JPS62500423A - Determining the identity of two species - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Determining the identity of two species Ll, double plate 11 The present invention can determine whether a first and second living organism, one of which is known in identity, is the same. It concerns decisions based on probability. The invention particularly relates to three main categories of organisms: (1) In principle, organisms that reproduce asexually, especially microorganisms such as tIA bacteria, fungi, and viruses. Organisms, Organisms for which it is important to identify unique individuals and ■ Applicable to sexually reproducing inbred populations, such as some plant species.

11111 dishes Identification of genetically engineered microorganisms as an example of uniquely identifying organisms that reproduce asexually There may be problems with this. of microorganisms treated by introducing plasmids. In some cases, it is relatively easy to determine whether the specific microorganisms are the same. plasmid It can be clearly identified at the molecular level. However, this method In the case of microorganisms that do not exist, identification becomes a considerable problem. naturally occurring microorganisms including the chromosomes of objects and living organisms, by mutation in the laboratory or by genetic engineering techniques. There are particular difficulties in the case of improved systems of the microorganisms that have been developed.

special mutants that are functionally unrelated to the properties that make living things useful It is possible to label microorganisms by introducing ion) or plasmids. It is Noh. However, plasmids usually reduce the fitness of microorganisms. follow Such plasmids are often deleted during large-scale culture. earliest The type of mutation marker introduced is a mutant for drug resistance; These mutants also often have some disadvantages. In addition, both types of markers Both can be modified relatively easily.

One example of identifying unique organisms is the use of high-value racing horses, or stud horses. +1 bull) can be considered.

Classically, the animal was identified using natural marks such as color patterns, branding irons, and tattoos. They have relied on artificial marks such as . Natural color patterns for absolute identification Not unique enough, even branding irons and tattoos can be deliberately altered.

m1 indication of invention To solve the problem of identifying living things, we use the unique properties of living things that cannot be changed. A new method has been developed. The present invention utilizes natural genetic variation that occurs in scales. It is.

and the second organism are the same or not (toanascerta). provide a method for determining the availability of (+) Genomic DNA of the first organism is extracted using one or more ill restriction enzymes. let it digest; (ii) Separate the DNA fragments thus obtained by electrophoresis: (iii) Separate the DNA fragments thus obtained. Genomic DNA of an organism of the same species as the first or second organism of the fragment thus separated one or more labeled proteins consisting of randomly derived DNA fragments from A. The location where the lobe hybridizes (“hybridization pattern”, (iv) the thus determined The location of the fragment and the genomic DNA of the second organism using the or each till enzyme. Produced from the genomic DNA of the second organism by digesting △ and DNA that has been subjected to electrophoresis in the same manner as DNA fragments obtained from the genomic DNA of a human being It consists of comparing the position of the A fragment where it binds to the above or each probe. Before Steps m to (iv) to hybridization in step (city) The probe DNA and one or more A comparison of two organisms that are identical in step (ivl) is carried out using restriction enzymes. There is a very low probability (X) that the Let it be obtained by the notation.

X = Fq (1) In the formula, F is the genome of two organisms belonging to the same species as the first and second organisms obtained separately. Nomu DNA 1. The proportion of identical DNA fragments in the II restriction enzyme digest number, and q is the value obtained by probing in step (iii). is the number of locations indicated.

In other words, when two species have the same map, q is A mutual comparison of the first and second organisms of on) is the total number of common DNA digest fragment positions revealed.

Preferably, steps (i) to (iv) include (io) the genome D of the first organism; 'A and the second organism's genomic DNA were digested separately using the same restriction enzyme, and the Divide each digestate according to the requirements: (ii’) Gel electrophoresis is performed on the digested material or a portion of the digested material of each organism in parallel. ; (iii') Blowing the gel with said labeled probe and thus obtaining Compare the hybridization patterns of two species; (iv') if necessary, for one or more separate digestate parts of each organism; step (ii) and using a labeled probe consisting of a different DNA fragment each time. This is carried out by repeating (iii').

Repeat steps (io) to (iv') two or more times using different enzymes each time. It may be carried out above.

The present invention provides that no difference is detected between the hybridization patterns of two organisms. depends on the extent to which something different can be taken as evidence of identity. Two kinds of genomes Fraction of conserved fragments detected when digested with restriction enzymes The fraction of base pair differences (P) between the two genomes can be estimated from (F). (Wise.

In the formula, n is the number of base pairs in the restriction enzyme recognition site. Rearranging the above formula Then, F can be estimated by assuming a virtual value of P.

F=(1-P)　2'/(2-(1-P)　')　■F means that fragments are preserved. It is very likely. Therefore, FQ means all fragments when a digest produces q fragments. is the probability that will be preserved. Therefore, if the above formula is used, two types of organisms are suspected. In the present invention, several types of restriction enzymes and It can be set whether a DNA probe should be used or not.

Fragments produced by digesting genomic DNA with restriction enzymes are analyzed by electrophoresis. After separation of the sample DNA fragments and hybridization, a labeled DNA probe is applied. Vine detected using. Restriction enzymes that yield more probe DNA and are tested The greater the number of primary digests, the more DNA fragments are observed.

That is, q increases. Since F has a value less than 1, Fq decreases as q increases. It gets colder. Therefore, although they show the same hybridization pattern, they are actually confusing. The probability of not being able to distinguish between two completely different species becomes smaller.

Confidence with which it can be estimated that two lines are derived from the same clone ;C) is the following formula % formula % (4) (wherein F and q have the same meanings as above). Estimate C and In order to do this, the F value for a particular species of organism must be determined.

In fact, F contains many uniquely obtained organisms known to belong to the same species (the standard organism ) can be determined experimentally. Therefore, one or more labeled probes can be For a suitable number of non-clonally derived organisms of the same species by using For example, hybridization patterns can be obtained according to conventional classification criteria. Wear. In order to obtain a statistically significant F value for a species, that is, an F value that is representative of the species, A quantity of 5 standard'organism's is used.

To this end, preferably 10 or more species, for example about 12 species, are present. used. Compare all obtained hybridization patterns one after another By doing so, the amount of conserved rragments The F value can be calculated from the definition of the number F.

F=2N/(a+b) 0 In the formula, N is the total number of bands common between the compared organisms, and a and b are the from all the restriction enzyme digests and probes of the genomic DNA of each pair of two organisms. is the number of fragments detected.

By experimentally determining F for a given biological species, the user of the present invention can avoid erroneously determining the sameness. a given amount of blocks to obtain the required unbroken confidence (C) or probability (X) level. Decide in advance the number of genomic DNA digests required to perform O-buDNA. can be determined.

Therefore, we cannot determine the probability that two species are really different or the confidence level that they are the same. To determine this, two types of organisms will be analyzed. There are two types of Togi with different band patterns. No two organisms can be identical, so two organisms can be clonally derived or matched. Indeed, there is a constant probability that the altitude will be incorrectly determined. Get extremely accurate results (See Example 3).

The method of the present invention involves digesting the genomic DNA of two organisms to be analyzed with restriction enzymes. The method includes a step of separating DNA fragments in the digested product by electrophoresis. to the probe The mobility of the DNA fragments detected by the method is measured for each organism. Identical restriction enzyme Since the electrophoresis was carried out in the same manner using If the mobility (or band pattern) of the fragments is the same, then the organism is absolute. Although they are not, they can be the same. However, if the mobility of the fragments is different, the two types of Things must be separate things.

If the band patterns are the same, indicate whether the two species are the same or not. For more reliable determination, use restriction enzymes and/or one or more [)NA probes. can be done. The greater the number of restriction enzymes used to digest genomic DNA, the more The larger the number and the larger the amount of DNA used as a probe, the more Probability that two non-identical organisms appear to be identical when the objects are not identical will be lower. The method of the present invention is carried out using various 1111 restriction enzymes and various probes. If the band patterns of the two species are always the same after a sufficiently large number of times, the two species can undoubtedly be judged to be the same. The identity of one organism is, for example, a conventional classification. By scientific standards, the other organism must be the same.

Type II restriction enzymes cut DNA at specific sites depending on the DNA sequence. child This means that a specific enzyme cuts DNA from a specific organism into a specific set of DNA fragments. It means to sever (digest). Various enzymes with different recognition sequences have different recognition sequences. A sat of fragments will be produced. These breaks The average length of the pieces is required for recognition by specific restriction enzymes and depends on the NA length. example For example, a restriction enzyme that recognizes a 6-base sequence has an average length of 4096 base pairs (bp). However, the restriction enzyme that recognizes the 4-base sequence has an average length of 256 bD. produces fragments with However, when the recognition sites are distributed, the fragment length becomes large. produces a large actual array. electrophoresis As a result, much of the DNA in the cell is bound with varying fragment lengths, resulting in a recognizable pattern. This produces smears of shard size that have no or only a few smears.

However, under conditions where DNA specifically binds to DNA with the same sequence and produce patterns unique to particular organisms.

When labeled and labeled, the cloned fragment will hybridize with DNA having the same sequence. Acts as a probe that can be The fact that the probe is labeled location can be detected.

Comparison of two species according to steps (ii) to (iv) of the method of the present invention is as follows. It can be implemented. Using random DNA fragments from the first strain of an organism or from a specific individual. to create a labeled probe.

Total genomic DNA from the first strain (or individual) is digested using restriction enzymes.

DNA fragments are separated by gel electrophoresis. If visible, digested DNA will be recognized as just a point. Hybridize the digested DNA with the probe Soybean. The probe is directed against a fragment that shows some degree of homology to the sequence in the probe. Combine only with As a result, instead of dots, some bands represent homologous DNA sequences. can be detected using a label on the probe at the hybridization site of the probe. child The location of these bands is determined by the fragment size. from a second strain or individual total genomic DNA was digested with restriction enzymes and subjected to electrophoresis using the same procedure. hybridize using a tube. A second strain resulting from a past mutation or the restriction site in the hybridized DNA of the individual is the same as that of the first strain or individual. The position of the band will be different when the restriction site in the DNA is different. why Therefore, when genomic DNA is digested, the size of the DNA varies depending on the position of each restriction site. There will be some fragments. If the positions are the same, the probe is the same DNA fragment. When hybridized, they will appear at the same location in the gel. Therefore, if the stock or the two individuals may be identical. Using various restriction enzymes and/or various probes further testing reveals that the strains or individuals of 2IIliI are undoubtedly the same. Please be judged.

In step (i), the genomic DNA is digested with one or more restriction enzymes. Digest separately. Each part of the DNA sample is treated with various restriction enzymes.

Typically, a sample of genomic DNA is divided into five parts and each part is treated with a different restriction enzyme. Process using Any suitable restriction enzyme can be used in the present invention. Genome DN Restriction enzymes that recognize the 4-95- or 6-base sequence on A can be used. suitable The enzymes are Sat13A, Mr.sB, Pressure II, Fat■, Ribdl[I, Dantomo. R1, Kawaro ■, Dan o11. A animal ■.

3al I, Aval I, Asp 700 and Tate Ial.

The DNA fragments obtained by restriction enzyme digestion are subjected to gel electrophoresis in step (ii). Separate by DNA fragments derived from organisms to be compared with each other using the same method , for example, subject to downward time electrophoresis. Preferably fragments on the same gel. are subjected to side-by-side electrophoresis. any suitable electricity Uses electrophoresis method. For example, DNA fragments are run on an agar gel.

The probe used in step (ii) was made from randomly derived DNA fragments. Become. The fact that the probes are randomly guided is important. DNA for probe , under strong selection (stron27'5electionl) for preservation, Functional genes that are resistant to a relatively small number of base substitutions as mutations Non-specifically selected from among. Changing the base sequence of the recognition site for restriction enzymes Mutations provide the variability detected by the present invention. Under pressure for selection A given gene will likely contain some variation depending on its genetic function. Therefore, Such genes are conserved between two species of organisms, especially between two closely related organisms. and are not representative of the original genetic variation between the two strains. On the other hand, the present invention By randomly selecting the DNA to be used as a probe in A probe may contain genes that are not under strong pressure for selection. Therefore, these genes are resistant to more mutations and are less likely to be carried from strain to strain of the species. less representative of the original genetic variation of individual strains.

The probe DNA is the genomic DNA of the strain to be identified or a strain from the same species. It can be prepared by digestion, preferably partial digestion, using restriction enzymes.

The DNA fragment thus obtained is introduced into an appropriate vector plasmid to generate a latent protein. Banks of lobes can also be produced. Preferably screen the plasmid select a suitable number, e.g. 5, containing distinct sequences of the original genomic DNA. do. Typically, the length of the DNA fragment into the plasmid is about 5 kb or less. or more, preferably 10 kb or more. For example, the length of a DNA fragment is for example, depending on the length of DNA that can be satisfactorily inserted into a particular plasmid. . Furthermore, the more DNAfi in the probe and the number of probes used. The more there are, the higher the probability of being able to distinguish between two different strains will be. some When using probes, the total amount of DNA is typically about 30 kb or More than that, for example 50 kb (5 fragments of approximately 10 kb each). any vector Although tar systems can be used in the present invention, suitable examples are cosmids.

Randomly derive DNA fragments to be inserted into probes from the genome DNA of an organism However, the aforementioned organism is compatible with the organism to which the present invention is applied, and the DNA fragment in the probe is the same as the organism to which the present invention is applied. or derived from the genomic DNA of an organism of the same species as the second organism; Preferably one or the first of the standard organisms used in determining the F value experimentally. or derived from the genomic DNA of a second organism.

Label each probe. For example, a radiolabeled probe can be converted into [α-P]dAT Created by nick translation using P or [α-32P]dCTP You may. Alternatively, permanently biotinylated probes can be used with biotinylated dUTP. It can also be created by nick translation. biotinylated probe The advantage of using radioactive probes is that they can be stored for future use; Generally, products must be used within a short period of time after manufacture.

After electrophoresis, determine the position of the DNA fragments that bind to the probe using any suitable method. Ru. For example, after electrophoresis, the DNA fragments on each gel are collected using the 5Otlthern capillary method. (E, 5outhern, 1975. J, Mo1. Biol.

98, 503-517), nitrocellulose membrane or diazotized aminoben Dyloxymethyl (DBM) paper or nylon membrane (e.g. Mersham PLC) 's 1lybond N). In addition, DNA fragments are subjected to reverse electrophoresis. Zeta Prob from Biorad Laboratories Ltd. charge-mod Nied nylon membranes such as c. You can also have it run.

The DNA fragment is then contacted with a labeled probe. Hybridize with probe The position of the DNA fragment is detected by an appropriate means. The resulting pattern of these positions and then compare it with the pattern obtained for the second organism or even another organism. Each student Obtain several band patterns for a substance using various restriction enzymes and various probes. Plot. When the patterns of two types of organisms are different, the two types of organisms themselves Different things are found. If enough patterns are compared and all are the same, It is logical that the two species are the same. In reality, two types of organisms are separated. An indistinguishable probability has been determined (Equation (1)). Sufficient amount of probe DNA and When using fractional control @ enzymes, the probability that two types of organisms cannot be distinguished is 10 or less than, such as 10-15 or less, 10 or less, and can be 10-40 or less.

The F value for a species of organism is determined experimentally as follows.

(a) A sufficient number of the first and second organisms to have a representative F value of 1q. The genomic DNA of many organisms obtained separately from one species can be separated using the same restriction enzyme. and then divide each digestate as necessary. fb) Separately harvest 1q of digestate or a portion of digestate for each organism in parallel. electrophoresis, (C) Randomly derived from the genomic DNA of the organism of the above species. Blobbing the gel with a labeled probe consisting of a DNA fragment 1(d), another 41 '1tlit engineered pairs of organisms, preferably many pairs and combinations of all pairs. Compare the hybridization patterns thus obtained on the gel, (e) If necessary, separate each time using a labeled probe consisting of a different DNA fragment. step (b) for one or more separate digestate portions of each organism obtained; - Repeat (d).

Steps (a) to (e) may be performed more than once, each time using a different restriction enzyme. can.

Preferably, the F value used in steps (i) and (iv) in determining the F value of Use the same restriction enzyme set as However, this is not required and another A restriction enzyme set may be used in step (a), The organisms used in step (a), the reference organisms, must be different strains of the same species. . These organisms are independently obtained, i.e., non-clonally derived. There must be. These organisms are the two species that will be analyzed for identification. Must belong to the same species. In fact, one of the two creatures is a standard creature set. included in. A sufficient number of standard organisms are statistically significant and representative of the species. is used to obtain Flli, which can be considered as

Restriction enzyme digests are made from genomic DNA samples of standard organisms. mentioned above Restriction enzymes can be used. The genomic DNA of each organism is digested using the same restriction enzymes. let A panel of samples digested with restriction enzymes according to the genome D of each organism is and can be established.

The digested product is subjected to electrophoresis to separate the DNA fragments therein.

This is accomplished as described above. (one or more) same restriction enzymes The digested material is subjected to electrophoresis under the same conditions. Preferably, the digestate Run in parallel on the same gel.

The labeled probe or each probe used in step (C) is one of the standard organisms or preferably consists of a genomic [)NA fragment from one of the two test organisms. Bu The O-b can be constructed as described above. Apply the same standards. one or more probes When used, the probe must contain various DNA fragments.

The probe is subjected to electrophoresis and has a DNA sequence homologous to the probe. Hybridize with A fragment. For a given restriction enzyme digest, each standard organism Hybridization patterns are shown. This allows you to compare patterns with each other Can be done. For a given restriction enzyme digest, each and every possible pair of standards Hybridization of the combination (pair C0IIlbinatiOn) Examine the pattern and find out by blobbing that the same hybridization occurs in many pairs. Determine the fraction of DNA fragments produced. This fraction is the F value for that pair. The above formula Please refer to 〇. Using all pairs of FfIi allows for specific restriction enzyme digests. The weighted average Fillill can be estimated for All restriction enzyme digests Obtaining the average F value for from the weighted average F value of the F values of the individual restriction enzyme digests. Can be done.

Once an F value that is considered to be representative of a particular species is obtained, this value can be used to develop the present invention. Two species of the same species that appear to be identical when analyzed according to steps m to (iv) of the method can ascertain the probability that a distinct organism is in fact indistinguishable from another strain. Ru. using specific restriction enzymes and one or more specific probe sets. Therefore, a representative average F value (F', v) was obtained for the species to which the two organisms belong. Assume. Using the same restriction enzymes and the same probe set, steps m to (i When two types of organisms are compared according to v) and shown to be the same, the two are distinguished. The probability that they cannot be separated is F' (number of identical bands indicated by probe set) v That is, X-Fq (formula (1)).

The probability that two species of organisms cannot be distinguished will be lower.

The number of bands tested increases as the amount of probe DNA increases. Another limit If steps (i) to (iv) are repeated using enzymes, the number of bands will increase. all The flat bottom for each restriction enzyme is the weight average F value of each restriction enzyme digest, 2F pooled to more accurately predict whether one organism is indistinguishable or not. , the total number of bands in the hybridization pattern obtained for the restriction enzyme. can be obtained from. In other words, as the q value increases, identity is incorrectly judged. There is less chance of it happening.

When determining the F value according to steps (a) to (e) above, the following are evaluated: It will be done.

- The restriction enzyme or each restriction enzyme used in step (i) determines the F value. It does not have to be the same restriction enzyme as you want to use. However, both cases It is preferable to use the same restriction enzymes for both.

- the probe or each probe used in steps (iii) and (iv) is It does not have to be the same as each probe used in determining the F value. however , it is preferable to use the same probe in both cases.

- When averaging the average F value for a specific restriction enzyme, calculate the average F value for each restriction enzyme. A standard organism of the determined niche shall be used.

The invention can be applied to any organism. The term “organism” refers to clonally derived It also includes collections of individuals that are present, such as microorganisms such as bacteria, fungi, and viruses. I want to be understood. the usage of one of the first and second organisms to be compared; The taxonomic identity of the species must be known. This suggests that at least two species One species is appropriate Ff! Must be publicly known so that i can select/set it It means that.

The invention particularly applies to asexually reproducing organisms such as bacteria, fungi and 'DNA viruses. It can be applied to microorganisms such as bacteria. The present invention uses naturally occurring microorganisms or experimental microorganisms. Plasmids and other genetic elements (genetic man in vitro by inserting ipulations) into genomic DNA. It can also be applied to microorganisms that are engineered mutants. In the case of RNA viruses cDNA obtained using reverse transcriptase can be used as genomic DNA. Ru.

The present invention also applies to sexually reproducing isogenic populations, such as certain plant species. It is also applicable to organisms for which it is important to identify unique individuals. Unique In order to fully identify individual organisms, we identify them immediately after birth or at any time thereafter. Collect tissue samples. Genomic DNA is generated from tissue samples and used for identification. Save it until a related problem arises. When DNA is created from the individual, its D Compare the NA to the original DNA sample. In this case two DNA samples are digested with a series of restriction enzymes to generate random DNA from organisms of the same species. Two pieces to be compared by hybridizing with a probe set containing the A fragment The identity of the sample pattern is used to identify the individual in question as the original DNA sample. The probability that the pull is the same as the individual from which it was taken is established. In this way, the present invention Vine applied to humans and animals, such as horses (especially racing horses) and cattle (especially breeding cattle) . The invention can also be used by plant breeders.

Therefore, the present invention can be widely applied. A strain of an organism is considered to be the same as a particular strain. If possible, the present invention can be used to quickly and reliably prove it. Each student Patterns of products can be obtained by using various restriction enzymes and various probes according to the present invention. can be constructed. If the patterns are the same, two species can be distinguished. There is a possibility that you will not get it.

The invention can also be used to clinically identify unknown pathogens. This thing is , creating a series of probes containing random fragments of the pathogen's genomic DNA. This can be achieved by Various microorganisms that are thought to have the same genomic DNA as the pathogen The genomic DNA of a species is digested using restriction enzymes according to the present invention, and then digested under the same conditions. Apply to electrophoresis. Band patterns of pathogens and organisms using already created probes construct and compare for identity. Next, after determining Flifl, correctly identify the pathogen. The degree of confidence in identifying the body can be shown.

To extend the method of the invention, first and second biochemical Cellular enzymes may be subjected to electrophoresis.

Differences in electrophoretic mobility indicate differences between two organisms. Electrophoresis of enzymes in two organisms Two species can be identical if their dynamic mobilities are the same. About more enzymes If you test, you have a better chance of detecting whether or not two species are in fact identical. It becomes. However, this means of establishing identity between two species Restriction enzyme analysis of a person's genomic DNA is not very powerful.

The electrophoretic mobilities of enzymes from two organisms can be compared very easily. each organism's crude Apply an electric field to the cell extract in a gel, e.g. starch or polyacrylamide gel. put on. After electrophoresis, each gel is immersed in a solution containing a substrate for the enzyme in question. . When the substrate degrades, a band forms at the enzyme location within the gel. appears. If the enzymes from two organisms are different, one or more charges Different amino acids migrate to different positions in the gel, resulting in different band patterns. .

Another means to complement the present method of identifying organisms is to identify each species in a gel of two organisms. The purpose is to compare protein patterns.

Proteins can be separated by charge by isoelectric focusing.

Proteins can be separated by size, for example on an SDS gel. These two By combining the two techniques, a single protein can be separated into two proteins. tampa The texture appears as a pattern of spots on the gel. Two types of the same spot pattern When it occurs in an organism, the organisms are the same and 1q. However, the pattern If they are different, then the two species are different.

Brief description of the drawing 1 to 3 show the band patterns obtained in Example 3. lane for each individual One label λ/pB R322) was used as a standard (restriction enzyme EC0RI or λDNA (digested using P and Hindll) and (restriction P?f≦, juice 3A') (digested using pBR322 DNA).

Figure 1 shows the results of digestion with restriction enzyme A CCl, nylon filter (Sout hern blot) and nick translated [32P ] Labeled plasmid I) λ DNA probed with BTL 30 and 12 Genomic DNA of Lactobacillus plantarum fj strain This is the band pattern shown. Autoradiate the filter at -70℃ for 1 day. The X-ray film shown in the figure was prepared. The bands are genome fragments and proteins. This corresponds to the hybridization position of the lobe DNA.

Figure 2 shows that the restriction enzyme used is 2 to sp700 and buO-bu is 1]BTL2. This is a band pattern created in the same manner as in FIG. 1 except that it is number 9.

Figure 3 shows the results of digestion using $11 restriction enzyme convex 700 and a nylon filter (So utern blot) and nick translated [32 P] Labeled bacteriophage λ DNA and probed DNA of type C X-ray autoradiograph of the gel (section 8 of Example 3). ,Δ””7 A second control lane containing λ DNA digested with It was used as a standard. Is DNA digestion completed using this gel? Evaluate.

Embodiments of the present invention (including the best mode) Example 1 General procedure (a) 1. Create genomic DNA from A strain. Genomic DNA was extracted using 3A restriction enzyme. and partially digested to obtain fragments with an average size of approximately 10 kbo. these The fragment is ligated into the Ba1HI site of plasmid I) BR322. suitable host Transform the `` Select Ap transformants and isolate 10 to 20 Tc transformants. and identify it.

3. Create large quantities of these plasmids and use biotinylated dUTP. Generate a permanently otinylated probe by block translation. these Save the probe for future use.

4. Digest 10 genomic DNA parts from strain A using different restriction enzymes.

Five restriction enzymes have a 6-base recognition sequence and another five restriction enzymes have a 4-base recognition sequence. It had an array. Enzymes have randomly distributed recognition sites must be selected to the extent that is known. Similarly, we must compare it with A stock. Digest the genomic DNA from the B strain. For each 10 digests , place the digested product of strain A on an agarose gel in the lane next to the digested product of strain B, and perform electrophoresis. perform a movement. Run all 20 digests on a single gel. Run 5 identical gels .

5. For each gel, transfer the DNA to a nitrocellulose filter or DBM paste. Transfer to the parr using the Southern method.

6. Probe each plot with one of the five probes. A share high Comparison of hybridization pattern and hybridization pattern of B strain do. If the patterns are the same, the probability that stocks A and B cannot be distinguished is expressed as Statistically significant F value and hybridization pattern for the species to which strain B belongs can be determined from the number of bands shown in .

Tomoi Wataru - Uni general method <b) The general method (a) of Example 1 can be modified as follows.

1. Create genomic DNA from A strain. Genomic DNA using restriction enzyme Pstl and partially digested to obtain fragments with an average size of approximately 10 kb. Convert the fragment into plasmid p Connect to the P St I site of BR322. DNA piece of A strain A ligated plasmid containing Tc is transformed into a suitable host, and Tc Select transformants and isolate 10-20 separate AD transformants.

2. Identify the required number of recombinant plasmids using various A strain DNA inserts, and Size the insert.

3. Create large quantities of these plasmids and perform nick translation [ 32P]-labeled probes or permanently otinylated probes. Biochi Save the nylated DNA probe for future use. [32P]-labeled DNA must be used within a few days of creation.

Example 3 In this example, Lactobacillus plantarum was used. 2 illustrates the actual operation of the invention.

1. I sent 1 yen to Shikou. 12 independently isolated Lactobacillus Iantarum strains , not clonally derived, or each representative of the plantarLIll population. The 11 strains are listed in the National Collection of Industry Real Bacteria <N0IB) and the rest from Biotechnica φ Limited. Collection (8io Technica LiIIIited’s co Collection). When the latter was analyzed by NCIB, it was found that the conventional It was confirmed as a member of the species based on anthropological criteria. N018 strain is 5914.6105.63 76, 6461.7220.8016.8026.8102゜8299, 853 1 and 11974. The 12th stock will be referred to as BTLSI here. , this is NCI B (Tarry Re5earch 5tation, P, 0. Box 31. 135 Abbey Road, Aberdee NCI deposited with AB98DG, GB) on September 25, 1985 It was 812156. In this example, strains were used for the following purposes.

− Random mutations are fixed within the population (at separate times and locations) Therefore, various strains have different hybridization patterns as a result of polymorphism of restriction enzyme fragments. Ni to indicate having a turn - The F value, i.e., is conserved on a pairwise basis (in other words, Restriction enzymes that are the same between the 10-) digests of the two genomic DNA samples taken To estimate the fraction of disjoint fragments; - To estimate the probability (F') of not being able to distinguish between two distinct isolates: For this analysis, two other organisms are required at the appropriate steps of the procedure.

These are E. coli strain SJ 84R/DBR322 (for constructing probes). plasmid I) as a source of BR322) and E. coli RL57 (as a source of BR322). (as an organism that can be transformed and analyzed by the ligation probe).

2. Growth of microorganisms To obtain DNA, a sufficient amount of organisms must be grown.

The method does not matter as long as a sufficient amount of DNA is obtained. In this case, L, 1 strain of 1 antar LIj was added to 1/4 strength MRS broth (Ox (1) were grown separately overnight at 30° C. without agitation on .oid). 1/8 strength MRS Strain N0188299 grown in broth 400d and MRS buoy with normal strength. One exception was the BTLS strain grown in Yong 100Id. E, col i asJ 84R/pBR32tr, 110 to maintain the plasmid 0I1/d of ampicillin was added into the broth.

3. Isolation into dormant DN Genomic DNA was isolated as follows. Collect living things (7000 rpm, 10 ), cold TES buffer (30mM-Tris HCl), pH 8.0 ; Wash once in 5 mH-E D T A and 50 m M -N a Cj) Helen l-wo 0.15 H-N acj! , 0. IH-EDTA 1. 1 in 5d. : 704/d Pronase E (Sigma, Registered Trademark a>Solution 100111 and incubated for 1 hour at 37°C. Dried lysozyme Bear) 40#l! J and butanol buffer (13IIIHN a 2H PO4; 30mM-KH, PO4: 1mH-EDTA: 65 (v/v ) Butanol) was added for 3.5 d and incubated at 37°C for 20 minutes.

Then 20% (w/w) SDS IId was added and the lysate was placed on dry ice. /ethanol mixture and thawed at 65°C.

The method was repeated three times.

Add (freshly prepared) 5M NaCj’04 11d to the lysate and mix. match. CHCj the mixture! 3-isoamyl alcohol (24:1.v/v) Extracted with Gd and placed on ice for 30 min before clarifying the phase by centrifugation. . Remove the aqueous phase with a wide-bore pipette and remove the DNA using 2 volumes of ethanol at 20 °C. I took it out (spo.

off). DNA with 200 units of TI RNase (Boehringer) TE buffer containing p-(10mM-t!JSHC1', fltl 8.0J 3J: (FlmH-E DTA) Re-dissolved in ld at 37°C for 1 hour . Add 25η/d proteinase K (Boehringer) 10ρ and incubate at 37°C for 1 hour. Incubated for a while.

The DNA was extracted twice with CHCl3 on ice, the aqueous phase was removed and 3M sodium acetate was added. (ptl 5.2) Add 1 volume of Q, then 0.6mM isopropanol. (room temperature). The precipitated DNA was taken out.

Removed DNA (vinegar! to remove salt) in cold 70, 80, 90 and 100 χ Subsequently immersed in ethanol and dissolved in 500 pITE buffer; It was then dialyzed using TE buffer. Quantify purified DNA and probe appropriately. It was prepared for use in protein construction or digestion. This method uses MarIlur (1 961, J, Mo1. Rial.

3, 208-218).

4. The power of Blasmi DNA For small-scale production (so-called mini-preps), Hania tis et al. (1982) (Molecular Cloning: a Labo ratory Manual.

Co1d Spring Harbor Laboratory, Co1d Spring t The boiling method of Larbor, New York, USA) was used. So This was followed by isopropanol precipitation at room temperature. For large-scale manufacturing, Hansen and and the method of Olsen (1978, J. Bact, 135.227-238) It was used.

5. Limiting enzyme Controls used to construct probes or digest genomic DNA Limited enzymes are available from Bethesda Lithage Laboratory (BRL) or Berry. Obtained from Nger Man 1~Heim (BM) or Pharmacia. Either way was also used according to the manufacturer's instructions.

6. DNA gel electrophoresis Horizontal agarose gel electrophoresis to separate DNA fragments (1982, ibid.) in Tris-borate EDTA buffer. It was carried out in the L apparatus. Edge bromide in agarose gel and electrophoresis buffer (5II9/d). Haniatis et al. (1982, ibid.) DNA bands were visualized under medium wavelength U and V as described and photographically removed.

7. Probe construction Genome from Lactobacillus plantarum strain BTLS 1 The DNA was partially digested using the male restriction enzyme PStl to generate multiple 5-15 kb fragments. The 311-sized DNA fragment obtained was digested with Stl and digested with plasmid pBR32. I contacted 2DNA. Intestinal alkaline phosphatase that carries plasmid DNA ze(calf 1ntestinal alkalinephosphatas e) Using 5 units, 1.0IIIH-Zn C12 and 1, OmH-M GCR2 containing 0.018-t-, IJsu HCf) (ptl 9.0) Hatsu 7 Processed in F- for 60 minutes at 37°C. This pretreatment prevents self-coupling and Sumido l) B R322 DNA and LaCtObaCillllS p+anta The genomic DNA of rum strain BTLS1 could be successfully ligated.

This ligation step is performed according to the specific instructions for use. Performed using T4 ligase from BRL. Genomic DNA and plasmids After ligating the DNA, the ligation mixture was used to transform E. coli strain RL57. Ta.

The transformation step was performed using Hani The method of atiS et al. (1982, supra) was followed. Transformed E, col i strain RL57 cells were treated with a solidified plate containing 20 mcg/ld tetracycline (Tc). I placed it on Lee Yeon and selected it. As a result, it contains the introduced pBR322 DNA. We were able to grow only those cells that Ampiculture of separately obtained Tc cells Phosphorus tolerance (Ap) and sensitivity (Ap>) were determined using 50 n/ml ampicillin and would have plantarumDNA.

Plasmid DNA from transformed E. coli RL57 TCAp strain was prepared on a small scale and digested with the restriction enzyme PstI.

Load the digested DNA for electrophoresis in a 0.7% (w/v) agarose gel. Four plasmids were identified. These are l) B T LB, pBTL23 ．． They were named pBTL29 and pBTL30. Size of inserted DNA The cuts were made using a standard DNA digest with fragments of known size, namely HindlI and bacteriophage λ DNA digested with EC0RI and EC0RI restriction enzymes. It was estimated by The following sizes were obtained. In pBTL8, 8.8 kb and pl antarumDNA, ρBTL23 is 8. Okb's DNA, I) BTL 5.7 kb of DNA in 29 and 10.4 kb of D in I) B T L 30 NA has been inserted.

Using these four plasmids, all LaCtObaCillLIS pl Probes used to test the genomic DNA of S. antarum strains were constructed. .

To use the probe to interrogate genomic DNA, add the probe to biotin-11- dUTP (“biotinylated” probe) or deoxytidine 5-[α-” 2P　1-1~rifosphate (herein referred to as [“2P]-dCTP”) (for radiolabeled probes).

In this example, the Transmit the radioactive probe by the nick translation method using the supplied kit. [32P]-dCTP was used for the preparation. Preliminary 50mM-Tris HCρ , 1 mH-E D T A and H 0.1% (W/V) S D S Te equilibration The labeled protein is The lobe and unblended [32P]-dCTP were separated. (Geiger Mullermo Collect the leading radioactive peak (detected with a This includes the following.

The four plasmid probes were cloned with l]BR322DN△, Contains ntarumDNA. Nick translated [3 2P]-labeled pBR322 DNA is digested under the conditions shown here, It was shown that there was almost no hybridization with ntarumDNA. subordinate Therefore, the observed hybridization: l:/c, probe L, plan Genomic HarumD N A hybridization with tarum[) NΔ caused by.

8. L. Testing of plantarum DNA (obtained from BM) Damu lambda bacterium A sample of genome D, ;A (12,5 ㎒) containing teriophage DNA (150 ㎒) Samples were digested with 30-250 units of restriction enzyme. The amount depends on the enzyme used. Depends on. DNA samples were separately digested with a wide range of restriction enzymes. This implementation In the example, the following restriction enzymes were used. l’Jci, ■.

DannI, bile 1. S30-I, A■ [, A Tsuji 700 and Nipdan T0 enzyme post-digestion Divide the DNA sample into three aliquots.A.

Electrophoresis was performed on three types of 0.9% (W/V) agarose gels, B and C. gel Δ and B were loaded with 5 ml of lj-conjugated DNA per rune. Gel C contains - 2.5 IJ9 was loaded per tube. Glue C is loaded with sample DNA. In addition to the restriction enzymes used for the genomic DNA, digest with the same restriction enzymes used for the genomic DNA. A truck loaded with only λDNA was also prepared. In any of the three types of gels , one lane contains λ DNA (digested with restriction enzymes EC0RI and 1) BR322 DNA was digested with restriction enzyme 5au3A. child The DNA fragment in the lane was used as a molecular marker. Check the digestibility of DNA electrophoresis to check and future proofread the size of the fragments produced. All gels were photographed immediately after staining.

Agarose gels were subjected to electrophoresis using standard methods. (Haniatis et al. (198 2, ditto) for each gel A and B using the procedure shown in p. 385). double filter blot, gel C The method was modified to generate a single filter plot for child Five plots were generated using the method.

Each plot represents an average of approximately 2.5 μg of digested DNA per lane bound to the filter. Contains equivalent.

5 duplicate filter blogs! - separately supplied by 8mersham plc Probe and probe to nick-translated λDN according to the method described. and four pBTL probes. Filing using standard methods Hive the nick-translated probe and sinus DNA on a router. Redized. Hybridize the filter and add 6xSSC buffer (S SC buffer F-0,158-N a C1 and 0.0158- N a 3k Etrate, pH 7,0) and washed (extensively) at 65°C in lX5SC buffer did. Probes were denatured by boiling immediately before incubation with filters.

9. Auto Radio Roughy The probed and hybridized filter was once dried with an intensifying screen (f ast tungstate intensifying 5 screens) Autoradiograph the filters at -70°C for 4 hours to 2 days using Ta.

10. Band pattern The resulting band pattern is illustrated in FIGS. 1 to 3 of the accompanying drawings. bacteriopha The completeness of the genomic DNA restriction enzyme digestion was monitored using λDN.

[32P]-standard was nicked and 1-lanced from the complete λ DNA digest. A known number of restriction fragments will be generated by hybridization with the identifying lambda DNA. Dew. O1 ~ The appearance of the restriction fragment after radiography indicates that λDNA is incomplete. Or, partial digestion was indicated, thus LplantarUm genomic DN△ was incomplete. It was shown that it was completely digested. That is, Gel C indicates that the sample has been completely digested. It is used as a control to find out what happened. indicates incomplete digestion Lane ignored him.

["2P] - From the filter hybridized with the standard W & D NA probe The probe and hybridizer are placed on the resulting X-ray film (or autoradiograph). A series of bands corresponding to DNA fragments from the sized DNA△° sample were shown. . Hybridization fragments were determined based on the relative transfer rAJ degree in agarose gel. It was distinguished by Therefore, the largest hybridization fragment (hybrtdi sing fragments), (mark on the corresponding autoradiograph) The band on the autoradiograph is generated closest to the origin of the gel (as shown). It was. Conversely, the smallest hybrid pesion fragments have high mobility and therefore A band on the autoradiograph occurred furthest from lysine. gel origin The position of the band relative to the The graph was digested with restriction enzymes and coupled to filtered Southern blotting. 12 glues electrophoresed on a gel and hybridized with one probe; (obtained from the plantarum D N A sample). Therefore, one or more The presence of identical hybridized DNA fragments in a DNA sample is , the presence of bands that migrated the same distance from the gel origin on the corresponding autoradiograph. It was inferred from the existence.

is shown. For each probe, hybridize the labeled probe with the digested DNA. The total number of bands visualized as a result of dizization is recorded. LL! ■ and 7 (va) [The total number of bands for each probe set for the digests is shown in Table 1.

Reciprocate the band patterns for each plasmid probe and each restriction enzyme digest ( The number of fragments common between each pair of total a and b fragments and digests, respectively ( 2N) was determined. Tables 2.5-2.8 and 3, 5-3.8 below show the Genomic DNA 8 by I and N friends! Mutual comparisons regarding I conversion are shown. . From the individual intercomparisons (i.e. Tables 2.5-2.8 and 3.5-3.8), the total Six tables (two tables here, Tables 2.9 and 3. 9) was created. Tables 298 and 39 include B'C1I and % A summary of intercomparisons of genomic DNA digested with va[ is shown. Finally, the table 4 contains all 4 probes and 6 restriction enzyme digests per complete Sera I. The results of cross-comparison between strains are shown.

Fractional fractions of conserved fragments (F) for mutual comparison were to intercompare F for the complete set of four probes with the mu DNA digest. ) using the data in Tables 2.9 and 3.9, and (4 probes and 6 To intercompare F for a set of genomic DNA digests) the data in Table 4 Calculated using These F values are shown in Tables 5.66 and 7, respectively.

As expected, the <12IvA strain was isolated independently, so the F values were different and there was no correlation. It was shown that the degree is different. In some cases, the level of analysis of genomic DNA is low ( (i.e., a single restriction enzyme digest), the restriction enzyme, 4va [by Even when using four probes for genomic DNA digestion, common DNA fragments were clearly detected. (e.g. 6461 bacterium v BTLS 1; 8102 v B TLSl) (Table 6). In a broader study using six restriction enzymes, several A common band was shown (Table 4) and a very low F value was shown (Table 7). vice versa, Some stocks were highly correlated. For example, 4 probes and 1 genome eraser. The F value of strains 7270 and 11974 when tested for compounds was 0.963. There was (Table 5). Overall, Fla of the same pair of L, plantarum strains is 0 ．． There were 8041' (Table 7). In both mutual comparisons, an F value of 1.0 was obtained, and It was shown that the different strains were also not identical.

11. Theoretical analysis of the 13th strain of L. plantarum The 13th strain, designated strain X Tl, was compared with a standard set of 12gA organisms and previously Suppose that a strain is found to produce a banding pattern identical to one of the strains analyzed in . To confirm that the two strains are indeed identical and therefore clonally derived, In order to ensure that the two strains are derived from the same clone and are therefore identical (formula ■), confidence level (C), or the degree to which two strains are judged to be the same when they are actually different. (Equation (1)).

The confidence range based on this data is the maximum, minimum, and average F value determined from complete reciprocal analysis. Maximum, minimum and average measured for the 12 stocks analyzed. It is best to make a judgment based on the number of bands of test strain X, which is the value (Table 8). this table Therefore, the closer F for the group is to 1, the larger the F value becomes, and the more two observations If the identity between the identified patterns is from two DNA sources (strain X and the strain being compared to it) It is less reliable to be interpreted as indicating clonal identity between the two.

In other words, strain X was probed with the four probes used in this study, and genome D Assuming that only NΔ was analyzed using restriction enzymes 1 and 2, using the highest F value, the confidence level was C. will be 0.29. The chance of a true clonal identity being incorrectly determined is 7. It is 1%.

Using the average F value and under the same conditions, C = 0.999658, clonal The chance of erroneously determining identity is only 0.04%.

Table 8 shows that the probability of error decreases rapidly as more genome digests are attempted. is shown. For example, similar F, V = 0.142 value (same 4 probes) 410 (4 identical probes, 6 restriction enzymes) and Fav-o, 410 (4 identical probes, 6 restriction enzymes) ti11 restriction enzyme), the probability of error is 3.42x 10' to 4.67x It drops to 10-27. Therefore, in fact a population of uniquely isolated individuals of the same species Once the F value (and P value) for the number of microorganisms and DNA used to probe test and reference microorganisms. The required degree of probability can be obtained by appropriately selecting the suspension.

Actual analysis strain BTLS of the 13th strain of 12, L, plantartlffl I samples should be compared with “known” BTLS I samples “unknown” 13 It was used as the second strain. We decided to refer to the “unknown” stock as stock Y. stock Y, B TLS 1 was isolated again from the inoculated natural source. Game from stock Y and BTLS 1 Nomu DNA was digested using a restriction enzyme digest, and the probe pBTL was prepared as described above. 8. DBTL23. Pipe rider using I) BTL29 and I) BTL30 It made me cry. Several identical band patterns were obtained.

A total of 4 probes for each BQII digest of strain Y and BTLS I Twenty-four identically affected bands were observed (Table 1). L, Iantar LIl Based on F, F, and Fav values estimated for a standard set of 12 strains of (Table 5), formula (1) and 0) or ff1aX l1lln It can be expected that the probability that stock Y and BTLS I are actually different is as follows. .

F: 0.056”’=9.05x 10-3’an In order to show that the probability that two stocks are different is low (high reliability), stock Y is Genomic DNA from and BTLS 1 was digested with restriction enzyme L■■, and the probe pBT L 8. pBTL23. Hybrid with pBTL29 and psTL30 I made her cry. An identical banding pattern with 35 common bands per strain was obtained. Ta. estimated for a standard set of 12 strains of L-plantaruIII. FII lax.

From the F and Fav values (Table 6), it is clear that stock Y and BTLS I are actually different. It can be expected that the probability that

To obtain a higher level of confidence that stock Y and BTLS I are in fact identical. DNA from the strain was collected in 1qci1. AC+I.

Digested with AS+)700 and QIa and hybridized with 4 probes. Ta. An identical banding pattern was shown. 1 inhibition I and Qva [results related to digested products Considering this, 159 bands were shown (Table 1). For all 6 digested products F estimated for a standard set of 12 strains of IantarLIll. , Flo and ax Based on the FaV value, the probability that stock Y and BTLS 1 are actually different is as follows. Ta.

F: 0.898 -3.72x10-81lax It is also possible to perform conventional statistical processing on all the values in 10-12 above. The restriction enzyme that recognizes produces fragments with an average length of approximately 0.25 kbp, and G-JH3 Restricted R elements that recognize sequences produce fragments with an average length of about 4 kbp. probe If a DNA sequence with a total length of L kbp is used, a 4-base cutter (cutte rs) yields a q-4[ fragment, and the 6-base cutter produces a ( 1=0.25 [produces fragments. Assuming that the restricted sites are randomly distributed , F and F2 can be derived by assuming the values for P in equation ■. , the probability <X> that two different organisms cannot be distinguished is as follows.

(i) When using the y 4-base cutter to digest genomic DNA (ii) When using 6-base cutter 2 to digest genomic DNA (iii) 4-base cutter of y and 6-salt of 2 to digest genomic DNA. When using base cutter ×3=×1 ・×2 ■ From the above analysis, if the hybridization patterns of two species are the same, For example, a given number of base sequences (4-base sequence of y; - The fraction (P) of base pair differences between two species whose base sequences are recognized and the number of base pair differences between the two species. The total length of the DNA used in the probe to investigate the genomic DNA of an object (L) Estimate the probability that two truly different organisms will be incorrectly judged to be the same from the information can do. The probability of incorrectly determining identity is the same as the genomic DNA of two organisms. Increase the DNAff1 used to probe A, or use various restriction enzymes. This can be enhanced by increasing the number of digests of genomic DNA attempted using It can be done. One such analysis is shown in Table 9. This table shows the given error probabilities. The amount of probe DNA required is indicated. If the P value becomes small (i.e., two types of If the conservation of bases in the DNA of a substance increases, identity will be incorrectly judged. The number of probes and the number of identical genomic digests required to achieve a high degree of confidence are small. Ru.

Table 2.1 Digest with restriction enzyme Bgl I and use pBTL 8 as a probe. DNA 0 Bands are numbered sequentially from the origin of the agarose gel.

DNA sample pool /Zend　8531　8299　8026　6376　6461　7220 Number BTLSI 610559148016119748102 Table 2.2 Restriction enzymes Lactoba was digested with Bgl and caught using pBTL23 as a probe. DNA derived from 12 strains of cilLus plantarum 0 The bands are numbered in order from the origin ds of the agarose gel.

DNA sample pool ζnd 8531 8299 8026 6376 6461 7220 Table 2 ．． 3. The restriction enzyme was once digested with Genko I and pBTL 29 was used as a probe. DN derived from 12 strains of Lactobacillus plantarum A0 Bands are numbered sequentially from the origin of the agarose gel.

DNA sample pool No Znp 8531 8299 8026 6376 6461 7220 number BTLSI 610559148016119748102 Table 2, 4 Restriction enzymes Lactoba digested with Bgl and caught using pBTL30 as a probe DNA derived from 12 strains of cillus plantarum 0 Bands are numbered sequentially from the origin of the agarose gel.

DNA sample pool No. 8531 8299 8026 6376 6461 7220 number BTLSI 6105591480161197481022 +++++++++ ＋＋ ＋ 5 ++ ＋＋＋＋ ＋＋ 6 + + +++ 7 ＋ ＋＋＋＋＋＋＋ ＋＋＋＋ 1o +, ++ +++++++++ , ff 2.5: Pairwise comparison of band pattern data in Table 2.1, a number of fragments For a unique pair of DNA digests in which and b fragments have N bands in common, Each value is shown as 2N/(a+b).

Table 2.6 = Band pattern of Table 2.2 determined as described for Table 25 Paired comparison of data Table 2.7 The pantograph numbers in Table 2.3 determined as described in Table 2.5 Paired comparison of sample data Band pattern in Table 2.4 determined as described in Table 2.8 and Table 2.5 Paired comparison of data Table 2.9: This table shows the denominator DNA digested with restriction endonuclease Bgl. Sum of ratios 2N/(a+b) for the four types of BTL probes used in the analysis of A It shows.

Table 3.1 Restriction enzyme Δ! Digested with ■ and used pBTL 8 as a probe. DN derived from 12 strains of Lactobacillus plantarum A.

Bands are numbered sequentially from the origin of the agarose gel.

14 +++ +++++++++ Table 3.2 Digested with restriction enzyme Aval and fished with pB’rL 23 as probe Raised band 8351 8299 8026 6376 6461 722 014 +++ +++++++++ 24 +++ +++++++ 27 ＋＋＋＋＋＋＋＋ 28 ＋＋＋＋＋＋ Table 3.3 Digested with restriction enzyme Aval and used pBTL 29 as a probe The results are derived from 12 strains of Lactobacillus llus plantarum. DNA 0 Bands are numbered sequentially from the origin of the agarose gel.

DNA sample pool Band -□--11---□-□-゛□□1 number BTLS183516105 829959148026801663761197464618102722 03 ＋＋＋＋＋＋＋ 20 + 10 + + + + + Table 3.5 I wrote about Table 2.5. Pairing comparison of band pattern data determined in Table 3.1 Table 3.8 = Band of Table 3.4 determined as described for Table 2.5, 11 Paired comparison of turn data Comparison of band and Q-turn pairings for data in Table 3.9 and Tables 3.5-3.8 Engraving (no changes to the content) G cheer 1: +8531 , eye 5914 ,, Wealth ε105 ' 1 8299 @6rin, 8026 @8016 Procedure Ne City Official Book (Method) %formula% 2. Title of the invention: Determination of the identity of two species of living things 3. Person making the amendment Relationship to the incident: Patent applicant Name: Biotechnica Limited 4. Manager Yamada Building (1), 1-14 Shinjuku, Shinjuku-ku, Tokyo (1) Proper drawing Add the translated text on the page as shown in the attached sheet.

(No change in content) international search report m1elRUle+tal Amkml@. 9゜pcT/cB　8510045 0

Claims (21)

[Claims] 1. What is the probability of determining whether the first and second living organisms, the identity of which is known, are the same? A method for determining whether (i) the genomic DNA of a first organism is one or more species; Digested using the above restriction enzyme; (ii) Separating the DNA fragments thus obtained by electrophoresis; (iii) separating them by electrophoresis; genomic DNA of an organism of the same species as the first or second organism of the fragments separated by one or more labeled proteins consisting of randomly derived DNA fragments from Determine the position to hybridize with the probe, (iv) The position of the fragment thus determined and the genomic DNA of the second organism are a DNA fragment obtained from the genomic DNA of the first organism by digestion with a limiting enzyme; produced from the genomic DNA of a second organism by subjecting it to electrophoresis in the same manner as comparing the positions of the DNA fragments that bind to the probe, The above steps (i) to (iv) are combined with hybridization in step (iii). An amount of probe DNA and one species or By using more restriction enzymes and comparing in step (iv), two types of restriction enzymes were used. When the organisms appear to be the same, the formula X=Fq(1) (where F is the genome of an independently obtained pair of organisms of the same species as the first and second organisms) It is a fraction representing the proportion of identical DNA fragments between restriction enzyme digests of DNA, and q is is the number of positions indicated by the blobbing in step (iii)). Two species of organisms can be clearly distinguished from each other as distinct and unrelated entities, as determined by How to achieve a low enough success rate (X) that it is not possible. 2. Steps (i) to (iv), (i') Genomic DNA of the first organism and genomic DNA of the second organism using the same restriction enzyme. digest each digestate separately as necessary; (ii') Electrolyze the digestate or a portion of the digestate for each organism in parallel on the gel. Subjected to electrophoresis; (iii') Blowing the gel with said labeled probe and thus Compare the hybridization patterns for the two organisms: (iv') If necessary, test each sample using a labeled probe consisting of a different DNA fragment each time. step (ii) and (for another one or more digestate parts of the product) The method of claim 1 carried out by repeating iii'). 3. Steps (i') to (iv') are repeated twice, each time using a different restriction enzyme. or more. 4. The probe or each probe is derived from the genomic DNA of the first or second organism. 2. The method of claim 1, comprising randomly derived DNA fragments. 5. The F value is (a) belong to the same species as the first and second organisms sufficient to obtain an F value representative of the species; The genomic DNA of multiple organisms obtained separately is digested separately using the same limiting oxygen. and divide each digestate as necessary; (b) Sort the digestate or part of the digestate for each organism obtained separately on a gel. (c) randomly derived from the genomic DNA of the organism of the species; blobbing the gel with a labeled probe consisting of a DNA fragment; (d) Hypothesis on gels thus presented for pairwise combination of separately obtained organisms. compare the hybridization patterns; (e) if necessary, compare different DNA fragments each time; A further species of each organism obtained separately using labeled probes consisting of fragments or Repeat steps (b) to (d) for further digestate portions; 2. The method of claim 1 which is determined empirically. 6. Repeat steps (a) to (e) twice or more, each time using a different restriction enzyme. The method according to claim 5, which is carried out at least once. 7. hybrids for each separately obtained organism and for all possible pairing combinations. 6. The method of claim 5, wherein the ization patterns are compared in step (d). 8. The probe or each probe used when determining the F-value is obtained separately. the genomic DNA of one of the organisms that has been identified, or the genomic DNA of the first or second organism; 6. The method according to claim 5, comprising DNA fragments randomly derived from . 9. Claims in which one of the separately obtained organisms is the first organism or the second organism Method 5. 10. The restriction enzyme or each restriction enzyme used in step (i) determines the F value. The method according to claim 5, which is the same as the restriction enzyme or each restriction enzyme used in the process. . 11. The probe or each probe used in steps (iii) and (iv) The probe is the same as the probe or probes used to determine the F value. The method according to claim 5. 12. If the comparison in step (iv) shows that the two species are the same, then The claim further comprises step (v) of calculating the degree of probability (X) that the objects cannot be distinguished. Scope 1 method. 13. Claim 1, wherein the first and second organisms are organisms that reproduce asexually in principle. Method. 14. 14. The method of claim 13, wherein the organism is a bacterium. 15. 14. The method of claim 13, wherein the organism is a fungus. 16. The method according to claim 13, wherein the organism is a virus. 17. The method according to claim 1, wherein the living organism is a plant. 18. Steps (i) to (iv) are performed such that X is 10-15 or less. 2. The method of claim 1, which is carried out using a limited amount of probe DNA and a limited number of oxygen. 19. 19. The method of claim 18, wherein X is 10-25 or less. 20. 19. The method of claim 18, wherein X is 10-40 or less. 21. It is not possible to determine whether the first and second living organisms, one of which is known, are the same. (A) the probability that the first and second organisms are the same species; DNA that is the same between restriction enzyme digests of genomic DNA of a pair of organisms obtained separately belonging to Determine the fraction (F) representing the proportion of A fragments; (B) Genomic DNA of the first organism is digested using one or more restriction enzymes. to become; (C) The DNA fragments thus obtained are separated by electrophoresis; (D) The DNA fragments thus obtained are separated by electrophoresis. from the genomic DNA of an organism of the same species as the first or second organism of the separated fragment; one or more labeled second proteins consisting of randomly derived DNA fragments; Determine the position to hybridize with the probe; (E) The position of the fragment thus determined and the genomic DNA of the second organism Digested using an enzyme or each restriction enzyme and obtained from the genomic DNA of the first organism. The genome of a second organism is extracted by electrophoresis in the same manner as the DNA fragments obtained. Binding to the probe or each probe of a DNA fragment produced from DNA Compare with the position; (F) If the comparison in step (E) shows that the two species are the same, X=Fq(1) (wherein F has the same meaning as above, and q is expressed by the blobbing in step (D)) the number of positions in which the two species are distinctly distinct and unrelated. consists of calculating the probability (X) that cannot be distinguished by Determination of fractions for step (A) involves (a') obtaining a representative F value for the species. The genomic DNA of a large number of separately obtained organisms belonging to said species, sufficient to (b') Digest each digestate separately as necessary; The digested material or a portion of the digested material for each organism was electrophoresed in parallel on a gel. Attachment; (c') DNA fragment randomly derived from the genomic DNA of the organism of the above species; blobbing the gel with a labeled first probe consisting of a piece; (d') Hatch on gels thus shown for pairwise combination of separately obtained organisms. Compare the hybridization patterns; (e') If necessary, use a different DN each time. Yet another species of each organism obtained separately using the labeled first probe consisting of the A fragment. or repeat steps (b') to (d') for more digestate parts. The method according to.