be widely used in industrial fields such as chemical, medical, food, electronics (high-density integrated circuit), environment, etc. where it is necessary to rapidly detect contaminating bacteria or useful bacteria.

[0038]

[Sequence Listing] (1) SEQ ID NO: 1 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Origin Biological name Strain name Sequence Features Symbols representing features: Location: 1. ． 18 sequence ^{5 'CAGCAGCCGC GGTAATAC3' 18 (2} ) SEQ ID NO: length of the two sequences: the number of a nucleic acid strand:: 15 sequence types single strand Topology: linear sequence type: other nucleic acid synthetic DNA origin Organism Strain name Sequence features Characteristic symbols: Location: 1. ． 15 (3) SEQ ID NO: 3 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Escherichia coli Strain name: K12 Sequence features Symbols that represent features: primer bind Location: 1. ． 18 sequence ^{5 'CAGCAGCCGC GGTAATAC3' 18 (4} ) SEQ ID NO: 4 sequence Length: 15 Type of sequence: number of nucleic acid strands: double-stranded Topology: linear sequence type: Genomic DNA Origin Organism: Escherichia coli (Escherichia coli) Strain name: K12 Sequence features Characteristic symbol: primer bind Location: 1. ． 15 Sequence 5 ^′ GTACACACCG CCCGT3 ^′ 15 (5) SEQ ID NO: 5 Sequence length: 1542 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Escherichia coli (Escherichia coli) Strain name: K12 Sequence features Characteristic symbols: rRNA DNA encoding 16S ribosomal RNA of Escherichia coli Location: 1. ． 1542 SEQ AAATTGAAGA GTTTGATCAT GGCTCAGATT GAACGCTGGC GGCAGGCCTA 50 ACACATGCAA GTCGAACGGT AACAGGAAGA AGCTTGCTTC TTTGCTGACG 100 AGTGGCGGAC GGGTGAGTAA TGTCTGGGAA ACTGCCTGAT GGAGGGGGAT 150 AACTACTGGA AACGGTAGCT AATACCTCAT AACGTCGCAA GACCAAAGAG 200 GGGGACCTTC GGGCCTCTTG CCATCGGATG TGCCCAGATG GGATTAGCTA 250 GTAGGTGGGG TAACGGCTCA CCTAGGCGAC GATCCCTAGC TGGTCTGAGA 300 GGATGACCAG CCACACTGGA ACTGAGACAC GGTTTAGACT CCTACGGGAG 350 GCAGCAGTGG GGAATATTGC ACAATGGGCG CAAGCCTGAT GCAGCCATGC 400 CGCGTGTATG AAGAAGGCCT TCGGGTTGTA AAGTACTTTC AGCGGGGAGG 450 AAGGGAGTAA AGTTAATACC TTTGCTCATT GACGTTACCC GCAGAAGAAG 500 CACCGGCTAA CTCCGTGCCA GCAGCCGCGG TAATACGGAG GGTGCAAGCG 550 TTAATCGGAA TTACTGGGCG TAAAGCGCAC GCAGGCGGTT TGTTAAGTCA 600 GATGTGAAAT CCCCGGGCTC AACCTGGGAA CTGCATCTGA TACTGGCAAG 650 CTTGAGTCTC GTAGAGGGGG GTAGAATTCC AGGTGTAGCG GTGAAATGCG 700 TAGAGATCTG GAGGAATACC GGTGGCGAAG GCGGCCCCCT GGACGAAGAC 750 TGACGCTCAG GTGCGAAAGC GTGGGGAGCA AACAGGATTA GATACCCTGG 800 TAGTCCACGC CGTAAACGAT GTCGACTTGG AGGT TGTGCC CTTGAGGCGT 850 GGCTTCCGGA GCTAACGCGT TAAGTCGACC GTCTGGGGAG TACGGCCGCA 900 AGGTTAAAAC TCAAATGAAT TGACGGGGGC CCGCACAAGC GGTGGAGCAT 950 GTGGTTTAAT TCGATGCAAC GCGAAGAACC TTACCTGGTC TTGACATCCA 1000 CGGAAGTTTT CAGAGATGAG AATGTGCCTT CGGGAACCGT GAGACAGGTG 1050 CTGCATGGCT GTCGTCAGCT CGTGTTGTGA AATGTTGGGT TAAGTCCCGC 1100 AACGAGCGCA ACCCTTATCC TTTGTTGCCA GCGGTCCGGC CGGGAACTCA 1150 AAGGAGACTG CCAGTGATAA ACTGGAGGAA GGTGGGGATG ACGTCAAGTC 1200 ATCATGGCCC TTACGACCAG GGCTACACAC GTGCTACAAT GGCGCATACA 1250 AAGAGAAGCG ACCTCGCGAG AGCAAGCGGA CCTCATAAAG TGCGTCGTAG 1300 TCCGGATTGG AGTCTGCAAC TCGACTCCAT GAAGTCGGAA TCGCTAGTAA 1350 TCGTGGATCA GAATGCCACG GTGAATACGT TCCCGGGCCT TGACCACACC 1400 GCCCGTCACA CCATGGGAGT GGGTTGCAAA AGAAGTAGGT AGCTTAACCT 1450 TCGGGAGGGC GCTTACCACT TTGTGATTCA TGACTGGGGT GAAGTCGTAA 1500 CAAGGTAACC GTAGGGGAAC CTGCGGTTGG ATCACCTCCT TA 1542 (6) SEQ ID NO: 6 SEQ length: 888 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: Ge Origin of nomic DNA Organism name: Escherichia coli Strain name: K12 Sequence features Characteristic symbols: rRNA Location: 1. ． 888 sequence CAGCAGCCGC GGTAATACGG AGGGTGCAAG CGTTAATCGG AATTACTGGG 50 CGTAAAGCGC ACGCAGGCGG TTTGTTAAGT CAGATGTGAA ATCCCCGGGC 100 TCAACCTGGG AACTGCATCT GATACTGGCA AGCTTGAGTC TCGTAGAGGG 150 GGGTAGAATT CCAGGTGTAG CGGTGAAATG CGTAGAGATC TGGAGGAATA 200 CCGGTGGCGA AGGCGGCCCC CTGGACGAAG ACTGACGCTC AGGTGCGAAA 250 GCGTGGGGAG CAAACAGGAT TAGATACCCT GGTAGTCCAC GCCGTAAACG 300 ATGTCGACTT GGAGGTTGTG CCCTTGAGGC GTGGCTTCCG GAGCTAACGC 350 GTTAAGTCGA CCGTCTGGGG AGTACGGCCG CAAGGTTAAA ACTCAAATGA 400 ATTGACGGGG GCCCGCACAA GCGGTGGAGC ATGTGGTTTA ATTCGATGCA 450 ACGCGAAGAA CCTTACCTGG TCTTGACATC CACGGAAGTT TTCAGAGATG 500 AGAATGTGCC TTCGGGAACC GTGAGACAGG TGCTGCATGG CTGTCGTCAG 550 CTCGTGTTGT GAAATGTTGG GTTAAGTCCC GCAACGAGCG CAACCCTTAT 600 CCTTTGTTGC CAGCGGTCCG GCCGGGAACT CAAAGGAGAC TGCCAGTGAT 650 AAACTGGAGG AAGGTGGGGA TGACGTCAAG TCATCATGGC CCTTACGACC 700 AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGACCTCGCG 750 AGAGCAAGCG GACCTCATAA AGTGCGTCGT AGTCCGGATT GGAGTCTGCA 800 ACTCGACTCC ATGAAGTCGG AATCGCTAGT AATCGTG GAT CAGAATGCCA 850 CGGTGAATAC GTTCCCGGGC CTTGTACACA CCGCCCGT 888 (7) SEQ ID NO: 7 Sequence length: 132 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Escherichia coli (Escherichia coli) Strain name: Features of K12 sequence Characteristic symbol: rRNA Cleavage fragment of DNA encoding 16S ribosomal RNA of Escherichia coli by restriction enzyme ALU I Location: 1. ． 132 sequence CAGCAGCCGC GGTAATACGG AGGGTGCAAG CGTTAATCGG AATTACTGGG 50 CGTAAAGCGC ACGCAGGCGG TTTGTTAAGT CAGATGTGAA ATCCCCGGGC 100 TCAACCTGGG AACTGCATCT GATACTGGCA AG 132 (8) SEQ ID NO: 8 Sequence length: 211 strands Sequence type: Nucleotide type: Nucleotide type: Nucleotide sequence type: Nucleic acid type Type: Genomic DNA Origin organism name: Escherichia coli Strain name: K12 Sequence features Characteristic symbols: rRNA Cleavage fragment of Escherichia coli 16S ribosomal RNA by restriction enzyme ALU I Location : 1. ． 211 sequences CTTGAGTCTC GTAGAGGGGG GTAGAATTCC AGGTGTAGCG GTGAAATGCG 50 TAGAGATCTG GAGGAATACC GGTGGCGAAG GCGGCCCCCT GGACGAAGACAG TG: 211 sequences 207: GC sequences 207 : Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Escherichia coli Strain name: K12 Sequence characteristics Characteristic symbol: rRNA DNA encoding 16S ribosomal RNA of Escherichia coli Cleavage fragment by restriction enzyme ALU I of 1. ． 207 sequence CTAACGCGTT AAGTCGACCG TCTGGGGAGT ACGGCCGCAA GGTTAAAACT CAAATGAATT GACGGGGGCC CGCACAAGCG GTGGAGCATG TGGTTTAATT CGATGCAACG CGAAGAACCT TACCTGGTCT TGACATG: 10 TG: 3 sequences: GGAAGAACCT TACCTGCGTG GGAG: 3 sequences Type of linear sequence: Genomic DNA origin Organism name: Escherichia coli Strain name: K12 Sequence features Characteristic symbols: rRNA Escherichia coli 16S ribosomal RNA cleaving with restriction enzyme ALU I Location of fragment: 1. ． 338 sequence CTCGTGTTGT GAAATGTTGG GTTAAGTCCC GCAACGAGCG CAACCCTTAT 50 CCTTTGTTGC CAGCGGTCCG GCCGGGAACT CAAAGGAGAC TGCCAGTGAT 100 AAACTGGAGG AAGGTGGGGA TGACGTCAAG TCATCATGGC CCTTACGACC 150 AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGACCTCGCG 200 AGAGCAAGCG GACCTCATAA AGTGCGTCGT AGTCCGGATT GGAGTCTGCA 250 ACTCGACTCC ATGAAGTCGG AATCGCTAGT AATCGTGGAT CAGAATGCCA 300 CGGTGAATAC GTTCCCGGGC CTTGTACACA CCGCCCGT 338 (11) SEQ ID NO: 11 Sequence length: 9 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Escherichia coli Strain name: K12 Sequence features Characteristic symbols : RRNA Cleavage fragment of Escherichia coli 16S ribosomal RNA cleaved by restriction enzyme ACC II Location: 1. ． 9 Sequence CAGCAGCCG 9 (12) SEQ ID NO: 12 Sequence length: 340 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: Genomic DNA Origin organism name: Escherichia coli Strain name: Characteristics of K12 sequence Characteristic symbol: rRNA Cleavage fragment of DNA encoding 16S ribosomal RNA of Escherichia coli by restriction enzyme ACC II Location: 1. ． 340 sequence CGGTAATACG GAGGGTGCAA GCGTTAATCG GAATTACTGG GCGTAAAGCG 50 CACGCAGGCG GTTTGTTAAG TCAGATGTGA AATCCCCGGG CTCAACCTGG 100 GAACTGCATC TGATACTGGC AAGCTTGAGT CTCGTAGAGG GGGGTAGAAT 150 TCCAGGTGTA GCGGTGAAAT GCGTAGAGAT CTGGAGGAAT ACCGGTGGCG 200 AAGGCGGCCC CCTGGACGAA GACTGACGCT CAGGTGCGAA AGCGTGGGGA 250 GCAAACAGGA TTAGATACCC TGGTAGTCCA CGCCGTAAAC GATGTCGACT 300 TGGAGGTTGT GCCCTTGAGG CGTGGCTTCC GGAGCTAACG 340 (13) SEQ ID NO: 13 Sequence length: 104 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Escherichia coli Strain name: K12 Sequence features Characteristic symbols : RRNA Cleavage fragment of Escherichia coli 16S ribosomal RNA cleaved by restriction enzyme ACC II Location: 1. ． 104 sequence CGTTAAGTCG ACCGTCTGGG GAGTACGGCC GCAAGGTTAA AACTCAAATG 50 AATTGACGGG GGCCCGCACA AGCGGTGGAG CATGTGGTTT AATTCGATGC 100 AACG 104 (14) SEQ ID NO: 14 Sequence length: 295 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear topology Genomic DNA Origin organism name: Escherichia coli Strain name: K12 Sequence features Characteristic symbols: rRNA Cleavage fragment of Escherichia coli 16S ribosomal RNA encoding enzyme ACC II Location: 1. ． 295 sequence CGAAGAACCT TACCTGGTCT TGACATCCAC GGAAGTTTTC AGAGATGAGA 50 ATGTGCCTTC GGGAACCGTG AGACAGGTGC TGCATGGCTG TCGTCAGCTC 100 GTGTTGTGAA ATGTTGGGTT AAGTCCCGCA ACGAGCGCAA CCCTTATCCT 150 TTGTTGCCAG CGGTCCGGCC GGGAACTCAA AGGAGACTGC CAGTGATAAA 200 CTGGAGGAAG GTGGGGATGA CGTCAAGTCA TCATGGCCCT TACGACCAGG 250 GCTACACACG TGCTACAATG GCGCATACAA AGAGAAGCGA CCTCG 295 (15) SEQ ID NO: 15 Length of sequence: 140 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Escherichia coli Strain name: K12 Sequence features Characteristic symbols: rRNA Escherichia coli Cleavage fragment of DNA encoding 16S ribosomal RNA by ACC II restriction enzyme. Location: 1. ． 140 sequences CGAGAGCAAG CGGACCTCAT AAAGTGCGTC GTAGTCCGGA TTGGAGTCTG 50 CAACTCGACT CCATGAAGTC GGAATCGCTA GTAATCGTGG ATCAGAATGC 100 CACGGTGAAT ACGTTCCCGG GCCTTGTACA CACCGCCCGT 140 (16) SEQ ID NO: 16 Sequence length: Nucleic acid strands: Nucleotide sequence number: 58 sequences Number of sequences: 58 sequences Type: Genomic DNA Origin Biological name: Escherichia coli Strain name: K12 Sequence characteristics Characteristic symbols: rRNA Cleavage fragment of Escherichia coli 16S ribosomal RNA cleaved by restriction enzyme HHA I : 1. ． 58 sequence CAGCAGCCGC GGTAATACGG AGGGTGCAAG CGTTAATCGG AATTACTGGG 50 CGTAAAGC 58 (17) SEQ ID NO: 17 Sequence length: 531 Sequence type: Nucleic acid Strand number: Double strand Topology: Linear Sequence type: Genomic DNA origin Organism name: Escherichia coli strain name: K12 Sequence features Characteristic symbol: rRNA Cleavage fragment of DNA encoding 16S ribosomal RNA of Escherichia coli by restriction enzyme HHA I Characteristic symbol: rRNA Location: 1. ． 531 sequence GCACGCAGGC GGTTTGTTAA GTCAGATGTG AAATCCCCGG GCTCAACCTG 50 GGAACTGCAT CTGATACTGG CAAGCTTGAG TCTCGTAGAG GGGGGTAGAA 100 TTCCAGGTGT AGCGGTGAAA TGCGTAGAGA TCTGGAGGAA TACCGGTGGC 150 GAAGGCGGCC CCCTGGACGA AGACTGACGC TCAGGTGCGA AAGCGTGGGG 200 AGCAAACAGG ATTAGATACC CTGGTAGTCC ACGCCGTAAA CGATGTCGAC 250 TTGGAGGTTG TGCCCTTGAG GCGTGGCTTC CGGAGCTAAC GCGTTAAGTC 300 GACCGTCTGG GGAGTACGGC CGCAAGGTTA AAACTCAAAT GAATTGACGG 350 GGGCCCGCAC AAGCGGTGGA GCATGTGGTT TAATTCGATG CAACGCGAAG 400 AACCTTACCT GGTCTTGACA TCCACGGAAG TTTTCAGAGA TGAGAATGTG 450 CCTTCGGGAA CCGTGAGACA GGTGCTGCAT GGCTGTCGTC AGCTCGTGTT 500 GTGAAATGTT GGGTTAAGTC CCGCAACGAG C 531 (18) SEQ ID NO: 18 Sequence length: 136 Sequence type: Nucleotide type: Nucleotide type: Nucleotide type: Nucleotide type: Nucleotide type: Nucleotide type: Nucleotide type: Nucleotide type: Nucleotide type: Nucleotide type: Nucleotide type : Genomic DNA origin Biological name: Escherichia coli Strain name: K12 Sequence characteristics Characteristic symbol: rRNA Escherichia coli 16S ribosomal RNA Cleavage fragments present position by the restriction enzyme HHA I de be DNA: 1. ． 136 sequences GCAACCCTTA TCCTTTGTTG CCAGCGGTCC GGCCGGGAAC TCAAAGGAGA 50 CTGCCAGTGA TAAACTGGAG GAAGGTGGGG ATGACGTCAA GTCATCATGG 100 CCCTTACGAC CAGGGCTACA CACGTGCTAC AATGGC 136 (19) SEQ ID NO: 19 Sequence length: 163 linear sequences: Nucleotide sequence: Nucleotide type: Nucleotide sequence number: 163 Type: Genomic DNA Origin organism name: Escherichia coli Strain name: K12 sequence characteristics Characteristic symbol: rRNA Cleavage fragment of Escherichia coli 16S ribosomal RNA by HHA I : 1. ． 163 Sequences GCATACAAAG AGAAGCGACC TCGCGAGAGC AAGCGGACCT CATAAAGTGC 50 GTCGTAGTCC GGATTGGAGT CTGCAACTCG ACTCCATGAA GTCGGAATCG 100 CTAGTAATCG TGGATCAGAA TGCCACGGTG AACGTTCC CGGGCCTTGT 150 ACACACCGCC 163 Type of linear sequence: Genomic DNA origin Organism name: Escherichia coli Strain name: K12 Sequence characteristics Characteristic symbol: rRNA Escherichia coli 16S ribosomal RNA by TAQ I restriction enzyme of DNA encoding 16S ribosomal RNA Location of the cleaved fragment: 1. ． 305 sequence CAGCAGCCGC GGTAATACGG AGGGTGCAAG CGTTAATCGG AATTACTGGG 50 CGTAAAGCGC ACGCAGGCGG TTTGTTAAGT CAGATGTGAA ATCCCCGGGC 100 TCAACCTGGG AACTGCATCT GATACTGGCA AGCTTGAGTC TCGTAGAGGG 150 GGGTAGAATT CCAGGTGTAG CGGTGAAATG CGTAGAGATC TGGAGGAATA 200 CCGGTGGCGA AGGCGGCCCC CTGGACGAAG ACTGACGCTC AGGTGCGAAA 250 GCGTGGGGAG CAAACAGGAT TAGATACCCT GGTAGTCCAC GCCGTAAACG 300 ATGTC 305 (21) SEQ ID NO: 21 sequence length Length: 53 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Escherichia coli Strain name: K12 Sequence features Characteristic symbols: rRNA Escherichia Cleavage fragment of DNA encoding Escherichia coli 16S ribosomal RNA by the restriction enzyme TAQ I. Location: 1. ． 53 Sequence GACTTGGAGG TTGTGCCCTT GAGGCGTGGC TTCCGGAGCT AACGCGTTAA 50 GTC 53 (22) SEQ ID NO: 22 Sequence Length: 86 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Genomic DNA Origin Organism Name: Escherichia coli strain name: K12 Features of sequence Characteristic symbol: rRNA Cleavage fragment of DNA encoding 16S ribosomal RNA of Escherichia coli by restriction enzyme TAQ I Location: 1. ． 86 sequence GACCGTCTGG GGAGTACGGC CGCAAGGTTA AAACTCAAAT GAATTGACGG 50 GGGCCCGCAC AAGCGGTGGA GCATGTGGTT TAATTC 86 (23) SEQ ID NO: 23 sequence length: 360 sequence type: number of nucleic acid strands: double strand topology: linear sequence type: Genomic DNA origin Organism name: Escherichia coli Strain name: K12 Sequence features Characteristic symbols: rRNA Cleavage fragment of Escherichia coli DNA encoding 16S ribosomal RNA by restriction enzyme TAQ I Location: 1. ． 360 sequence GATGCAACGC GAAGAACCTT ACCTGGTCTT GACATCCACG GAAGTTTTCA 50 GAGATGAGAA TGTGCCTTCG GGAACCGTGA GACAGGTGCT GCATGGCTGT 100 CGTCAGCTCG TGTTGTGAAA TGTTGGGTTA AGTCCCGCAA CGAGCGCAAC 150 CCTTATCCTT TGTTGCCAGC GGTCCGGCCG GGAACTCAAA GGAGACTGCC 200 AGTGATAAAC TGGAGGAAGG TGGGGATGAC GTCAAGTCAT CATGGCCCTT 250 ACGACCAGGG CTACACACGT GCTACAATGG CGCATACAAA GAGAAGCGAC 300 CTCGCGAGAG CAAGCGGACC TCATAAAGTG CGTCGTAGTC CGGATTGGAG 350 TCTGCAACTC 360 (24) arranged Number: 24 Sequence length: 84 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Biological name: Escherichia coli Strain name: K12 Sequence characteristics Features Symbol for: rRNA Cleavage fragment of the DNA encoding 16S ribosomal RNA of Escherichia coli cleaved by the restriction enzyme TAQ I Location: 1. ． 84 sequences GACTCCATGA AGTCGGAATC GCTAGTAATC GTGGATCAGA ATGCCACGGT 50 GAATACGTTC CCGGGCCTTG TACACACCGC CCGT 84 (25) SEQ ID NO: 25 Sequence length: 96 Sequence type: Nucleic acid Strand number: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism name: Escherichia coli Strain name: K12 Features of sequence Characteristic symbol: rRNA Cleavage fragment of DNA encoding 16S ribosomal RNA of Escherichia coli by restriction enzyme HAP II Location: 1. ． 96 sequences CAGCAGCCGC GGTAATACGG AGGGTGCAAG CGTTAATCGG AATTACTGGG 50 CGTAAAGCGC ACGCAGGCGG TTTGTTAAGT CAGATGTGAA ATCCCC 96 (26) SEQ ID NO: 26 Sequence length: 106 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear DNA sequence type: Genomic Origin Organism name: Escherichia coli Strain name: K12 Sequence characteristics Characteristic symbols: rRNA Cleavage fragment of DNA encoding 16S ribosomal RNA of Escherichia coli by restriction enzyme HAP II Location: 1. ． 106 Sequence GGGCTCAACC TGGGAACTGC ATCTGATACT GGCAAGCTTG AGTCTCGTAG 50 AGGGGGGTAG AATTCCAGGT GTAGCGGTGA AATGCGTAGA GATCTGGAGG 100 AATACC 106 (27) SEQ ID NO: 27 Sequence length: 137 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Genomic DNA Origin organism name: Escherichia coli Strain name: K12 Sequence features Characteristic symbols: rRNA Cleavage fragment of DNA encoding 16S ribosomal RNA of Escherichia coli by restriction enzyme HAP II Location: 1. ． 137 Sequences GGTGGCGAAG GCGGCCCCCT GGACGAAGAC TGACGCTCAG GTGCGAAAGC 50 GTGGGGAGCA AACAGGATTA GATACCCTGG TAGTCCACGC CGTAAACGAT 100 GTCGACTTGG AGGTTGTGCC CTTGAGGCGT GGCTTCC 137 (28) SEQ ID NO: 28 Nucleotide Sequence Number: 28 Nucleotide Sequence Number: 28 Nucleotide Sequence Number: 28 Nucleotide Sequence Number: 28 Nucleotide Sequence Number: 28 Type: Genomic DNA Origin organism name: Escherichia coli Strain name: K12 Sequence features Characteristic symbols: rRNA Cleavage fragment of Escherichia coli 16S ribosomal RNA by HAP II restriction enzyme : 1. ． 280 sequence GGAGCTAACG CGTTAAGTCG ACCGTCTGGG GAGTACGGCC GCAAGGTTAA 50 AACTCAAATG AATTGACGGG GGCCCGCACA AGCGGTGGAG CATGTGGTTT 100 AATTCGATGC AACGCGAAGA ACCTTACCTG GTCTTGACAT CCACGGAAGT 150 TTTCAGAGAT GAGAATGTGC CTTCGGGAAC CGTGAGACAG GTGCTGCATG 200 GCTGTCGTCA GCTCGTGTTG TGAAATGTTG GGTTAAGTCC CGCAACGAGC 250 GCAACCCTTA TCCTTTGTTG CCAGCGGTCC 280 (29) SEQ ID NO: 29 SEQ Length: 4 sequence Type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: Genomic DNA Origin organism name: Escherichia coli Strain name: K12 Sequence characteristics Characteristic symbol: rRNA Escherichia coli 16S ribozo -Cleavage fragment of DNA encoding mulRNA by restriction enzyme HAP II Location: 1. ． 4 Sequence GGCC 4 (30) SEQ ID NO: 30 Sequence Length: 162 Sequence Type: Nucleic Acid Number of Strands: Double-Strand Topology: Linear Sequence Type: Genomic DNA Origin Biological Name: Escherichia coli Strain name: Features of K12 sequence Characteristic symbol: rRNA Cleavage fragment of DNA encoding 16S ribosomal RNA of Escherichia coli by restriction enzyme HAP II Location: 1. ． 162 sequences GGGAACTCAA AGGAGACTGC CAGTGATAAA CTGGAGGAAG GTGGGGATGA 50 CGTCAAGTCA TCATGGCCCT TACGACCAGG GCTACACACG TGCTACAATG 100 GCGCATACAA AGAGAAGCGA CCTCGCGAGA GCAAGCGGAC CTCATAAAGT 150 GCGTCGTAGT CC 162 (31) SEQ ID NO: 162 (31) Sequence: SEQ ID NO: 162 (31) Type of linear sequence: Origin of genomic DNA Organism name: Escherichia coli Strain name: K12 Characteristic of sequence Characteristic symbol: rRNA Escherichia coli 16S ribosomal RNA by the restriction enzyme HAP II of DNA encoding RNA Location of the cleaved fragment: 1. ． 81 sequence GGATTGGAGT CTGCAACTCG ACTCCATGAA GTCGGAATCG CTAGTAATCG 50 TGGATCAGAA TGCCACGGTG AATACGTTCC C 81 (32) SEQ ID NO: 32 Sequence type: 22 Sequence type: Nucleic acid Strand number: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism name: Escherichia coli Strain name: K12 Sequence characteristics Characteristic symbol: rRNA Cleavage fragment of Escherichia coli 16S ribosomal RNA-encoding DNA with restriction enzyme HAP II Location: 1. ． 22 Sequence GGGCCTTGTA CACACCGCCC GT 22 (33) SEQ ID NO: 33 Sequence Length: 373 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Genomic DNA Origin Biological Name: Escherichia coli coli) Strain name: Features of K12 sequence Characteristic symbol: rRNA Cleavage fragment of DNA encoding 16S ribosomal RNA of Escherichia coli by restriction enzyme AFA I Location: 1. ． 373 sequence CAGCAGCCGC GGTAATACGG AGGGTGCAAG CGTTAATCGG AATTACTGGG 50 CGTAAAGCGC ACGCAGGCGG TTTGTTAAGT CAGATGTGAA ATCCCCGGGC 100 TCAACCTGGG AACTGCATCT GATACTGGCA AGCTTGAGTC TCGTAGAGGG 150 GGGTAGAATT CCAGGTGTAG CGGTGAAATG CGTAGAGATC TGGAGGAATA 200 CCGGTGGCGA AGGCGGCCCC CTGGACGAAG ACTGACGCTC AGGTGCGAAA 250 GCGTGGGGAG CAAACAGGAT TAGATACCCT GGTAGTCCAC GCCGTAAACG 300 ATGTCGACTT GGAGGTTGTG CCCTTGAGGC GTGGCTTCCG GAGCTAACGC 350 GTTAAGTCGA CCGTCTGGGG AGT 373 (34 ) SEQ ID NO: 34 Sequence length: 502 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Biological name: Escherichia coli Strain name: K12 Sequence Features Cleavage fragment of DNA encoding Escherichia coli 16S ribosomal RNA by restriction enzyme AFA I Characteristic symbol: rRNA Location: 1. ． 502 sequence ACGGCCGCAA GGTTAAAACT CAAATGAATT GACGGGGGCC CGCACAAGCG 50 GTGGAGCATG TGGTTTAATT CGATGCAACG CGAAGAACCT TACCTGGTCT 100 TGACATCCAC GGAAGTTTTC AGAGATGAGA ATGTGCCTTC GGGAACCGTG 150 AGACAGGTGC TGCATGGCTG TCGTCAGCTC GTGTTGTGAA ATGTTGGGTT 200 AAGTCCCGCA ACGAGCGCAA CCCTTATCCT TTGTTGCCAG CGGTCCGGCC 250 GGGAACTCAA AGGAGACTGC CAGTGATAAA CTGGAGGAAG GTGGGGATGA 300 CGTCAAGTCA TCATGGCCCT TACGACCAGG GCTACACACG TGCTACAATG 350 GCGCATACAA AGAGAAGCGA CCTCGCGAGA GCAAGCGGAC CTCATAAAGT 400 GCGTCGTAGT CCGGATTGGA GTCTGCAACT CGACTCCATG AAGTCGGAAT 450 CGCTAGTAAT CGTGGATCAG AATGCCACGG TGAATACGTT CCCGGGCCTT 500 GT 502 (35) SEQ ID NO: 35 Sequence length: 13 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Genomic DNA sequence Origin Organism name: Escherichia coli Strain name: K12 Sequence characteristics Characteristic symbol: rRNA Restriction enzyme of DNA encoding 16S ribosomal RNA of Escherichia coli Cleavage fragment by AFA I Location: 1. ． 13 Sequence ACACACCGCC CGT 13 (36) SEQ ID NO: 36 Sequence Length: 216 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Genomic DNA Origin Organism Name: Escherichia coli ) Strain name: Features of K12 sequence Characteristic symbol: rRNA Cleavage fragment of Escherichia coli 16S ribosomal RNA encoding fragment with restriction enzyme HAE 3 Location: 1. ． 216 sequences CAGCAGCCGC GGTAATACGG AGGGTGCAAG CGTTAATCGG AATTACTGGG 50 CGTAAAGCGC ACGCAGGCGG TTTGTTAAGT CAGATGTGAA ATCCCCGGGC AG: GCAACT37CATG: GGAACTGGCA AG TG: AGGATGGC216 AG: GGAACTAGGCG AG: GCGAGAGCAG: GCGAGAGCAG: GCGAGAGCAG: GCGAGAGCAG: GCGAGAGCAG: GCGAGAGCAG: CGGAGAGCAG: CGGAGAGCAG: CGGAGAGCAG: CGGAGAGCAG: CGGAGAGCAG: CGGAAG: CGGAAG: CGGAAG: GGGAGCAG : Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Escherichia coli Strain name: K12 Sequence characteristics Characteristic symbol: rRNA DNA encoding 16S ribosomal RNA of Escherichia coli Cleavage fragment by restriction enzyme HAE 3 of 1. ． 161 sequence CCCCCTGGAC GAAGACTGAC GCTCAGGTGC GAAAGCGTGG GGAGCAAACA 50 GGATTAGATA CCCTGGTAGT CCACGCCGTA AACGATGTCG ACTTGGAGGT 100 TGTGCCCTTG AGGCGTGGCT TCCGGAGCTA ACGCGTTAAG TCGACCGTCT 150 GG38AG sequence: number of two sequences: GG38 series: number of GG38 chains Type of linear sequence: Origin of genomic DNA Organism name: Escherichia coli Strain name: K12 Characteristic of sequence Characteristic symbol: rRNA Escherichia coli 16S ribosomal RNA by restriction enzyme HAE 3 Location of the cleaved fragment: 1. ． 34 sequence CCGCAAGGTT AAAACTCAAA TGAATTGACG GGGG 34 (39) SEQ ID NO: 39 Sequence length: 210 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: Genomic DNA Origin organism name: Escherichia coli ( Escherichia coli) Strain name: K12 Sequence characteristics Characteristic symbol: rRNA Cleavage fragment of DNA encoding 16S ribosomal RNA of Escherichia coli by restriction enzyme HAE 3 Location: 1. ． 210 sequence CCCGCACAAG CGGTGGAGCA TGTGGTTTAA TTCGATGCAA CGCGAAGAAC 50 CTTACCTGGT CTTGACATCC ACGGAAGTTT TCAGAGATGA GAATGTGCCT 100 TCGGGAACCG TGAGACAGGT GCTGCATTCC CTGTGACA number CTAA sequence: number of sequences: Double-stranded topology: Linear Sequence type: Genomic DNA Origin organism name: Escherichia coli Strain name: K12 Sequence characteristics Characteristic symbol: rRNA Escherichia coli 16S ribosomal RNA encoding DNA Cleavage fragment by the restriction enzyme HAE 3 Location: 1. ． 68 sequence CCGGGAACTC AAAGGAGACT GCCAGTGATA AACTGGAGGA AGGTGGGGAT 50 GACGTCAAGT CATCATGG 68 (41) SEQ ID NO: 41 Sequence length: 180 Sequence type: Nucleic acid Strand number: Double strand Topology: Linear Sequence type: Genomic DNA Origin Biological name : Escherichia coli Strain name: K12 Sequence features Characteristic symbols: rRNA Cleavage fragment of DNA encoding 16S ribosomal RNA of Escherichia coli by restriction enzyme HAE 3 Location: 1. ． 180 sequences CCCTTACGAC CAGGGCTACA CACGTGCTAC AATGGCGCAT ACAAAGAGAA 50 GCGACCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG TAGTCCGGAT 100 TGGAGTCTGC AACTCGACTC CATGAAGTCG GAATCGCTAG TAATCGTGGA 150 TCAGAATGCC 42 sequences : Linear sequence type: Genomic DNA origin Organism name: Escherichia coli Strain name: K12 Sequence characteristics Characteristic symbol: rRNA Restriction enzyme HAE 3 of Escherichia coli 16S ribosomal RNA Location of the fragment cleaved by 1. ． 19 sequences CCTTGTACAC ACCGCCCGT 19

[Brief description of drawings]

1] Ultraviolet absorption spectrum of chromosomal DNA extracted from Escherichia coli

FIG. 2 is a digestion diagram of DNA encoding Escherichia coli 16S ribosomal RNA with seven kinds of restriction enzymes.

FIG. 3 16S ribosomal RNA of Bacillus subtilis
Diagram of DNA encoding DNA by 7 restriction enzymes

FIG. 4: 16S ribosomal R of Bacillus licheniformis
Cleavage diagram of DNA encoding NA with 7 restriction enzymes

FIG. 5: 16S ribosomal RNA of Bacillus confusus
Diagram of DNA encoding DNA by 7 restriction enzymes

FIG. 6 is a digestion diagram of the DNA encoding 16S ribosomal RNA of Bacillus stearothermophilis with seven kinds of restriction enzymes.

FIG. 7 is a diagram showing separation by high performance liquid chromatography after digestion of DNA encoding Escherichia coli 16S ribosomal RNA with a restriction enzyme.

FIG. 8: 16S ribosomal R of contaminated bacteria derived from almond
Cleavage diagram of DNA encoding NA with 7 restriction enzymes

FIG. 9: 16S ribosomal R of contaminant bacterium A derived from fruit juice raw material
Cleavage diagram of DNA encoding NA with 7 restriction enzymes

FIG. 10: Cleavage diagram of DNA encoding 16S ribosomal RNA of contaminated bacterium B derived from fruit juice raw material by seven kinds of restriction enzymes

Claims (12)

[Claims] 1. A PC using a DNA base sequence as a primer
After R amplification, the obtained DNA base sequence is cleaved with a specific restriction enzyme, the cleaved DNA fragment is separated by a desired separation means, and the separation pattern of the cleaved DNA fragment obtained is used as an index to determine the DNA base sequence. A method for specifying a DNA base sequence, which is characterized by specifying. 2. The method for identifying a DNA base sequence according to claim 1, wherein the means for separating the cleaved DNA fragment is electrophoresis. 3. The method for identifying a DNA base sequence according to claim 1, wherein the means for separating the cleaved DNA fragment is a chromatography method. 4. The method for identifying a DNA base sequence according to claim 1, wherein the DNA base sequence is derived from a microorganism. 5. The DN according to claim 4, wherein the microorganism is a bacterium.
A base sequence identification method. 6. A microorganism is heat-treated with a surfactant to extract DNA, PCR amplification is performed with a primer using the DNA base sequence as a template, and the resulting DNA base sequence is cleaved with a specific restriction enzyme, and then the digestion is performed. A method for classifying or identifying a microorganism, which comprises classifying or identifying the microorganism using the separation pattern of the cleaved DNA fragment obtained by separating the DNA fragment by a desired separation means as an index. 7. A microorganism having a DNA base sequence as a template
7. The method for classifying or identifying a microorganism according to claim 6, which is a specific region of DNA encoding 6S ribosomal RNA. 8. The method for classifying or identifying a microorganism according to claim 6, wherein the microorganism is a bacterium. 9. The method for classifying or identifying a microorganism according to claim 8, wherein the bacterium is a bacterium belonging to the genus Lactobacillus or a genus Bacillus. A primer-DNA capable of PCR-amplifying a specific region of DNA encoding microbial 16S ribosomal RNA. 11. The primer-DNA according to claim 10, wherein the primer-DNA has the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2. 12. The primer according to claim 10, wherein the primer DNA is a DNA capable of hybridizing complementarily to the nucleotide sequence of SEQ ID NO: 3 and / or SEQ ID NO: 4, respectively.
DNA.