JPH06189800A - Identification and characterization of organism - Google Patents

Abstract

PURPOSE: To obtain a gene diagnosis kit including a vessel in which a probe organism, etc., having information the sequence of a stock gene in a concordance sequence is put, and a hybridized material, etc., of plural different known organism species, and capable of rapidly and accurately identifying an unknown organism.

CONSTITUTION: In a method for identifying pronucleus cell- and eucaryotic cell-organisms such as bacteria, plants and animals, the kit includes a carrier partitioned to tightly hold one or more vessels, one primary vessel contains a nucleic acid (no ribosome RNA information-containing nucleic acid) containing information of a stock gene material sequence of a probe organism, derived from the probe organism or of a concordance sequence and the kit includes a catalog having hybridized or reassociated pattern concordance sequences of chromatograph bands of at least 2 different known organism species or a gene material of at least 2 known organism species or derived therefrom and is able to rapidly and accurately identify the unknown organism.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for rapidly and accurately characterizing and immobilizing organisms, including prokaryotic and eukaryotic organisms such as bacteria, plants and animals.

[0002]

Background of the Invention The classification of living organisms has traditionally been done more or less voluntarily and along some artificial line. For example, the world of living beings is divided into two kingdoms: plants and animals.
ia). This classification is convenient for generally known organisms, but for organisms such as unicellular organisms (eg,
It is difficult for P. chinensis, bacteria, and cyanobacteria. This is because these are basically different from "plants" and "animals".

It has been proposed to simply divide the organism by the internal structure of its cells. In this way, all cell organisms are either prokaryotic or eukaryotic. Prokaryotes are eukaryotes (eukar)
less complex than they are, they lack internal compartments by unit membrane tissue and lack a clear nucleus. The genetic information of prokaryotic cells is carried in the cytoplasm on double-stranded circular DNA. No other DNA is present in the cell (unless there is a phage, bacterial virus, and circular DNA plasmid capable of autonomous replication). On the other hand, eukaryotes have a wide variety of unit membrane tissues, which serve to separate many functional components into specialized and isolated regions. For example, genetic information (DNA) is found in well-partitioned nuclei, and also in organelles glomeruli (mitochondria) and (in photosynthetic organisms) chloroplasts. The replication, transcription and translation of the eukaryotic genome occurs in two or three distinct sites within the cell, the nuclear cytoplasmic part, the glomerulus and the chloroplast.

However, the difference between prokaryotes and eukaryotes is crushed when comparing glomeruli and chloroplasts in prokaryotes. These organelles are now considered to be derived from free-living prokaryotes, which are prokaryotic primitive and endosymbiotic organisms.
c) entered into a relationship and ultimately became so closely integrated with the structure of the host cell that it became impossible to be independent (see Reference, Fox, GE et al. "Science").
209; 457-463 (1980) p. 462; St
anier, R .; Y. et al. "The Micr
"Obial World" 4th Edition, Prentice-
Hall, 1976, p. 86). For example, DNA obtained from mouse L cell filaments that carry the ribosomal RNA gene region is Escherichia coli.
i) Demonstrated significant sequence homology with ribosomal RNA, strongly supporting the endosymbiotic model (Van
Etten, R .; A. et al, "Cell" 2
2: 157-170 (1980)). Also, 23S type ribosome D of corn chloroplast (Zea mays)
The nucleotide sequence of NA has also been shown to have 71% homology to E. coli 23S ribosomal DNA (Edwards, K. & Kossel, H., "N.
ucleic Acids Research "9: 2
853-2869 (1981); and other related studies (Bonen, L. & Gray, MW, ibid. 8:
319-335 (1980)). It further supports the general idea.

In this model, eukaryotic cells are phylogenetic "chimeras" with organelles that are clearly prokaryotic in nature. The “pronuclear-eukaryotic” dichotomy also has drawbacks, even as a broad class of methods. The classification of organisms is more than a scientific task because of crossing and breeding (b
Reeding purposes are to identify plants or animals, and microorganisms that may infect so-called "higher" organisms or other media are accurately and reliably identified. For example, plant growers, livestock farmers or fish farmers will want to have a quick and reliable way to identify heterologous and atypical strains.
Veterinarians, doctors or horticulturists will want to accurately identify infectious organisms (parasites, fungi, bacteria, etc.) and viruses in test plants and animal tissues. Correct identification of these organisms and species of Willes is of particular importance.

This problem is best understood by explaining the identification of bacteria. The name of a bacterial species usually represents many strains, and one strain is considered to be a population derived from one cell. Bacterial species are generally defined by describing the degree of homogeneity and diversity of attributes in a typical strain of the species. Expressing an accurate definition of a bacterial species is difficult because it is necessary to define boundaries for strain diversity within a species in order to establish the boundaries of the species (Buch.
anan, R.A. E. , "International
Bulletin of Bacteriological
al Nomenclature and Taxon
Omy ", 15: 25-32 (1965)). To actually apply the definition of species to the identification of unknown bacterial strains, the selection of appropriate probes such as substrates and conditions for detecting phenotypic attributes, and radioactively labeled DNA from the same species is necessary. Due to the diversity of bacterial species, screening methods are the first tools used in classical, progressive methods for strain identification. The results of the screening method are used to predict which laboratory methods and reagents are suitable for the definitive identification of strains. The identification is ultimately based on certain phenotypic and genotypic similarities between the unidentified strain and the characterized species. The challenge is to define the boundaries of the species tightly, and preferably to do this using standard probes that provide species-specific information. By doing so, species definition can be applied directly and equally to the identification of unknown strains.

Vergie's Manual of Determinating Bacteriology (Buchanan, R .;
E. and Gibbons, N .; E. Edit, 197
4, 8th Edition, Williams & Wilkins
(Baltimore, Inc.) has shown the most easy-to-understand taxonomy, especially nomenclature, basic strains, and related literature. However, this is only the starting point for species identification. This is especially because it is obsolete and space limited so the description of the species is too simple. (Reference example: Brenner DJ (Brenner
D. J) "Manual of Clinical
Microbiology "3rd edition, American Society of Microbiology,
Washington D.C. C. , 1980, pp. 1-6).

When used in bacteria, the term "species" generally resembles each other as distinct species of organic matter, with distinctive characteristics, and in terms of essential organic tissue characteristics. It has been defined as a group of organisms with sex. The problem with these definitions is that they are subjective (Brenner, ibid., P. 2).
Species have also been defined based on criteria such as host range, pathogenicity, whether or not to produce gas during fermentation of certain sugars, and whether sugar fermentation is fast or slow.

[0009] In the 1960s, a numerical bacterial taxonomy (also called computer taxonomy or genetic phenotypic taxonomy)
Became widely used. Numerical taxonomies are based on testing as much as possible the genetic potential of an organism. By classifying on the basis of a number of characteristics, groups of strains with a certain degree of similarity can be formed and these can be considered as species. However, this definition of a species is not directly and practically applicable to the identification of unknown strains, as a test that is valid for characterizing one species may not be useful for the next. Even if these attributes are used for identification of unknown strains and can be partially overcome by choosing attributes that appear to be species-specific, this type of definition is indirectly applied. (See Brenner, ibid., Pp. 2-6). Moreover, the general method has several problems when it is used as the sole basis for defining species, among them the number and nature of the tests to be used and how well the tests are evaluated. What should be done to reflect the relevance, how much similarity should be chosen, and whether the same similarity criteria can be applied to all groups. Hugh R. H (Hugh, RH) and Gilliage G.H. L (Gilliardi, GL) "Manua
l of Clinical Microbiolog
y "2nd edition, American Microbial Society, Washington, D.Y. C. ，
1974, p. 250-269, list the minimal phenotypic traits as a means of defining bacterial species using genomic fractions. By studying multiple and randomly selected strain samples from within a single species, it is possible to select and define the attributes that are the most highly conserved or common to so many strains. it can. The use of minimal properties is an advance, starting with screening methods to tentatively identify strains and allowing the selection of appropriate additional media. The known attributes conserved in the species are then examined, expecting the strain to have most of the minimal characteristics. Some of the minimal characteristics do not appear in all strains of the species. Related concepts include comparative studies of species types, neotypes, or established control strains of the genus. This collation is necessary because each laboratory has different media and methods, and the strain is the standard of the species, not the method.

A molecular-level approach to bacterial taxonomy is to use two genomes for DNA-DNA reassoc.
iation). The genetic definition of a species includes the assumption that strains of the species are 70% or more related. Strains can be identified by DNA-DNA repairing only if the radiolabeled DNA probe and the unknown DNA are of the same species. However, the practical application of this 70% species-definition is limited by the choice of the appropriate probe. This may be overcome, in part, by selecting phenotypic attributes that appear to be interrelated with the repair group, but when they are used alone, species due to DNA-DNA repairing may occur. The definition of still applies only indirectly.

Brenner, supra, page 3, the ideal means of identifying bacterial species is to isolate the gene and immediately transfer the nucleic acid sequence in the strain to that of any known species.
It is described as a "black box" similar to mass spectrophotometric analysis as compared to a standard pattern for es-something).

However, although Brenner is able to perform restriction endonuclease analysis to determine the general sequence of isolated genes, "We have found that suitable black boxes, especially those suitable for use in clinical laboratories. Is unlikely to be available. " His words apply equally to every species of organism.

From this brief review of the prior art, unknown bacteria and other organisms are identified and rapidly classified, particularly pathogenic organisms or organisms that carry on biochemical reactions available. We conclude that there is now a need for a fast, accurate and reliable way to The method must be universally and readily available in the clinical laboratory, must not depend on the number of trials conducted, the clinician's subjective prejudice, and must Or inevitable trial and error method (Trial and error method)
Don't depend on od). In addition, it helps identify and distinguish the genus and species of any living organisms, by veterinarians, plant growers, toxicologists, animal keepers, entomologists, and in other relevant areas where such identification is required. It is also necessary to have a method that can be used easily and reliably.

[0014]

SUMMARY OF THE INVENTION The object of the present invention is therefore to provide organisms, in particular but not exclusively, microorganisms,
It is to provide a rapid, accurate and reliable method for objective identification.

[0015] Another object of the present invention is to provide a method for identifying an organism such as a bacterium by utilizing the genome of the organism.

Another object of the present invention is to provide a method for characterizing and identifying the species and genera of pathogenic organisms in clinical laboratories so that the cause of animal or plant diseases can be characterized and identified. It is to be.

[0017]

Another object of the present invention is to provide various products useful in the taxonomy described above. These and other objects of the invention include
This was accomplished by providing the following method, as will be more readily found below. That is, a method for characterizing a species of an unknown organism is provided (hybridization or repairing with a ribosomal RNA information-containing nucleic acid obtained from the unknown organism, from a known probe organism, or derived from the organism). Combined restriction endonuclease-digestion D
Of the evolutionary conserved sequence of the genetic material of the unknown organism corresponding to the known position of the restriction endonuclease cleavage site in the genetic material of the unknown organism (as opposed to the method by determining the chromatographic pattern of NA). The position of some or all is determined, thereby obtaining the genetic characterization for identification of the unknown organism, and at least two sets of genetic characterization for identification from the conserved sequence (of known identity). The method comprises comparing information from each of the pairs defining the airframe) with the characterization.

Another object of the present invention has been achieved by providing the following method. A method of diagnosing a pathogenic organism infection in a sample, the method comprising identifying an organism in the sample by the method described above.

The present invention provides a strain which objectively delimits species, despite dispersal, if the species is a collection of individually separated strains associated with a common species formation event. It is based on the inventors' recognition that there should be shared similarities and that strains of species should have structural information that provides clues to their common roots. The past history of organisms is the most abundant semantide (se
mantises), remaining in DNA and RNA (Zuckerhandle, E. and Paul.
ing, L.D. "Journal of Theoret
ical Biology "Volume 8: 357-366
(1965)).

European Patent Application (EP-A-) No. 0
In 076123 (added here as a reference),
The inventors have described a system for species definition and characterization of organisms that utilizes the information contained in ribosomal RNA (rRNA). Ribosomal RNA has structural and functional roles in protein synthesis (Schaup, “Jo
urinal of Theoretical Biol
, 70: 215-224 (1978)), and general conclusions drawn from studies of rRNA-DNA hybridisation, that the basic sequence of the ribosomal RNA gene is evolutionary compared to most other genes. It is said that it is less likely to change during the process and will be more preserved (Mo
ore, R.M. L. Written by "Current Topics
InMicrobiology and Immun
"Obiology", Volume 64, 105-128 (197)
4), Spring-Verlag, New York). For example, 16S type rR obtained from many bacterial species
The primary structure of NA was deduced from oligonucleotid analysis (Fox, GE et al, "Inter.
national Journal of System
"Matter Bacteriology", Volume 27:
44-57 (1977)). The 16S oligomer catalogs of several strains of E. coli have negligible differences (Uc
hida T. et al; “Journal of
Molecular Evolution ", Volume 3:
63-77 (1974), but substantial differences between species can be used to generate a bacterial phylogenetic taxonomy (Fo).
x, G. E. "Science" 209: 457-4
63 (1980)). Different strains of a given bacterial species are not always identical, and the restriction map shows different Ec
It has been shown that the oR I site occurs in the rRNA gene of two strains of E. coli (Boros, IA et.
al., “Nucleic Acids Research”
ch ”Volume 6: 1817-1830 (1979)). Bacteria appear to share conserved rRNA gene sequences, and other sequences may vary (Fox, 197).
7, ibid.).

Thus, the present inventor has found that the restriction endonuclease digestion of DNA is dependent on certain organisms (eg bacteria).
Strains of other species have similar combinations of strains, but strains of organisms of other species have different combinations of conserved sequences,
That is, despite the mutation of the strain, it has a high occurrence frequency,
We have found that the specific combination of enzymes, restriction fragments with minimal genotypic traits, determines species. This is EP-
It is the essence of the invention described in A-0076123 and the essence of the invention described herein.

The present invention is an extension of the concept developed in EP-A-0076123, and it was further discovered that, in addition to the sequence of rRNA, there are highly conserved sequences throughout evolution. , It would be as useful as an rRNA sequence as an identification system. In other words, the present invention provides a method for performing the identification and characterization technique of EP-A-0076123 by using a conserved probe rather than rRNA.

The present invention also provides additional embodiments of methods that may be used in the identification process. The present inventor intends to identify whether the method is prokaryotic or eukaryotic regardless of whether they are the same or different in taxonomy (classical). It has also been found to be general in that it can be applied to both prokaryotic and eukaryotic DNA using the described nucleic acid probes. The present invention provides an objective method of defining organisms based on conserved sequences of DNA or other genetic material corresponding to known positions such as restriction endonuclease sites. Detection of restriction fragments containing conserved sequences is carried out by hybridizing or repairing DNA fragments with nucleic acids containing conserved sequence information from the probe organism.

"Organism" characterized by the process of the present invention (this term is taken to include "identify")
By the term is meant by definition virtually any organism that contains DNA or RNA in its genome. It is useful to mention the traditional taxonomy for reference in this regard.

Monera kingdom, plants (Pla
ntae) and all organisms belonging to animals (Animalia). For example, monera
a) In the world, mycobacterium (myxobacterium)
ia), spirochetes, eubacteria, ricketttsiae divisions (bacteria) and cyanobacteria: cyanophytha
Can be mentioned. In the plant kingdom, euglenoides (Euglenophta), green algae: Chlorophyta, chlorophyceae (Chl)
orophyceae) and Carophyceae (Char
chrysota (chryso)
phta) xanthophyce (xanthophyc)
eae), chrysophysea
e), bacillarioph
Yersae; Pyrrophyt
a) Dinoflagella
tes), brown algae: phaeophy
ta); red algae: Rhodopt
a); slime mold: Myxomyco
phyta), myxomycete (myxomycete)
s), acrasiaes, plasmodiophoreae, labyrinthuleae; Yumycophyta: fungi (Eumycophyt)
a), phycomycetes, ascomycetes, basidomycetes; bryophytes, hepaticae, anthotes
cerotae), Musci class; vascular plant Tracheophyta, psilopsida, licocida (l)
ycopsyda), shenopsida (sphenops)
ida), pteropsida, spermopsida subspecies, cycadae, zincgoe (gingkoa)
e), coniferae, gnete (gn)
eteae) and angiolospermae (angios)
permae class, decotyledone
doneae), monocotyroedone (monocoty)
loedoneae) subclass is described. In the animal kingdom,
Protozoa sub-world, Protozoa Protozoa
a), Plasmodroma subphyla, flagellata, sarcodina and sporozoa
ozoa class; siliophor
a) Subphylum, Ciliata; Parazoa, Polyhera (Spongea porifera), Calcarea, Hexacinerida (hex)
actinellida) and Desmos Poguier (d)
esmospongiae; Mesozoa subdivision, Mesozoa; Metazoa subdivision Radiata division, coelenterata phylum, hydrozoa, siphozoa (scip)
hozoa, authozoa class, ctenophora phyla, tentaculata and nuda.
Class; protostmia division Platyhelminte
s), tubellana, trematoda and sesto.
da class; nemertina gate, acanthucephala gate; aschelmintles gate, rotifera, gastrotrica (gast)
rotricha), quinorhynka
ha), preaplida, nematoda and nematomorpha (n)
genus ematomorpha; ent proctor (ent
Oprocta gate; ectoprocta
cta gate, gymnolaemat
a) and phylactolata
emata) class; phoronida
Phylum; braciopoda phyla, inarticulata and articulata class; morska molluscata, amfinula (amph)
ineura), monoplacophora (monoplac)
opora), gastropod
a), scaphopoda, perecypoda and cephalopoda classes; sipuncrida (s)
Gate of ipunculida; echiuda
Rida Gate, Anelida Gate, Polychaeta, Oligolator (ol
igochaeta) and hirudineah
inea); onychophor
a) gate; tardigrada gate;
Pentastoimida
Gate; Arthropoda Gate; Trilobita Gate, Keri Serrata (c
helicerata submonium, xipho
sura, arachmida, pycnogomida, Mandibulata subphylum, krustae.
stacea), chiropoda, dipropoda, polopoda (p)
auropoda), symphyla
Rope, collembola, protura, diplura, thisanura, ephemerida (eph)
emerida, odonata, orthoptera, dermaputera (d)
ermaptera, embini
a), plecoptera, zoraptera, condentia (cor)
rodentia), mallofaga
ga), anoplura, thysasnoptera, hemiptera (h)
emiptera, neuropter
ra), choleoptera, hymenoptera, mecoptera (m)
ecoptera, siphonaptera (siphonap)
utera, diptera, trichoptera and lepidoptera, insects of the order Denterostomia; (Astero
dea), opiuroide
a), echinoidea and holoturoidea, Pogonophora phylum, hemicordata phylum, enteropneusta and pterobranchia (pterobranchia)
Hordata Gate, urochor data (urochor)
data) Submonium, ascidiacia
e), Thaliaaceae, Larvacea; Cephalocordata (c)
subdivision of ephalochordata, subdivision of vertebrata, agnat
ha), chondrichthye
s), osteichthyes, saccopteiigii subclass, crosopterygii and dipnoi, amphivia (amph)
ibia), lepitiglia, aves and mammalia
Class, Prototheria subclass, Theria subclass, Marsa pierris (mars)
upialis), Insecti Bora (insecti)
vora), Dermoptera,
Chiroptera, primatis (p
rimates), edentata, polidota, lagomorpha (l)
agomorpha), rodentia
a), cetaceae, carnivore (c)
arnivora), Tubuli dentata (tubuli)
dentata), provosided
ea), hiracoidea, sirenia, perisodactyla (peris)
sodactyla) and altiodachira (arti)
odactyla) can be mentioned. It is known that there are still families, families, genera, species and subspecies under the eyes, as well as taxa, strains or individuals other than subspecies. Moreover, cultured cells (plants or animals) can be identified in the same way as viruses. These classifications are used in this application for descriptive purposes only and not as a limitation. The organism to be identified may be known or unknown, but is most commonly unknown. Functionally, it is convenient for the purposes of the present invention to divide all organisms into eukaryotic and prokaryotic populations. When identifying prokaryotic organisms, the DNA is that which is present in the cell or in an uncompartmented chromosome. When identifying a eukaryotic organism, nuclear DNA or DNA within organelles (filament body DNA or chloroplast DNA) may be used.

Briefly, conserved sequences (and perhaps subclass subclass or subclass subclasses (infras).
High molecular weight DNA and / or small circular DNA are isolated and identified from the organism in order to analyze (sequences that can be used to generate ubspecific subdivision). DNA is extracted by methods well known to those skilled in the art.

The DNA is analyzed to confirm both 1) the presence and location of conserved sequences and 2) the location with respect to endonuclease restriction sites. The most convenient way to analyze the presence of conserved sequences is to utilize polynucleotide probes that can hybridize with conserved DNA sequences. However, direct sequence information such as that obtained by chemical sequence measurement and analysis is also utilized. EP-
The probe used in A-0076123 is r
Although it was a probe containing RNA information, any other probe having a conserved sequence can be used in this case. In a similar manner, the simplest way to find a given set of endonuclease restriction sites is to cleave the DNA with appropriate restriction enzymes (in fact this was taught in EP-A-0076123 and carried out Was done). However, sequence information linked to known restriction site sequences or other methods such as truncation and partial sequence may also be used.

Most commonly, DNA is cleaved into fragments by restriction endonucleases at specific sites. The fragments are separated by size in a chromatographic system. In EP-A-0076123 gel chromatography was used as an example of a useful chromatographic system. However, other systems such as high pressure liquid chromatography, capillary zone electrophoresis, or other separation techniques can also be used. When using gel chromatography, the fragments are separated, the gel is stained and standardized for fragment size using a standard curve made of fragments of known size, in a manner well known to those skilled in the art. The separated fragments are then separated from Southern
rn) spot method (Southern, EM “J.
individual of Molecular Biolo
gy ", 38: 503-517 (1975), incorporated herein by reference), and transfer to covalent bonds thereto by heating. Fragments containing conserved sequences are then located by their ability to hybridize to nucleic acid probes that contain conserved sequence information.
Alternatively, hybridization may occur after digestion but prior to separation, or a restriction fragment may occur after hybridization and then the fragments separated.

The nucleic acid probe is labeled with a non-radioactive substance or preferably with a radioactive substance.
When labeled with a radioactive substance, the probe may be RNA, or DNA complementary to RNA (cDNA that is synthesized on the clone fragment that can be synthesized by reverse transcription or labeled by, for example, nick translation). ) Is okay. In addition, synthetic oligodeoxyribonucleotides may be synthesized from labeled nucleotides.

Well-defined probes may be obtained from arbitrarily selected organisms, as will be seen later, or may be obtained from matched sequences. Once hybridization has taken place, the hybridized fragments can be detected by selectively detecting double-stranded nucleic acid (non-radioactively labeled probe) or made visible, for example by radioautograph. (Radiolabeled probe). The size of each hybridized fragment is related to the restriction site and can be determined from the migration distance using the standard curve described above. The amount hybridized, the hybridization pattern, and the size of the hybridized fragments are related to the restriction sites and are used to identify organisms individually or in combination. The genetic characterization that results from this technique can be easily compared to equivalent characterizations obtained from at least two, or otherwise a large number of known standard organisms, genera or species. After a preliminary broad classification had already been performed (eg using classical classification), it was hybridized (as in EP-A-0076123) by visual inspection and matching with the appropriate chromatographic pattern. Comparison can be performed by comparing the sizes of the restriction fragments, by the band intensity (hybridization amount), or by combining these. Ideally, point-of-sale
e) a one-dimensional computer, such as used for processing-
It is better to compare with a pattern recognition device.

When using the above method, the inventors have found that the genetic characterization of organisms of the same species is very similar, the slight difference is only the difference between subspecies due to strain variation, but The differences between and among genera (and even at higher levels of classification) are very large.

The variability of enzyme-specific fragments between strains of one species can be used to type strains for various purposes, eg in the case of bacteria, epidemiological purposes. In fact, restriction enzymes can be chosen that can distinguish between strains within a species.

“Probe organism” used in this study
(And from which the nucleic acid probe is obtained) may be any of the organisms listed above, either a eukaryotic organism or a prokaryotic organism. The only limitation is that the conserved sequence-containing probe
Given by the fact that it must hybridize maximally with DNA of an unknown organism.

Conserved sequence information-There are four types of probes containing: 1) pronuclear probes (especially obtained from bacteria), 2)
Eukaryotic glomerular probe, 3) eukaryotic chloroplast probe, 4) eukaryotic non-organic probe.

There are also four sources of DNA (digested by endonuclease): 1) prokaryotic DNA, 2) eukaryotic glomerular DNA, 3) eukaryotic chloroplast DNA, 4) eukaryotic nuclear DNA. ..

The following hybridization table was thus created (Table 1).

[0037]

[Table 1]

The table generally shows which probes are capable of maximally hybridizing to DNA of unknown organism. For example, in order to identify certain eukaryotic organisms, species-specific glomerular or chloroplast DNA is extracted and digested with an endonuclease, which digest is used as a pronuclear probe or organelle. It may be hybridized with the eukaryotic probe extracted from. Similarly, to identify pronuclear organisms, species-specific cellular DNA is extracted, digested with endonucleases, and the digest is hybridized with pronuclear probes or eukaryotic probes extracted from organelles. You can change it. Eukaryotic organisms can also be identified by extracting and digesting species-specific nuclear DNA and hybridizing it with non-organic eukaryotic probes. Eukaryotic cells could be defined by one or some combination of nuclei, glomeruli, or in some cases the chloroplast system. These cross-hybridizations show that nucleic acids from eukaryotic organelles have broad homology with evolutionary conserved sequences from prokaryotic nucleic acids, but not nuclear-extracted eukaryotic DNA and prokaryotic DNA.
Between and is based on the fact that the homology does not generally exist as such.

The choice of the DNA to be digested and the associated probe pair is arbitrary and will depend on the organism to be identified. That is, it will depend on the question asked.
For example, when detecting a prokaryotic species (eg bacteria) present in or together with a eukaryotic cell (eg animal or plant) for the purpose of detecting and identifying infectious agents, a pronuclear probe should be selected, The DNA derived from the organ may be treated under the condition that it is not extracted or the minimum amount is extracted. Using this approach, it can be believed that the interference between organelle-derived DNA and pronuclear DNA is minimal. When identifying a eukaryote (which is not infected by prokaryotic cells) with a prokaryotic probe, DNA from the organelle is isolated, for example by separating the organelle from the nucleus and then extracting only the organelle's DNA. It is best to maximize the concentration. When it is desired to identify prokaryotic infected eukaryotic organisms, it is best to use probes from non-organ, non-prokaryotic cells. Because this probe generally does not hybridize well with DNA from prokaryotic cells.

Same world, same world, same department, same gate, same subgate, same class, same subclass,
Alternatively, it is preferred to use pairs (DNA and probe) from the same order, or the same family, or the same family, or the same genus. It is particularly preferred to use a prokaryotic probe (eg a bacterial probe) to form a hybrid with prokaryotic DNA. In this way, genus, species and strains of pronuclear organisms could be detected, quantified and identified. One of the most preferred pronuclear probes is obtained from bacteria, and among them, E. coli is particularly preferable because it is easy to use and is easily available. The probe obtained from E. coli can be used to identify any organism, especially all prokaryotic organisms, and is most preferred for the identification of strains of any bacterium. Another, particularly preferred embodiment is the use of eukaryotic probes from a given family to identify eukaryotic cell organisms of the same family (eg, mammalian organisms to identify mammalian organisms). Using a probe). Most preferred is the use of probes and DNA obtained from organisms of the same subfamily and / or order and / or family (eg in the case of identifying certain species of mouse, probes of mouse origin are used). Is good).

The most sensitive and effective pairing system is the one in which the source of the probe and the source of the unknown DNA are evolutionarily less distanced or less dispersed.

In the present invention, the phrase "an evolutionarily conserved genetic material sequence" refers to a sequence of genetic material, for example DNA, which shows homology between at least two different species of plants, animals or microorganisms. Used to represent Homology between two conserved sequences means that two single-stranded DNA molecules or fragments are hybridized to each other under hybrid conditions if one of the sequences of such a DNA molecule is detectably labeled. Hybridisation or annealing occurs when it is placed in, which results in a duplex that is sufficiently stable to be detectable by standard methods (ie radioactive labeling, enzymatic labeling, etc.).

The evolutionary conserved sequence exemplified in EP-A-0076123 was that of the ribosomal RNA gene. This is still a highly desirable gene sequence. However, it has been discovered that there are other useful gene sequences that are well conserved across evolutionary distances.

Examples of such additional sequences are given in Dayhoff's "Atlas o", incorporated herein by reference.
f Protein Sequence and St
structure ", Volume 5, Supplem
ent 3,1978, NBR, 1979, pages
9-24, Superfamily, Family, and preferably, Subfamily.
ly) or a sequence of a gene or part encoding a transfer RNA or protein shown as belonging to the Entry. The family of proteins is one in which any two proteins are distinguished by 50% or less amino acid residues of their sequences. A subfamily of proteins is one that distinguishes any two proteins by 20% or less of the amino acid residues in the sequence. The phylum distinguishes any two proteins by amino acid residues of 5% or less of the sequence.

Specific examples of gene sequences or suitable parts thereof that may be used are cytochrome C-related genes, cytochrome C ₃ -related genes, cytochrome C ₁ -related, cytochrome b ₅ -related, ferrodoxin-related, libredoxin-related, flavodoxin-related, alcohols. Dehydrogenase related, peroxidase related, adenylate kinase related, phospholipase A ₂ related, tryptophan operon related, carboxypeptidase related, subtilisin related, penicillinase related, protease inhibitor related, somatotropin related, corticotropin related, lipotropin related, glucagon related, snake Vein toxin (snak)
e venom toxin-related, plant toxin (pla)
nt toxin-related, antimicrobial toxin (antiba)
cterial toxin-related, immunoglobulin (immunoglobulin) -related gene, ribosomal-related gene other than ribosomal RNA, heme carrier gene, chromosomal protein gene,
A fibrous protein gene and the like.

Conservation of some of these additional DNA sequences is not as prevalent throughout the animal, plant or microbial kingdom as is the case for ribosomal RNA (rRNA) genes. (Thus, the use of rRNA is still preferred). However, this does not constitute a significant impediment to its use as additional sequences may be available to identify or characterize the organism within a more limited range or subfield. Absent. For example, trp
It is possible to utilize the trpD sequence from a microorganism to generate this microorganism probe and then use this probe for testing in the microbial kingdom. In fact, trp D probes of the same species, family or genus may be used in a narrower world (eg, Enterobacter).
teriaceae) or Bacillus
It is possible to use the trp D probe within the test for the presence of s) etc.). Thus, although the range of applicability of some additional probe sequences is not as wide as the rRNA probe, its applicability will still be very effective within the narrow world.

The probe containing the conserved DNA sequence information is E
It is produced by the same operation as the method for producing the rRNA information-containing probe exemplified in PA-0076123. Thus the probe may be RNA, DNA or cDNA and the like.

The individual steps involved in the technique refer to both eukaryotic cells and prokaryotic cells (where applicable), or, if there are technical differences, each type of cell. I'll talk about each separately.

The first step is the extraction of DNA from unknown organisms. Nuclear DNA from eukaryotic cells can be selectively extracted by standard methods known to those of skill in the art (as an example, by Drohan, Wet al., "Bioche."
m. Biophys. Acta ", 521 (197)
8), 1-15, hereby incorporated by reference). Since organelle DNA is small and circular, the spooling method helps separate non-circular nuclear DNA from circular, organelle-derived DNA. As a result, the unspooled material contains DNA from organelles that can be individually separated by density gradient centrifugation. Alternatively, the glomeruli (or chloroplasts) are separated from a mixture of crushed cells and the purified fraction of glomeruli (or chloroplasts) is used to prepare DNA from organelles and purified. The prepared nuclear fraction is used for preparation of nuclear DNA. (As an example, Bonen
L. and Gray M.D. W "Nucleic Ac
ids Research "8: 319-335 (19)
80)).

Pronuclear DNA extraction is also well known to those skilled in the art. For example, unknown bacteria present in media such as industrial fermentation suspensions, agar, plant or animal tissues or samples etc. are treated under well-known conditions designed to extract high molecular weight DNA. . For example, cells of an unknown organism are suspended in extraction buffer, lysozyme is added to it and the suspension is incubated. Cell disruption can be further promoted by the addition of detergents and / or increased temperature. After protease digestion, chloroform / phenol extraction and ethanol precipitation are performed to complete DNA extraction. Another extraction method, which is much faster than phenol / chloroform extraction, is the rapid separation of DNA using ethanol precipitation. This method is preferably used to separate DNA directly from colonies or small volumes of liquid medium. This method is described by Davis, R .; W et al "A Manual for Genetic Eng"
inering, Advanced Bacteri
al Genetics "(hereinafter referred to as" Davis "), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1980, p. 120-121 (written here as a reference).

DNA (prokaryotic or eukaryotic (nuclear or non-nuclear)) is lysed in physiological buffer for the next step.

There is diversity in the possible steps that follow after isolation of the desired DNA. One of these steps is endonuclease digestion.

Digestion of the extracted DNA is carried out with a restriction endonuclease enzyme. Any restriction endonuclease enzyme can be used. It is preferably not obtained from the same species of organism as the one to be identified. Otherwise the DNA will remain completely intact. (This will eventually identify the organism.
Because it is unlikely that the enzyme will cleave DNA from its own source species). Since the organism species to be characterized will be unknown, a minimum amount of trial and error will be required to obtain a fragment digest, which will be routinely performed by a skilled person without undue experimentation. It is a work that can be performed. Examples of potential restriction endonuclease enzymes are Bgl I, BamHI, EcoR
I, Pst I, Hind III, Bal I, Hga
I, Sal I, Xba I, Sac I, Sst
I, Bcl I, Xho I, Kpn I, Pvu I
I, Sau IIIa, and others. Davis, ibid,
p. 228-230, see also (incorporated herein by reference) one or more endonuclease mixtures can also be used for digestion. Usually, the DNA and endonuclease are combined in a suitable buffer for a suitable time (1 to
Incubation within the range of 48 hours, temperature range 25 ° C-65 ° C, preferably 37 ° C).

The resulting identifying gene characterization will depend on the type of one or more endonucleases used and will be specific to the endonuclease. Therefore, it is necessary to pay attention to which enzyme (one kind or several kinds) was used for digestion. This is because the comparative characterization used in the catalog must have been made with the same enzyme or enzymes.

Another step is to reveal the endonuclease sites on the desired DNA molecule without digesting the desired DNA molecule, for example by reference to a sequencing and restriction site library. Obviously, digestion is a more effective way of indicating the site, but should not be limited to this. The essence of the invention is that the DN is related to the position of the endonuclease restriction site.
The finding is that the position of the conserved sequence along A constitutes a characteristic set for each species. Therefore, any technique for obtaining desired information (relationship between site position and gene position) will be useful in the invention.

Moreover, the location of conserved sequences along the DNA molecule is best indicated by the use of hybridized probes. This probe anneals a restriction fragment of unknown DNA. However, other methods of determining conserved DNA sequences, such as sequencing, are also useful. When using hybridization probes, it is preferred to first digest and separate the DNA fragments according to size, and then hybridize the separated fragments. However, it is also possible to first digest and anneal the DNA with excess molar probe and / or a sequence complementary to the probe before separating the mixture. For example, unknown D
NA is digested with restriction endonucleases, denatured,
Small detectable DNA fragments in liquid, or
It may be hybridized with one or more molar excesses of synthetic oligodeoxyribonucleotides complementary to some or all of the conserved sequences of interest. In most cases one or more double-stranded regions of the hybrid will be small compared to the size of the restriction fragment, as most restriction enzymes cut quite rarely in DNA. The hybridization reaction is performed under conditions such that only oligodeoxyribonucleotides hybridize. Single chain DN
DNA fragments containing unreacted products such as the A fragment and hybridized oligodeoxynucleotides are separated by traditional chromatographic techniques. Labeled DNA fragments will appear in the expected sized fractions. It is also possible to first anneal the DNA with an excess molar amount of probe, then digest and then separate the mixture. When the solution is incubated for a short time or at low Cot, the restriction sites will be restricted to the hybridisation or duplex regions. When the solution is incubated for a long time or at high Cot, the unknown DNA will anneal, resulting in a labeled duplex susceptible to restriction endonuclease cleavage. Single-stranded ends without recombination are removed by a nuclease such as S1.
Unpaired bases are DNA polymerase I or T4
Filled with polymerase.

Also, stored sequence information (for example, 20-, 30
-Or 50), some subsequences are more highly conserved than the rest of the conserved regions selected as probes. These "short" sequences may optionally be made synthetically or enzymatically and incorporate labeled nucleotides. Single chain from unknown, predigested D
NA can be incubated with and hybridized to these short, highly conserved fragments. Separation is then performed on the short labeled probe with a digestion mixture containing the partially annealed fragments.
(Thus, separation occurs after hybridization). For all practical purposes the digestion mixture behaves like a mixture of essentially single-chain fragments, so that the separation is carried out by liquid chromatography.

As pointed out, the preferred method is to first digest, then separate and then hybridize. Therefore, after endonuclease digestion, the incubation mixture containing fragments of different sizes is preferably separated by a suitable chromatographic method. If hybridization is the last step, any method that allows the nucleic acid digests to be separated by size and allowing subsequent hybridization with nucleic acid probes can be used. For example, gel electrophoresis, high pressure liquid chromatography or capillary zone electrophoresis can be used. (Jorgenson, J .;
W. J. of HRC and CC, 4: 230-
231 (1981)). Presently, gel electrophoresis is preferred, and agarose gel electrophoresis is particularly preferred. In this device, the DNA digest is usually electrophoresed in a suitable buffer and the gel is usually
Immerse in ethidium bromide solution and place in UV-light-box to see possibly added standard marker fragments. It is also possible to use labeled standard marker fragments for detection.

After separation and visualization, the D
The NA fragment was subjected to the Southern method by nitrocellulose filter paper or a charge-modified nylon membrane (charge-modify).
dnylon membranes) (“Jou
rnal of Molecular Biolog
y "38: 503-517 (1975)). This transfer can be done after the denaturation and neutralization steps and is usually transferred from the gel to the filter paper over a long period of time (about 10-20 hours) or by applying electrical force. Devices that facilitate transfer from gel to filter paper are commercially available. The received nitrocellulose filter paper is then heated at elevated temperature (60-80 ° C) for several hours to bind the DNA to the filter paper.

In addition, Purrello, M. et al. Et al (Ana
l. Biochem. , 128: 393-397 (19
By using the recent method of direct hybridization of 83)), transposition is avoided.

The probe used for the hybridization of the DNA digested fragment bound to the filter paper is a nucleic acid probe,
It is preferably derived from a given well-known organism or a known base sequence.

The probe sequence has a natural counterpart (c
Those having no outer part) are preferable. That is, the probe sequences may be heterologous, with the bases being the same sequence in each of the many commonly occurring residues of the same sequence. The same sequence will generally form a more stable hybrid than one of any naturally occurring sequences. The probe may be a synthetic oligodeoxyribonucleotide molecule made by covalently linking individual nucleotides to a predetermined sequence. Synthetic molecules are, for example, the triphosphate method in the synthesis (Alvarado-Orbina et a.
1, Science 214: 270-274 (198).
1)). Probe molecules of any size are useful and need only be one or more sequences in the probe solution.
For example, several 20 base sequences are used to detect several highly conserved portions of the rRNA gene. It is either detectably labeled or unlabeled, but it is preferably detectably labeled. In this case, the nucleic acid probe is a detectable labeled RNA, preferably a truncated translationally labeled DNA, a cloned DNA or a detectable DNA (cDN) which is complementary to the RNA from the probe organism.
Any one of A), which contains highly conserved DNA sequence information. Since synthetic oligodeoxynucleotides are prepared with detectable labeled nucleotides, the molecule is labeled by incorporating labeled nucleotide residues. Depending on the choice of combination, the probe can be from prokaryotic cells or eukaryotic cells (cytoplasm-derived,
Or derived from an organelle). Most preferably, the detectable label is a radioactive label such as radioactive phosphorus (eg ³² P, ³ H or ¹⁴ C), or based on biotin / avicin (biotin / avidin).
-Based) system. The nucleic acid probe may be labeled with a metal atom. For example, uridine and cytidine nucleotides can form covalently bound mercury derivatives. Nucleoside triphosphate bound to mercury is a good substrate for many nucleic acid polymerases, including reverse transcriptase (Dale et al, “Proceedings”).
of the National Academy o
f Sciences "70: 2238-2242, 1.
973). Direct covalent mercuration of natural nucleic acids has been reported. (Dale et al, "Biochem
Istry "14: 2447-2457). The reannealing property of the mercuric polymer is
Similar to that of the corresponding mercury-free polymer (Dale
and Ward, "Biochemistry" 1
4: 2458-2469). Metal-labeled probes can be detected, for example, by photo-acoustic spectroscopy, X-ray spectroscopy, such as X-ray fluorescence, X-ray absorption or photon spectroscopy.

Isolation and preparation of the desired conserved DNA sequence containing probe is within the skill of the art. For example, separation of rRNA from eukaryotic and prokaryotic cells is well known to those of skill in the art. Therefore, from the eukaryotic cytoplasmic ribosome to rRN
To prepare A, RNA can be extracted from whole cells or ribosomes and separated by sucrose gradient centrifugation and the 18S and 28S fractions can be collected using markers of known molecular weight (eg P
erry, R.A. P. And Kelly, D .; E. , 5S RNA when production of 28S and 18S ribosomal RNA is inhibited by small amounts of actinomycin D.
Persistent Synthesis of "J. Cell. Physiol. , 7
2: 235-246 (1968), hereby incorporated by reference). As a corollary, organ-derived rR
NA is isolated and purified from organelle fractions in a similar manner (eg Van Etten RA, e.
Tal, "Cell" 22: 157-170 (198).
0), or Edwards, K .; et al, "Nuc
Leic Acids Research "9: 2853
-2869 (1981)).

If a radiolabeled probe is used, it may be a probe organism grown or cultivated in a nutrient containing a compound with suitable radioactivity, or in a culture medium containing such a compound. Separated from. When the probe is complementary DNA (cDNA), it separates RNA separated from the probe organism into radioactive nucleoside triphosphates (eg ³² P-nucleosides or
³ H-nucleoside) in the presence of reverse transcription.

The labeled probe is also a transcribed translation DNA
It may be derived from a molecule, in particular a whole circular DNA from an organelle. Specifically, the chloroplast or filamentous circular DNA is cut and translated in the presence of a radioactive label to obtain a labeled DNA probe. The chloroplast-labeled probe will hybridize best with chloroplast DNA and the glomerular-labeled probe will hybridize best with glomerular DNA. A chloroplast (or glomerulus) incision translation labeling probe is a glomerulus (or chloroplast)
Will hybridize second best with DNA. It will also hybridize to the DNA of the whole plant (or animal), although it is generally less preferred. The probe may be obtained from the nuclear DNA of a eukaryotic cell by incision translation even if this method is not good in consideration of practicality. A more useful approach to achieve this is to excise a highly conserved gene from the nuclear DNA of eukaryotic cells (with a restriction enzyme), separate the fragments and identify the gene sequence (by hybridization),
Then, the gene sequence is separated (by electrophoresis). The isolated sequences can then be religated into a plasmid or other vector and cloned in a ³² P-containing medium after transformation of the appropriate host. Another method is to grow a transformed host and then separate the DNA and label it by truncation translation or separate the DNA,
The sequence is excised and then labeled. The resulting ribosomal probe hybridizes in the same state as cDNA (see below).

A preferred nucleic acid probe is radiolabeled DNA complementary to RNA from the probe organism.
The RNA is usually a messenger RNA that decodes a conserved gene and is essentially the carrier DNA (tRNA).
Or (unless rRNA is used) ribosomal R
Other RNA such as NA (rRNA) is not included. If rRNA is used, prokaryotic rRNA is usually 3
Contains subgroups of types. So-called 5S, 16S, and 23S-fragments. Reverse transcription to cDNA is done with a mixture of all three or else with a mixture of 16S and 23S fragments. Reverse transcription with only one of these rRNA components, although possible under certain conditions, is less preferred. Eukaryotic rRNA
Is usually in two subgroups, 18S and 28
Contains S. And reverse transcription into cDNA is 18S and 2
Either a mixture of 8S fragments or each of these.

Pure rRNA, depleted in other types of RNA, is incubated with reverse transcriptase capable of reverse transcribing to cDNA. More preferred in this case is the presence of avian myeloblastosis virus (AM) in the presence of a primer such as calf thyroid DNA hydrolyzate.
Incubation with reverse transcriptase from V). The mixture must contain a suitable deoxynucleoside triphosphate, at least one of the nucleosides being radiolabeled, for example with ³² P. For example, deoxycytidine 5 ′-( ³² P), deoxythymidine 5 ′-( ³² P), deoxyadenine 5 ′-(
³² P), or deoxyguanidine 5 ′-( ³² P) .triphosphate is used as the radioactive nucleoside. 30 minutes to 5
After incubation at 25 ° C-40 ° C for a period of time, extraction with chloroform and phenol, centrifugation and chromatography, the radiolabeled fractions are combined and used as a cDNA probe. A radiolabeled cDNA probe containing substantially pure form of conserved DNA information, ie a cDNA that is free of unlabeled molecules and is complementary to other forms of RNA.
A radiolabeled cDNA probe that has no NA, no proteinaceous substances, and no cellular components such as membranes and organelles also constitutes one aspect of the present invention. Preferred probes are labeled with prokaryotic cells c
The most preferred DNA is bacterial labeled cDNA. The species of probe is a bacterial microorganism, for example Enterobacteriacea.
e), Brucella, Bacillus (Ba)
Cillus), Pseudomonas
as), Lactobacillus
s), Haemophilus,
Microbacterium
m), Vibrio, Neisseria (Nei)
sseria), Bactroides
s) Species within the family and other anaerobic groups such as Legionella are used. Although the prokaryotic example in this application is limited to the use of E. coli as a bacterial pronuclear probe organism,
It is by no means limited to this microorganism. The use of radiolabeled cDNA as a probe results in greater stability during hybridization of the DNA and therefore radiolabeled R
It is preferable to use NA.

It is important to recognize that the labeled cDNA probe must be a faithful copy of the RNA, ie that the entire nucleotide sequence of the template RNA is transcribed at every time synthesis takes place. The use of primers is essential in this respect. That the cDNA is a faithful copy can be evidenced by the fact that it has two properties after hybridization: The cDNA must protect 100% of the labeled rRNA from ribonuclease digestion; and The labeled cDNA must anneal specifically to rRNA as evidenced by resistance to S1 nuclease. Belgiansky M. M. et al., “CR Acad Sc”
Paris' t286, Series D.P. p. 1825-
1828 (1978) derived from E. coli rRNA ³
H-radiolabeled cDNA has been described. The cDNA in this study was not prepared with reverse transcriptase in the presence of a primer as in the present invention, but was rRN pre-divided with ribonuclease U _2.
It was prepared with DNA polymerase I using A as a template. Beljansky et al.'S rRNA digestion product (R
NA se U ₂ ) has a different base ratio than the original rRNA, indicating that bases and / or short fragments have been lost. CDNA thus obtained
Is not a faithful copy. Moreover, the use of DNA polymerase I used by Belgiansky is known to accelerate the predominance of homopolymeric transcription over heteropolymeric transcription of rRNA (see Sarin PS et al.
al, "Biochem, Biophys, Res. C.
omm. , 59: 202-214 (1974)).

In summary, the probes are: a) cloned and / or truncated translated from genomic DNA containing conserved sequences, eg, genes,
b) from RNA itself, or c) from cDNA, RN
Obtained by reverse transcription of A.

[0070] Typically, the next step in the process of the invention is the unlabeled or (preferably) radiolabeled RNA or DN of isolated DNA digests from unknown organisms.
Hybridization with A probe. Hybridization is performed by contacting a paper containing a covalently labeled DNA digest from an unknown organism with a hybridization mixture containing the probe. Incubation is carried out at elevated temperature (50-70 ° C) for an extended period of time, after which the filter paper is washed to remove unbound radioactivity (if required) and then air dried to prepare for detection. Another highly preferred hybridization, which is much faster than the above method, is Corne D. et al. E (Kohne,
D. E) et al., "Biochemistry" 16:53.
29-5341 (1977).
Room temperature phenol emulsion re-pairing method.

After hybridization, this method requires the selective detection of appropriately hybridized fragments. This detection takes advantage of the fact that the hybridized fragment has a double-stranded structure, which allows a selective method (in the case of unlabeled probe), radio-autograph method or computerized or not. Well, this can be done by any suitable radiation scanner method (in the case of labeled probes) which will increase the detection speed.
These methods are well known to those skilled in the art,
There is nothing more to say in this regard.

The final product of this method is an identifying gene characterization, such as a chromatographic band pattern, with peaks and valleys of varying intensity at specific positions, or preferably light and dark regions. These positions are EcoR
By subjecting a marker such as I-digested λ bacteriophage DNA to isolation techniques, one can easily match to a particular fragment size (kilobase pair). In this way, the relative positions of the bands and the absolute size of each band can be easily ascertained. The identifying genetic characterization of the unknown organism is compared to the characterization found in the catalog or library. The catalog or library may consist of at least two, and almost infinite number of books listing the genus and species characterization of various identifiable organisms. For example, the pathologically relevant bacteria that cause human disease are estimated to be about 100, so it is estimated that a standard catalog of pathogenic bacteria will include 50-150 such characterizations. A catalog of bacterial strain types for epidemiological judging systems may also be included.

The characterization depends on the type or group of endonuclease enzymes chosen and is probably applied to the particular organism used as the source of the radiolabeled probe (probe organism) and also for probe preparation. Composition of stored conserved DNA sequence information nucleic acid (eg 5S of prokaryotic cells, 1
6S or 23S type subtypes, 16S and 23S types only or matching sequences, etc.). Thus
The catalog may include various enzyme-specific characterizations with recorded band sizes and relative intensities for each probe. As the concentration of bound DNA bound to the filter paper decreased, only the strongest bands became visible, and the size of these or these bands can identify the species.

The above changes or permutations are, of course, all applied to the library. Moreover, for eukaryotic organisms, the library may contain patterns of one type of DNA, or organelles and / or combinations of each-DNA. The pattern of each DNA digest will depend on the probe composition. The catalog is
If one or more strains or species are present in the extracted sample and are detected by the probe, they are arranged so that the characterization resulting therefrom can be interpreted. The user can compare the resulting characterizations, for example the swath patterns visually, or by a one-dimensional computerized digital scanner programmed for pattern recognition. These computer scanners are time-of-sale.
It is well known to those skilled in the art of-sale processing (a commonly used "supermarket" checkout barcode or pattern reader). Ideally, the library or catalog should reside in computer memory with the relative characterization of multiple organisms and the absolute value of the molecular weight or size of the fragments. If so, the catalog comparison is by matching the unknown characterization with one of the characterizations present in the library by one or both of the stored information elements (relative characterization and / or absolute size element). Done. The intensity of each band when compared to the standard is the hybridized bound DNA
Can be expressed as the amount of
For example, it can be used to estimate the degree of presence of prokaryotic cells in eukaryotic cells.

If the user wants to confirm the properties of a given organism and further identify, such a user can digest an unknown organism with a second different endonuclease and obtain The resulting characterization may be compared to the catalog characterization of the organism in the case of the second chosen endonuclease. This process can be repeated as many times as necessary to make an accurate identification. However, it is usually sufficient in most cases to perform a single analysis with a single probe.

The present invention and its variants have numerous applications. The present invention may be used by plant growers or animal keepers to correctly identify their objects, and in clinical and microbiology laboratories, bacteria present in any medium, including eukaryotic cells, parasites. It may be used to identify insects or fungi. In this latter case, this method is used as a standard microbial assay. This is because there is no need for microbial isolation and growth. In vitro
Proliferation and characterization is currently performed by Mycobacterium leprae.
Impossible for some microorganisms (pathogens of Ley's disease), essential intracellular bacteria (eg rickettcher,
Some microorganisms, such as G. glamdia, are either impossible on standard media or, if not impossible, are very dangerous (eg B. anthracis).
(Pathogens of anthrax disease). This method can eliminate these problems because it is based on the isolation of nucleic acids, which do not perform conventional bacterial isolation and characterization. It seems that this method can detect microorganisms that have not been formally described. Moreover, the method can distinguish between different strains, which is useful for epidemiological typing, for example in bacteriology. This method can be used in forensic laboratories to correctly and unambiguously identify plant or animal tissue in criminal investigations. It is also used by entomologists to quickly identify insect species when ascertaining the nature of crop damage.

Further, this method is applied to a taxon other than subspecies (for example, plant root nitrogen enzyme gene; see Hennecke H).
In connection with the identification of 291 "Nature" 354 (1981)), this methodology can be used to probe and identify the genotype of individual strains.

The method of the invention is preferably used for identification of microorganisms wherever they are found. These microorganisms will be found in physiological as well as non-physiological substances. They will be found in industrial growth media, culture broth, etc. and will be concentrated, for example by centrifugation. It is preferred that the microorganism be found in physiological media, most preferably it is found in the infected animal source. In this latter embodiment, the method is used to diagnose bacterial infections in animals, particularly preferably humans. The detection and identification of bacterial DNA with pronuclear probes is highly selective and can be performed without hindrance even in the presence of animal (eg mammalian) DNA. If a pronucleus probe is used, conditions can be chosen that minimize hybridization to the filament DNA or the filament bands can be subtracted from the pattern. In this way, this technique can be used in clinical laboratories, fungal depositary institutions, industrial fermentation laboratories and the like.

Of particular interest is the possibility, in addition to identifying the species and strain of the infecting organism, the possibility of discovering the presence of some unusual gene sequence in the organism. For example, the presence of antibiotic resistance sequences found on transmissible plasmid R factors that mediate drug resistance can be detected. By adding labeled factor R DNA or clone-labeled antibiotic resistance sequences to the hybridization mixture, the presence or absence of antibiotic resistance of the organism can be correctly determined (one or more bands that are not normal appear). Alternatively, the filter paper once hybridized in the presence of the added antibiotic resistance sequence probe (one or several kinds) may be rehybridized. As another method, unknown DNA
Into aliquots, the first aliquot for identification, the second aliquot for the presence or absence of the drug resistance sequence,
A third aliquot can also be tested, such as used for the toxin gene. As another method, hybridization of a probe containing conserved genetic information labeled with one radionuclide (for example, ³² P) and an R-factor probe labeled with another radionuclide (for example, ³ H or ¹⁴ C) is added. It could also be used in a mixture. After hybridization,
The presence of R-factor DNA in unknown DNA can be tested by scanning with two scanners. One is for species and strain identification (eg ³² P), the other is for drug resistance etc. (eg ³ H or
¹⁴ C). In this way the experimenter can identify genera and species, type strains, detect drug resistance, toxin production or other characteristics, or labeled nucleic acid sequences or probes without the need to isolate and characterize the microorganism. All tests for sub-species taxa can be done in one experiment.

Since the R-factor is ubiquitous and crosses the boundaries of the species, the identification is in any bacterial genus or species.
It can be done with the same R-factor probe (see: To
mkins, L .; S. et al. J. Inf. Di
s. , 141: 625-636 (1981)).

In addition, the presence of virus or virus-related sequences in eukaryotic or prokaryotic cells can also be detected and identified in conjunction with the method of the invention. "Manu
alof Clinical Microbiolog
y ”third edition (publisher Lennette, EH Ame
r. Soc. Microb. , 1980, 774-77
All viruses listed in 8) can be identified. For example, Picornaviride
aviridae), calicivide
iridae, reovirida
e), togaviridae, orthomycoviridae
e), paramyxoviri
dae), rhab dovirida
e), retroviride,
Arenaaviride, coronaviridae, bunyaviridae, parvoviridae, papovaviride
povaviridae), adenoviride (adeno)
viridae), herpesvide (herpesv)
iridae), vidovirid
ae) and poxviridae
etc. A) When the viral genome is integrated into the host DNA (DNA virus such as papovillide member, RNA virus such as retroviride member)
Extracts high molecular weight DNA from tissue and digests it with restriction enzymes. The overall procedure is similar to that for bacteria. The choice of viral probe will again depend on the task sought, and will depend on the degree of homology between the "probe virus" and the viral-related sequence to be detected. To obtain convenient sequence homology, it will be necessary for the probe and tissue sequences to be related to the same family or species of virus. In addition to the degree of conserved sequence, whether or not the virus probe hybridizes to the virus-related sequence of the host DNA depends on the hybridization conditions, for example, stringent condition or relaxed condition. Let's do it. The result of the hybridization will be a band or band pattern indicating that there are viral sequences incorporated into the host DNA. This information helps predict carcinogenesis. Any of the labeled complementary nucleic acid probes containing cloned virus sequences can be used as a probe. In the case of RNA virus, DNA can be produced by reverse transcriptase using, for example, virus RNA, and in the case of DNA virus, for example, virus DNA labeled by cut translation can be used. Again, multiple probes can be used, especially those with different labels.

The same general characteristics apply equally to DNA and RNA viruses. The virus genome is relatively small and the precipitated nucleic acids are preferably collected by centrifugation. All operations may be performed using all nucleic acids, or various operations may be performed separately. It is believed that the viral nucleic acid can be concentrated by removing cellular DNA by spooling before centrifugation. This can be used to determine if the viral genome is integrated.

In order to hybridize a virus probe, it is necessary, and at least most preferable, that the probe belongs to the same family, genera or species as the unknown organism. The reaction conditions, stringent or relaxing, determine whether a given probe will hybridize to a less related genome. The probe may be a labeled cloned viral sequence or the complete genome or part thereof.

The method described in Southern, supra, is useful for transferring large DNA fragments (about 0.5 kilobases or more) to nitrocellulose paper after alkaline denaturation. This method is useful for DNA viruses,
May not be useful in the case of RNA viruses. RNA can be transferred to activated cellulose paper (diazobenzyloxymethyl paper) and covalently attached and used for RNA viruses. A modification of the Southern method by Thomas (Thomas, P.,
"Proc. Nat. Acad. Sci" USA 7
7: 5201-5205 (1980)) can be used to efficiently transfer RNA and small DNA fragments to nitrocellulose paper for hybridization. RNA and small DNA fragments are denatured with glyogizal and dimethylsulfoxide and electrophoresed in an agarose gel. In this procedure, DNA fragments that are 100-2000 nucleotides, and RNA migrate efficiently and remain on the nitrocellulose paper during hybridization. It is also useful for small ribosomal DNA fragments. Therefore, it is most preferable to divide a sample digested with a restriction enzyme and denature part of the nucleic acid with glyoxal. The Southern and Thomas maneuvers will give the greatest amount of information.

B) In the case of DNA virus, double strand (D
S) The presence of virus can be identified by performing a restriction analysis on the virus DNA. Single chain (S
S) DNA viruses will have different length genomes. Hybridizing probes (sequence information can be converted to DS-DNA), patterns of hybridizing fragments and / or one or more sizes can be used to identify viruses. Again, there are numerous ways to obtain complementary nucleic acid probes. For example, DS-
In the case of DNA, truncated translation can be used and S
In the case of S-DNA, DNA polymerase is cDNA
Used for synthesis.

C) In the case of RNA viruses, RNA is not digested by restriction endonucleases (sequence information may have been converted into DS-DNA). Different RN
The genome of A-wheels has different sizes and some R
There are more than one molecule in the NA viruses genome.
As a result, RNA viruses can be identified along the base sequence detected by a certain probe or a coalescing probe. One example of a probe is the Wheels R
It is a cDNA synthesized using NA.

When searching for infectious pathogens in a sample, nucleic acid is extracted from the sample, or the cells are first cultured in a medium or cells to increase the number of pathogens, or a concentration process such as centrifugation is used. Or try all the approaches directly.

From the present invention, it is easy to make a "kit" containing the necessary elements for carrying out the method. Such a kit would consist of a carrier which is partitioned to tightly pack one or more containers, such as test tubes or vials, therein. One of the containers is a non-labeled nucleic acid probe or a detectably labeled nucleic acid probe, such as radiolabeled cDNA for RNA from an organism probe (in the case of a kit for identifying bacteria, the pronuclear Cell cDNA is more preferred). The labeled nucleic acid probe will be in lyophilized form or, if necessary, in a suitable buffer. One or more containers are DNA from an unknown organism
It will contain one or more endonuclease enzymes utilized for digestion of The enzymes are present alone or as a mixture in lyophilized form or in a suitable buffer solution. Ideally, the enzymes used in the kit are those for which catalogs are prepared. However, there is nothing that prevents the user from making their own comparison standard during the experiment, so if the user suspects that an unknown is actually of a given genus or species. For example, the person may make an identified characterization of a known one and compare it to the characterization of an unknown. Thus, the kit may include all the elements necessary to carry out this subprocess. These elements include one or more known organisms (such as bacteria) or DNA isolated from known organisms. In addition, the kit includes a booklet, or a "catalog" or computer tape or disk, or computer access number, etc., which is defined as a book or pamphlet, which includes plant species, mammalian species, bacterial species,
It incorporates the identified characterizations of a group of different organisms such as bacteria, insect species, etc., of particular pathological importance. In this way the user prepares the characterization of the unknown organism,
This only needs to be compared visually (or computer) with the characterizations in the catalog. The kit may also include probe RNA for probe synthesis in one container, radiolabeled deoxyribonucleoside triphosphate in another container, and a primer in another container. good. In this way, the user can create his own probe cDNA.

Finally, the kit comprises the technology of the present invention, such as buffers, growth media, enzymes, pipettes, plates, nucleic acids, nucleoside triphosphates, filter papers, gel materials, transfer materials, autoradiographic supplements. It may include all of the additional elements needed to do It also
It may also include antibiotic resistance sequence probes, viral probes or probes with other specific properties.

Although the present invention has been generally described above,
The present invention will be better understood by reference to certain reference experiments and examples. It should be noted that the examples are described merely for the purpose of explanation, and are not intended to limit the present invention unless otherwise specified.

Materials and Methods A. Extraction of Bacterial High Molecular Weight DNA Bacterial broth cultures were spun down and cells were washed with cold saline. The cells were loaded with about 10 times the gram weight of packed cells in ml volume of extraction buffer (0.15M sodium chloride, 0.1M EDTA, 0.03M Tris).
It was suspended in pH 8.5). Lysozyme 10 mg / ml was added to give a final concentration of 0.5 mg / ml. 37 suspension
Incubated at 0 ° C for 30 minutes. Cell destruction is 25
%, SDS was added to a final concentration of 2.5%, and the temperature was raised to 60 ° C. for 10 minutes. After cooling in a water bath, mercaptoethanol was added to a final concentration of 1%. Pronase ^™ 20mg / ml 0.02M
Tris buffer (pH 7.4) was pre-digested for 2 hours at 37 ° C and then added to a final concentration of 1 mg / ml. The solution was incubated at 37 ° C for 18 hours. Phenol was redistilled phenol 1 liter, double-distilled water 2.
5 liters, 270 ml of saturated Tris base, 12 ml of mercaptoethanol and a final concentration of 10 ^-3 M EDT
It was prepared by mixing A and allowing the mixture to separate at 4 ° C. The phenol was added to the washing buffer (10 ^-1 M
Sodium chloride, 10 ^-3 M EDTA, 10 mM Tris pH
It was washed with 8.5). Then an equal volume of fresh buffer was added. Mercaptoethanol was added to a final concentration of 0.1%. The solutions were mixed and stored at 4 ° C. The prepared half volume of phenol and half volume of chloroform were added to the lysed cell solution. This was shaken for about 10 minutes and spun down at 3400 × g for 15 minutes. The aqueous phase was removed with a 25 mg glass pipette. This extraction operation was repeated until almost no precipitate was found at the boundary. 1/9 volume of 2N sodium acetate (pH
5.5) was added to the aqueous phase. Two volumes of −20 ° C. 95% ethyl alcohol were slowly poured along the wall of the flask. The tip of a Pasteur pipette was melted and closed and used to spool the precipitated DNA. High molecular weight DNA was dissolved in buffer (10 ⁻³ M EDTA, 10 ⁻² M Tris pH 7.4). The DNA concentration was measured by the absorbance at 260 nm, using 30 μg per unit of absorbance as a conversion coefficient.

Restriction endonuclease digestion of DNA The EcoRI restriction endonuclease reaction is 0.1M
Tris-HCl pH 7.5, 0.05M NaCl,
Performed in 0.005 M MgCl ₂ and 100 μg / ml bovine serum albumin. The EcoR I reaction mixture contained 5 units of enzyme per DNA / μg, which was incubated for 4 hours at 37 ° C. The PSTI restriction endonuclease reaction is 0.006M Tris-HCl pH
7.4, 0.05M sodium chloride, 0.006M magnesium chloride, 0.006M 2-mercaptoethanol, and 100 μg / ml fetal calf serum albumin. The PST I reaction mixture contained 2 units of enzyme per DNA / μg, which was incubated at 37 ° C for 4 hours. Usually, 10 μg of DNA was digested to a final volume of 40 μl. A 10 × buffer was added. Sterile distilled water was added depending on the DNA volume. λ-Bacteriophage D
NA was EcoR I restricted to create a marker band for fragment size determination. Usually 2 μg λ DNA is digested with 20 units of EcoRI to give a final volume of 20 μl
Became.

Gel Electrophoresis and DNA Transfer DNA digests were spiked with up to about 20% glycerol and bromophenol blue dye. In the case of λ DNA digest, 20 μl of 1 × EcoRI buffer is added to each 20 μl
Added to the reaction mixture. Usually 75% glycerol 15
4 μl each and 5 μl of 0.5% bromophenol dye
0 μl was added to the reaction mixture. Digested bacterial DNA 10μ
g and 2 μg of digested λDNA were added to a per well (pe
R well) and cover the surface with agarose melted. Digested material is 0.02M sodium acetate, 0.002M
EDTA, 0.018M Tris base, and 0.02
Electrophoresis was performed in 0.8% agarose containing 8M Tris-HCl pH8.05 at 35V until the dye migrated 13-16 cm. The gel was then dipped in ethidium bromide (0.005 mg / ml) and placed in a UV light box to visualize the lambda fragments. The DNA was transferred to nitrocellulose filter paper by the method of Southern described above. The gel was denaturing solution (1.5M sodium chloride, 20 min on a vibrating table).
0.5 M sodium hydroxide). Denatured solution is neutralized solution (3.0M sodium chloride, 0.5 Tris HCl pH
Substituting 7.5) and after 40 minutes the gel was checked with pH paper. After neutralization, the gel was washed with 6 × SSC buffer (SSC =
0.15M sodium chloride, 0.015M sodium citrate) for 10 minutes. Pass the gel and nitrocellulose paper through 6x SSC with a bundle of paper towels for 15
The DNA fragments were transferred from the gel to nitrocellulose paper by aspirating for a period of time. A filter was placed between two pieces of 3 mm chromatograph paper, wrapped with an aluminum wheel with the shiny side facing outward, and dried in a vacuum oven at 80 ° C. for 4 hours.

Synthesis of ³² P ribosomal RNA complementary DNA
( ³² P-rRNA and DNA) Escherichia coli R-13 23S and 16S type ribosomal RN
³² P-labelled DNA complementary to A was synthesized using reverse transcriptase from avian myeloblastosis virus (AMV). The reaction mixture was 5 μl of 0.2 M dithiothreitol,
25 μl of 1 M Tris pH 8.0, 8.3 μl of 3 M potassium chloride, 40 μl of 0.1 M magnesium chloride, 7
0 μg actinomycin, 14 μl 0.04 M d
It contained ATP, 14 μl 0.04M dGDP, 14 μl 0.04M dTTP, and 96.7 μl water. The following were added to the plastic tube:
137.5 μl reaction mixture, 15 μl calf thoracic primer (10 mg / ml), 7 μl H20, 3 μl rR
NA (2.76 with 40 μg / OD unit concentration)
μg / μl), 40 μl of deoxycytidine 5 ′
-( ³² P) triphosphate (10 mCi / ml), and 13 μl AMV polymerase (6,900 units / μl). The enzymatic reaction was carried out at 37 ° C. for 1.5 hours. The solution was then extracted with 5 ml of chloroform and prepared phenol. After centrifugation (JS13,600RPM), the aqueous phase was directly applied to Sephadex ^™ G-50 column (1.5 × 22 cm).
Layered on top. A plastic 10 ml pipette was used as the column. Place one small glass bead on the tip, attach a rubber tube with a pinch metal fitting, and set it to 0.0
Degassed G-50 swollen by immersing in 5% SDS was added. The aqueous phase was poured directly along the wall into G-50, and then at 0.
Eluted with 05% SDS. Twenty 0.5 ml fractions were collected in plastic vials. The tubes containing the peak fractions were found by the Selenkov method, counting for 0.1 min for each sample using a ³ H-discriminator and recording the total count. The peak fractions were combined. Aliquots were added to Aquesol ^™ (commercially available) and CPM (counts per minute) of ³² P per ml was measured by a scintillation counter.

Hybridization and Autoradiograph
The fragment containing the I over ribosomal RNA gene sequences, was hybridized to DNA on the filters to ^{32 P-rRNAcDNA,} and detected by autoradiography. Filters were hybridized (3 x SSC, 0.
Soak in 1% SDS, 100 μg / ml denatured and sonicated dog DNA and Dynehardt's solution (0.2% calf serum albumin, Ficoll, and polyvinylpyrrolidine, respectively) at 68 ° C. for 1 hour. . ³² PrRNA cDNA was added to 4 × 10 ⁶ CPM / ml.
And the hybridization reaction was incubated at 68 ° C. for 48 hours. Then filter 3x SSC
And then 0.1% SDS for 15 minutes at intervals for 2 hours or until the wash solution contained about 3,000 cpm ³² P / ml. Air dry the filter, wrap in plastic wrap, and -7 with Kodak X-OMATR film.
Autoradiography was performed at 0 ° C for about 1 hour.

B. Mammalian experiment Mus musculus domesticus (mouse) rR
NA probes were synthesized from 18S and 28S and 28S only rRNA. Nucleic acid was extracted from mouse liver and precipitated. High molecular weight DNA was spooled and removed.
The remaining nucleic acids were collected by centrifugation and dissolved in 50 mM MgCl ₂ and 100 mM Tris pH 7.4 buffer. DNA
se (not including RNAse) was added to a concentration of 50 μg / ml. The mixture was incubated at 37 ° C for 30 minutes. The generated RNA is re-extracted, ethanol-precipitated,
It was dissolved in 1 mM sodium phosphate buffer pH 6.8. 0.1
5-2 of M Tris pH 7.4 and 0.01 M EDTA
A 0% scroll gradient solution was prepared. A sample was added and the gradient was spun at 35K RPM for 7 hours on a SW40 rotor. Fractions were collected by optical density. The 18S and 28S fractions were selected by comparison with known molecular weight markers. Relaxed hybridization conditions were used in all mammalian experiments. 54
° C. The washing treatment was carried out at 54 ° C., and the washing was separately performed 3 times for 15 minutes each in 3 × SSC containing 0.05% SDS.

Reference experiments 1 to 8 describe the experiments performed with the rRNA information-containing probe. In Examples 1 to 3,
Computer simulations using a histone gene information-containing probe, a tryptophan operon trpD gene information-containing probe, and an α-fetoprotein gene information-containing probe are described respectively.

Reference Experiment 1 Bacterial species are the restriction endonucleosides of the ribosomal RNA gene.
Determined by Lease analysis. The P. Several strains of Aeruginosa have minimal phenotypic characteristics that identify the species (Hugh RH, et al.
al. "Manual of Clinical Mi
"crobiology" 2nd edition, ASM, 1974, p.
p, 250-269) (Table 2). Another three Pseudomonas and two Acinetobacter (Acineto)
Bacterial) strains were selected to compare species and genera (Table 3).

[0099]

[Table 2]

The strains used for comparison of Pseudomonas and Acinetobacter species are listed in Table 3.

[0101]

[Table 3]

Acinetobacter species were chosen for genus comparison because they share certain attributes with many Pseudomonas species.

The size of the fragments (kilobase pair) in the EcoR I digest was P. P. stutzer
i) 16.0, 12.0, 9.4; Fluorescence (P. fluorescens) 16.0, 10.
0, 8.6, 7.8, 7.0; Puchida
ida) 24.0, 15.0, 10.0, 8.9;
A. anitratus 20.0, 1
5.0, 12.5, 9.8, 7.8, 6.1, 5.2,
4.8, 3.8, 2.8 (the size of the smallest 3 fragments was not calculated); Luoffi (A.lwoffi
i) 12.0, 10.0, 9.1, 7.0, 6.4,
5.7, 5.5, 5.3, 4.8, 4.4, 3.6,
3.2, 2.9 (the size of the smallest 3 fragments was not calculated). The size of the fragments in the PST I digest (kilobase pair) is shown below; Stowzeri 6.
7, 6.1, 5.5; Fluorescence 10.0,
9.4, 7.8, 7.0; Putida 10.5, 9.
9, 6.8, 6.3, 4.4; Anitratus 36.
0, 28.0, 20.5, 12.0, 10.0, 5.
8, 3.7, 2.6, 2.4; Luoffi 9.9,
8.7, 7.2, 5.7, 4.0, 3.2, 2.7.

P. Comparing hybridized restriction fragments obtained from seven strains of Aeruginosa, this species contains 10.1, 9.4, 7.6 and 5.9 kilobase pairs (KBP) of rRNA gene sequences. Fragment Ec
We come to the conclusion that it is defined by the oR I-specific combination (FIG. 1).

The 7.6KBP EcoR I fragment appears in 4 out of 7 strains in this sample. A similar situation occurs in some phenotypic traits of species strains. The fact that the EcoR I combination of fragments from 7 strains is used to divide the strain into two groups is described in P. It draws the inference that there are two species with the minimal phenotypic trait of Aeruginosa. From the experimental results of digestion of DNA with PST I (FIG. 2), it is concluded that the strain mutations exhibited by the EcoR I 7.6KBP fragment represent intra-species mutations. Because one conserved combination of PST I fragments, 9.4, 7.1, 6.6 and 6.4 KBP
Because it defines the species. 9.
4 and 6.6 KBP PST I fragments were isolated from P. It appears in 6 out of 7 aeruginosa strains; the 7.1 and 6.4 KBP PST I fragments appear in all of the sampled strains. Mutations in the PSTI fragment are
Occurs in strains that do not contain the I7.6KBP fragment; RH15
1 has 10.1 and 8.2 KBP fragments and has RH80
9 does not contain the 9.4 KBP fragment but has the 6.0 KBP fragment. And the type strain RH815 is 6.6
Does not include the KBP fragment. The pattern of hybridized fragments supports the conclusion that enzyme-specific conservative combinations can be used for species determination. Strains of one species probably have multiple fragments as conserved combinations. Fragment mutations in some strains do not interfere with identification and prove useful for epidemiological studies.

P. EcoR in aeruginosa strains
Mutagenesis of the I 7.6 KBP Fragment is a Hybridization Ec Found in Other Pseudomonas Type Strains.
It may be considered prospectively by examining the oRI fragment (Fig. 3). P. Stoweeri, P. Fluorescence and P. The type strain of Putida is 7.6 KB
It does not contain the P fragment but shares an EcoRI fragment of the same size; Aeruginosa and P. S. tusseri has a 9.4 KBP fragment and P. Stoweeri and P.P. Fluorescence has a 16 KBP fragment, Fluorescence and P. Petit is 10 KB
It has a P fragment. In general, fragment size is unique to each of the four Pseudomonas species type strains; and each species type strain has different size range fragments. These general explanations are also applicable to PST I digests (Figure 4).

When comparing the respective fragment patterns of the four Pseudomonas species and the two Acinetobacter species or their I strains, it can be concluded that the species of each genus are similar, but the comrades are different. The two Acinetobacter species have a larger range of hybridizing fragment sizes than the four Pseudomonas species.

E. coli, Bacillus thuringiensis (B
acillus thuringiensis) and B. Where without the help of a restriction map such as that obtained with B. subtilis, where the enzyme was rR
It is not possible to predict whether to cut the NA gene or whether the number of copies per genome and heterologous flanking regions between multiple genes or heterogeneous genes. RRNA of E. coli
The cRNA probe may not hybridize with some restriction fragments containing the rRNA gene sequence,
If so, this reflects the evolutionary distance or variance between the test organism and E. coli. rRNA
We can argue that this is not the case with the conservative property of. However, these are minor problems compared to the advantage of having a standard probe that is equally applicable to any unknown species.

Reference Experiment 2 Restriction Analysis and DNA-DNA Liquid Hybridization Ratio
Comparison: The strains used in this study are listed in Tables 4 and 5.

[0110]

[Table 4]

[0111]

[Table 5]

High molecular weight DNA was isolated from each strain. Liquid DNA-DNA hybridization data was collected using RH3021 and RH2990 labeled DNA. The results are shown in Table 6.

[0113]

[Table 6]

This data shows that there are two hybridization groups. Similar data can be found in B. Subtilis has been reported in the literature (Seki et al, "I.
international Journal of S
"ystematic Bacteriology" 2
5: 258-270 (1975)). RH of these two groups
3021 and RH2990. EcoRI digests (Fig. 5) showed 2 when restriction endonuclease analysis of the ribosomal RNA gene was performed.
I was able to divide into groups. The group represented by RH3021 has two strongly hybridizing fragments (2.15 and 2.1KBP). The group represented by RH2990 has two strongly hybridizing fragments (2.6 and 2.5KBP). EcoR I
B. using data Subtilis strains can be placed in appropriate locations in the DNA-DNA hybridisation group. DNA
According to the 70% DNA hybridization rule, B. Subtilis is actually two species. However, P
Considering the STI data (FIG. 6), those groups can be considered as two dispersed populations involved in a common ancestral or speciation event. B. The conclusion that Subtilis is a species correlates with phenotypic data. The strains listed in Table 5 are Gordon R. "T by E et al.
"He Genus Bacillus" Agricult
ure Handbook No 427 (US Department of Agriculture, Agricultural Research Service, Washington, DC) p.
36-41 B. It has been identified as Subtilis. Restriction analysis can provide data comparable to DNA-DNA hybridization data, and by selecting the appropriate enzyme, restriction analysis can be well-defined species despite variance. RH3061 lost the PSTI site. However, EcoRI data show that the strain is B. It is suggested to be Subtilis. The same can be concluded from the BglII data (Figure 7) and SacI data (Figure 8).

Reference Experiment 3 Stability Patterns of Restriction Analysis and Other Bacillus Poly
Mica (Bacillus polymyxa) experiment

[0116]

[Table 7]

B. Subtilis and B. Polymica is E
coRI data (Fig. 9), PSTI data (Fig. 10)
It can be distinguished by Bgl II data (Fig. 11 left) and Sac I data (Fig. 12 right). PST
From the large difference in the I-band pattern, it can be concluded that Bacillus polymica is in the wrong genus. Both species produce spores, but they are phenotypically dissimilar. B. cerevisiae from both ATCC and NRRL culture collections. Certainly, Polymica type strains have the same banding pattern. However, the important data is that asporic mutants can be identified. If they were unable to sporulate, identifying Bacillus species would be very difficult and probably impossible.

Reference Experiment 4 Swiss Mouse, Mus musculus domesticus, to Identify Bacterial Species in Mouse Tissue without Isolation
Streptococcus pneumonia (inbred strain)
e) 0.5 ml of a turbid suspension of RH3077 (ATCC 6303) was inoculated intraperitoneally. When the mice were moribund, the heart, lungs and liver were removed. High molecular weight DNA was isolated from these tissues, S. Eco for isolation of DNA from pneumoniae RH3077 and Swiss mouse organs and digestion of DNA
The RI was used to manipulate the restriction endonuclease analysis of the rRNA gene. In addition to washing the filter with 3 × SSC, 0.3 × SSC and 0.05 for 2 × 15 minutes.
Washed with% SDS. Autoradiography was performed for 48 hours. The data (FIG. 12) is S. It was shown that the Pneumonie was defined by seven hybridizing fragments (17.0, 8.0, 6.0, 4.0, 3.3, 2.
6 and 1.8 KBP). This bacterial cDNA probe contained two mouse DNA fragments (14.0 and 6.8 KB).
It does not hybridize with P). The hybridizing fragment is S. cerevisiae in infected tissue. It informs the existence of Pneumonier. All seven bands are found in the heart DNA extract. Although less intense in liver DNA extracts, they are all visible by autoradiography. Only the 6.0 KBP band appears in the lung DNA extract. The low number of bacteria in the lungs may be explained by the fact that the mice have sepsis rather than pneumonia. The lungs showed no solidification at culture. To determine the sensitivity of this assay, bacterial DNA was added to mouse DN.
It was diluted with A and electrophoresed. 0.1 μg bacterial DNA
With, all seven bands were visible on the autoradiograph. With 10 ⁻³ μg bacterial DNA, 17.
The 0, 8.0 and 6.0 KBP bands were seen. 10
⁶ S. Using the number of 5 × 10 ⁻³ μg DNA per Pneumoniae cell (Biochem Biophys
Acta 26:68), 10 ^-1 μg corresponds to 2 × 10 ⁷ cells. This method is useful for diagnosing infectious diseases at this sensitivity level.

This reference experiment also shows that the bacterial probe hybridizes to a mouse-specific EcoRI fragment (see Figure 9, fragments with 14.0 and 6.8 KBP). These fragments are mouse 18S and 28S
Detected by a ribosomal RNA probe
The bacterial probe corresponding to the RI fragment (FIG. 14, supra shows that the 6.8 KBP fragment contains the 28S type rRNA sequence) does not hybridize well with the mammalian ribosomal RNA gene sequences, so the bands are less intense. The smaller, bacterial probe and nuclear mammalian DNA system is less sensitive, with a clear selectivity for the DNA of infected prokaryotic cells. Bacterial probe in lane (la
In an experiment hybridized to 10 μg digested bacterial DNA per ne), no hybridization was found to 10 μg digested human or mouse DNA per lane even when the bacterial bands were clearly visible.

Reference Experiments 5-8 Mammalian Experiments These reference experiments demonstrate that the concept of confirming organisms by rRNA restriction analysis has been successfully applied to complex eukaryotic organisms as well as bacteria. To do.

FIG. 13 shows that the genus of mammals can be identified with the Mus musculus domesticus 18S and 28S rRNA probes and that several species of Mus are distinguishable. In this figure, the enzyme is PSTI,
The objects and their respective bands are as follows.

1. Mus musculus melosinus
(Mouse) 14.5, 13.5, 2.6 2. Mus musculus domesticus (mouse) 1
3.5, 2.6 3. Canis family
iaris) (dog) 12.0 4. Caviar porcellus
us) (guinea pig) 17.0, 14.0, 13.0,
4. One band of 8.8, 5.7, 4.7 and 3.0 or less 5. Crisetourus Griseus
s griseus) (hamster) 25.0, 4.7 6. Homo sapiens
(Human) 15.0, 5.7 7. Felis catus
(Cat) 20.0, 9.7 8. Ratus norvegicus
icus) (rat) 12.5 9. Mus musculus domesticus (mouse) 1
3.5, 2.6 10. Mus Cerbicolo
service cell color (mouse) 14.0, 2.7 11. Mus Serbicolo Papeus (Mus ser
vicolor papeus) (mouse) 13.5,
2.6 12. Mus pahari (mouse) 13.0, 3.7 13. Mus cookie (mouse) 13.5, 2.6

FIG. 14 shows that mouse and cat DNA is 28S.
It shows that they can be distinguished only by the type rRNA cDNA, and that the pattern of the hybridized band depends on the constitution of the probe sequence. In Figure 14, the enzyme is EcoR
In I, the objects and bands are as follows. 1. Mus musculus domesticus (mouse)
6.8 KBP 2. Feliz Catus (cat) 8.3KBP

In FIG. 15, the enzyme is Sac I and the objects and bands are as follows. 1. Erythrocebu pasta
s patas) (Patus monkey) 8.5, 3.7, <3.
0 2. Rats norvegicus (rat) 25.0, 9.
5, 3.6, <3.0 3. Mus musculus domesticus (mouse)
6.8, <3.0 4. Feliz Catus (cat) 9.5, 5.3, 4.0,
<3.0, <3.0 5. Homo sapiens (human) 10.5, <3.0 6. Macaca mulatta
a) (Laser monkey) 9.8, <3.0 When comparing FIG. 5 (Sac I digestion) with the other mammalian figures, it can be seen that the hybridization pattern is enzyme specific.

FIG. 16 shows that primates animals are distinguishable. Cultured cells have a band in common with the tissue of their original species, and different human cultures can be distinguished by the addition or lack of bands. In this figure, the enzyme is EcoRI and the objects and bands are: 1. Erythrosebas Patas (Patus monkey)> 22.0,
11.0, 7.6, 2.6 2. Macaka Murata (Laser monkey) 22.0, 11.
5, 7.6 3. Homo sapiens (human)> 22.0, 22.0,
16.0, 8.1, 6.6 4. M241 / 88 (Langer monkey cultured cells) 14.
0, 7.2, 5.7 5. HeLa (cultured human cells)> 8.1, 6.0 6. J96 (human cultured cells)> 22.0, 22.0, 1
6.0, 11.0, 8.1, 6.6 7. AO (human cultured cells) 22.0, 16.0, 8.
1, 6.6 8. X-3 8.1 (Laser monkey) 22.0, 11.
5, 7.6

Example 1 Use of H4 Histone Gene Probes Computer simulations for the identification and characterization of two animal species (sea urchin and mouse) were performed using conserved DNA sequences derived from the H4 histone gene. Sea urchin (Ps
The histone H4 gene sequence for Ammechinus miriaris) is shown below. Where A, T,
C and G represent known nucleotides, and N represents a currently unknown site (788 base pairs).

[0127]

Table 8 10 20 30 40 50 CAACATATTA GAGGAAGGGA GAGAGAGAGA GAGAGAGAGA GAGAGAGAGA GTTGTATAAT CTCCTTCCCT CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT 60 70 80 90 100 GGGGGGGGGG GAGGGAGAAT TGCCCAAAAC ACTGTAAATG TAGCGTTAAT CCCCCCCCCC CTCCCTCTTA ACGGGTTTTG TGACATTTAC ATCGCAATTA 110 120 130 140 150 GAACTTTTCA TCTCATCGAC TGCGCGTGTA TAAGGATGAT TATAAGCTTT CTTGAAAAGT AGAGTAGCTG ACGCGCACAT ATTCCTACTA ATATTCGAAA 160 170 180 190 200 TTTTCAATTT ACAGGCACTA CGTTACATTC AAATCCAATC AATCATTTGA AAAAGTTAAA TGTCCGTGAT GCAATGTAAG TTTAGGTTAG TTAGTAAACT 210 220 230 240 250 ATCACCGTCG CAAAAGGCAG ATGTAAACTG TCAAGTTGTC AGATTGTGTG TAGTGGCAGC GTTTTCCGTC TACATTTGAC AGTTCAACAG TCTAACACAC 260 270 280 290 300 CGCGGCCTCC AGTGAGCTAC CCACCGGGCC GTCGCGGAGG GGCGCACCTG GCGCCGGAGG TCACTCGATG GGTGGCCCGG CAGCGCCTCC CCGCGTGGAC 310 320 330 340 350 TGCGGGAGGG GTCATCGGAG GGCGATCGAG CCTCGTCATC CAAGTCCGCA ACGCCCTCCC CAGTAGCCTC CCGCTAGCTC GGAGCAGTAG GTTCAGGCGT 360 370 380 390 400 TACGGGTGAC AATACCCCCG CTCACCGGGA GGGTTGGTCA ATCGCTCAGC ATGCCCACTG TTATGGGGGC GAGTGGCCCT CCCAACCAGT TAGCGAGTCG 410 420 430 440 450 GAAACGTCCA GTCGTCAGCA TCGCACTAAG ACTCTCTCTC AATCTCCATA CTTTGCAGGT CAGCAGTCGT AGCGTGATTC TGAGAGAGAG TTAGAGGTAT 460 470 480 490 500 ATGTCAGGCC GTGGTAAAGG AGGCAAGGGG CTCGGAAAGG GAGGCGCCAA TACAGTCCGG CACCATTTCC TCCGTTCCCC GAGCCTTTCC CTCCGCGGTT 510 520 530 540 550 GCGTCATCGC AAGGTCCTAC GAGACAACAT CCAGGGCATC ACCAAGCCTG CGCAGTAGCG TTCCAGGATG CTCTGTTGTA GGTCCCGTAG TGGTTCGGAC 560 570 580 590 600 CAATCCGCCG ACTCNNNNNN NNNNNNNNNN NNNNNNGAAT CTCTGGTCTT GTTAGGCGGC TGAGNNNNNN NNNNNNNNNN NNNNNNCTTA GAGACCAGAA 610 620 630 640 650 ATCTACGAGG AGACACGAGG GGTGCTGAAG GNNNNNNNNN NNNNNNNNNN TAGATGCTCC TCTGTGCTCC CCACGACTTC CNNNNNNNNN NNNNNNNNNN 660 670 680 690 700 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 710 720 730 740 750 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNGGCCGAAC ACTGTACGGC NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNCCGGCTTG TGACATGCCG 760 770 780 TTCGGCGGCT AAGTGAAGCA GACTTGGCTA G AATAACG AAGCCGCCGA TTCACTTCGT CTGAACCGAT CTTATTGC

A similar mouse H4 gene sequence is shown below (968 base pairs).

Table 9 10 20 30 40 50 GAATTCTCCG AGGGACTTCG GCACCATAAT TAAGAAAATC GAAAATAAAA CTTAAGAGGC TCCCTGAAGC CGTGGTATTA ATTCTTTTAG CTTTTATTTT 60 70 80 90 100 AAATAAAGGC TTGAGACTGT AAGGAACCGG TAGAGGGCAG AGAAGAGAAA TTTATTTCCG AACTCTGACA TTCCTTGGCC ATCTCCCGTC TCTTCTCTTT 110 120 130 140 150 AGAAAAACAG GAAGATGATG CAACATCCAG AGCCCGGATA ATTTAGAAAG TCTTTTTGTC CTTCTACTAC GTTGTAGGTC TCGGGCCTAT TAAATCTTTC 160 170 180 190 200 GTTCCCGCCC GCGCGCTTTC AGTTTTCAAT CTGGTCCGAT CCTCTCATAT CAAGGGCGGG CGCGCGAAAG TCAAAAGTTA GACCAGGCTA GGAGAGTATA 210 220 230 240 250 ATTAGTGGCA CTCCACCTCC AATGCCTCAC CAGCTGGTGT TTCAGATTAC TAATCACCGT GAGGTGGAGG TTACGGAGTG GTCGACCACA AAGTCTAATG 260 270 280 290 300 ATTAGCTATG TCTGGCAGAG GAAAGGGTGG AAAGGGTCTA GGCAAGGGTG TAATCGATAC AGACCGTCTC CTTTCCCACC TTTCCCAGAT CCGTTCCCAC 310 320 330 340 350 GCGCCAAGCG CCATCGCAAA GTCTTGCGTG ACAACATCCA GGGTATCACC CGCGGTTCGC GGTAGCGTTT CAGAACGCAC TGTTGTAGGT CCCATAGTGG 360 370 380 390 400 AAGCCCGCCA TCCGCCGCCT GGCTCGGCGC GGTGGGGTCA AGCGCATCTC TTCGGGCGGT AGGCGGCGGA CCGAGCCGCG CCACCCCAGT TCGCGTAGAG 410 420 430 440 450 CGGCCTCATC TACGAGGAGA CCCGTGGTGT GCTGAAGGTG TTCCTGGAGA GCCGGAGTAG ATGCTCCTCT GGGCACCACA CGACTTCCAC AAGGACCTCT 460 470 480 490 500 ACGTCATCCG CGACGCAGTC ACCTACACCG AGCACGCCAA GCGCAAGACC TGCAGTAGGC GCTGCGTCAG TGGATGTGGC TCGTGCGGTT CGCGTTCTGG 510 520 530 540 550 GTCACCGCTA TGGATGTGGT GTACGCTCTC AAGCGCCAGG GCCGCACCCT CAGTGGCGAT ACCTACACCA CATGCGAGAG TTCGCGGTCC CGGCGTGGGA 560 570 580 590 600 CTACGGCTTC GGAGGCTAGA CGCCGCCGCT TCAATTCCCC CCCCCCCCCC GATGCCGAAG CCTCCGATCT GCGGCGGCGA AGTTAAGGGG GGGGGGGGGG 610 620 630 640 650 ATCCCTAACG GCCCTTTTTA GGGCCAACCA CAGTCTCTTC AGGAGAGCTG TAGGGATTGC CGGGAAAAAT CCCGGTTGGT GTCAGAGAAG TCCTCTCGAC 660 670 680 690 700 ACACTGACTT GGGTCGTACA GGTAATAACC GCGGGTTTAG GACTCACGCT TGTGACTGAA CCCAGCATGT CCATTATTGG CGCCCAAATC CTGAGTGCGA 710 720 730 740 750 ACTAGGTGTT CCGCTTTTAG AGCCATCCAC TTAAGTTTCT ATACCACGGC TGATCCACAA GGCGAAAATC TCGGTAGGTG AATTCAAAGA TATGGTGCCG 760 770 780 790 800 GGATAGATAG CATCCAGCAG GGTC TGCTCA CACTGGGAAT TTTAATTCCT CCTATCTATC GTAGGTCGTC CCAGACGAGT GTGACCCTTA AAATTAAGGA 810 820 830 840 850 ACTTAGGGTG TGAGCTGGTT GTCAGGTCAA GAGACTGGCT AAGATTTTCT TGAATCCCAC ACTCGACCAA CAGTCCAGTT CTCTGACCGA TTCTAAAAGA 860 870 880 890 900 TTAGCTCGTT TGGAGCAGAA TTGCAATAAG GAGACCCTTT GGATGGGATG AATCGAGCAA ACCTCGTCTT AACGTTATTC CTCTGGGAAA CCTACCCTAC 910 920 930 940 950 ACCTATGTCC ACACATCAAA TGGCTATGTG GCTGTGTCCC TGTGTTTCCA TGGATACAGG TGTGTAGTTT ACCGATACAC CGACACAGGG ACACAAAGGT 960 ATGAGTGGCT GTGCTTGA TACTCACCGA CACGAACT

The regions of homology with respect to the above sequences of both are shown below. Here, the asterisk indicates that it is not a homologous site. In the region shown, the first 118 base pairs have a homology of 80.5%, and are used as a conserved DNA sequence probe in this example (the base portion of sea urchin (upper row) is located at the 449th to 567th positions in the mouse ( The base portion of (lower row) is shown at the 257th to 375th positions).

[Table 10] % = 84.503 F (342,289) =. 000E + 00 E =. 00
0

The restriction endonuclease cleavage site was determined from two sequences. A list of cleavage sites in the sea urchin mouse sequence is shown below. If there is no part name in parentheses,
The numbers indicate the 5'side of the cleavage site, indicating that only the recognition site is known.

[0131]

[Table 11] sea urchin array out of the current part position AcyI (GPCGQC) 495 AluI (AGCT ) 147 267 AsuI (GGNCC) 277 514 AvaII (GGLCC) 514 CauII (CCMGG) 276 377 DdeI (CTNAG) 396 427 DpnI (GATC) 326 EcoRI * (PPATQQ) 184 EcoRII (CCLGG) 531 Fnu4HI (GCNGC) 254 FnuDII (CGCG) 125 253 285 FokI (GGATG) 148 FokI (CATCC) 324 515 HaeII (PGCGCQ) 498 HaeIII (GGCC) 256 279 459 HgaI (GCGTG) 491 HgiCI (GGQPCC) 494 HgiJII (GPGCQC) 483 HhaI (GC C) 125 253 295 497 HindIII (AGGCTT) 145 HinfI (GANTC) 200 431 561 HpaII (CCGG) 275 376 HphI (GGTGA) 368 HphI (TCACC) 195 365 M 532 MboI GCT3C2I (GACTC) 195 532 MboI GCT3C2I (GACTC) 195 532 MboI (GANTC3) (GANTC) ) 4 42 54 280 299 311 372 463 484 NarI (GGCGCC) 495 NspBII (GCMGC) 256 PvuI (CGATCG) 327 ScrFI (CCNGG) 276 537 Sfa27T40IQGCATCAT.

[0132]

TABLE 12 Mouse sequence out current part position AcyI (GPCGQC) 302 571 AflII ( CTTAAG) 731 AluI (AGCT) 234 256 648 815 855 AsuI (GGNCC) 184 540 611 622 AvaII (GGLCC) 184 BssHII (GCGCGC) 162 CauII (CCMGG) 135 DdeI (CTNAG) 803 840 DpnI (GATC) 190 [EcoB] (AGCANNNNNNNNTCA) 766 [Ecop1] (AGACC) 418 496 AT 882 COE 585G775 (Ecop1) (GGTCT) 285G775 (Eco1) 2 EcoRI * (PPATQQ) 4 790 845 EcoRII (CCLGG) 338 368 4 43 536 Fnu4HI (GCNGC) 366 543 574 577 FnuDII (CGCG) 162 164 380 461 682 682 FokI (GGATG) 526 905 511 GH 511 C 511 GH 511 C 511 H 532 C 711 322 C 411 322 C 411 322 C HR CAT 111 322 C HR CAT HI C HI C HI C HI C HI C HI C HI C HI HI C HI C HI C HI C HI C HI C HI C HI C HI C HI C HI C HI C HI C HI C HI C HI C HI C HI C HI C HI C HI C HI C HI C HI HI HI HI 612 624 HgaI (GACGC) 472 579 HgiAI (GLGCLC) 485 HgiCI (GGQPCC) 21 301 HgiJII (GPGCQC) 135 HhaI (GCGC) 164 166 304 311 380 395 494 536 HinfI (GANTC) 692 HpaII (CCGG) 78 135 401 HphI ( TCACC) 220 339 462 4 5 MboI (GATC) 188 MboII (GAAGA) 105 124 MboII (TCTTC) 629 MnlI (CCTC) 202 227 236 415 559 MnlI (GAGG) 3 76 261 407 555 MarI (GGCGCC) 302 NspBII (GCMGC) 368 545 576 579 PvuII ( CAGCTG) 234 RsaI (GTAC) 523 668 SacII (CCGCGG) 683 ScrFI (CCNGG) 135 340 370 445 538 SfaNI (GATGC) 127 SfaNI (GCATC) 385 751 TaqI (TCGA) 40 Tth111I (GACNNNGTC) 466 Tth111II (TGQTTG) 953 XmnI (GAANNNNTTC) 439

The sequence of the sea urchin and the mouse was Hha I (GC
GC) and the probe sequences described above. The sea urchin sequence has cleavage sites at positions 295 and 497 so as to form a 202 base pair (bp) fragment, and if denatured, hybridizes with the probe sequence. Hh of mouse sequence
The a I (GCGC) sites (166, 304, 311 and 380 sites) indicate that the 69 and 138 fragments can be detected with the probe sequence. Thus, the genetic characterization of sea urchins is 202, in the case of mice 69 and 138.

Example 2 The tryptophan operon trp D fragment as a probe
A computer simulation was performed in the same manner as in Example 1 using the trp D gene as a probe used for the gene. Now, E. coli and Salmonella typhimurium.
It is concluded that m) can be identified with restriction fragments containing conserved sequences.

Escherichia coli (E.co) of 684 base pairs (bp)
li) trp D gene is shown below.

Table 13 10 20 30 40 50 GAAGCCGACG AAACCCGTAA CAAAGCTCGC GCCGTACTGC GCGCTATTGC CTTCGGCTGC TTTGGGCATT GTTTCGAGCG CGGCATGACG CGCGATAACG 60 70 80 90 100 CACCGCGCAT CATGCACAGG AGACTTTCTG ATGGCTGACA TTCTGCTGCT GTGGCGCGTA GTACGTGTCC TCTGAAAGAC TACCGACTGT AAGACCACGA 110 120 130 140 150 CGATAATATC GACTCTTTTA CGTACAACCT GGCAGATCAG TTGCGCAGCA GCTATTATAG CTGAGAAAAT GCATGTTGGA CCGTCTAGTC AACGCGTCGT 160 170 180 190 200 ATGGGCATAA CGTGGTGATT TACCGCAACC ATATACCGGC GCAAACCTTA TACCCGTATT GCACCACTAA ATGGCGTTGG TATATGGCCG CGTTTGGAAT 210 220 230 240 250 ATTGAACGCT TGGCGACCAT GAGTAATCCG GTGCTGATGC TTTCTCCTGG TAACTTGCGA ACCGCTGGTA CTCATTAGGC CACGACTACG AAAGAGGACC 260 270 280 290 300 CCCCGGTGTG CCGAGCGAAG CCGGTTGTAT GCCGGAACTC CTCACCCGCT GGGGCCACAC GGCTCGCTTC GGCCAACATA CGGCCTTGAG GAGTGGGCGA 310 320 330 340 350 TGCGTGGCAA GCTGCCCATT ATTGGCATTT GCCTCGGACA TCAGGCGATT ACGCACCGTT CGACGGGTAA TAACCGTAAA CGGAGCCTGT AGTCCGCTAA 360 370 380 390 400 GTCGAAGCTT ACGGGGGCTA TGTCGGTCAG GCGGGCGAAA TTCTCCACGG CAGCTTCG AA TGCCCCCGAT ACAGCCAGTC CGCCCGCTTT AAGAGGTGCC 410 420 430 440 450 TAAAGCCTCC AGCATTGAAC ATGACGGTCA GGCGATGTTT GCCGGATTAA ATTTCGGAGG TCGTAACTTG TACTGCCAGT CCGCTACAAA CGGCCTAATT 460 470 480 490 500 CAAACCCGCT GCCGGTGGCG CGTTATCACT CGCTGGTTGG CAGTAACATT GTTTGGGCGA CGGCCACCGC GCAATAGTGA GCGACCAACC GTCATTGTAA 510 520 530 540 550 CCGGCCGGTT TAACCATCAA CGCCCATTTT AATGGCATGG TGATGGCAGT GGCCGGCCAA ATTGGTAGTT GCGGGTAAAA TTACCGTACC ACTACCGTCA 560 570 580 590 600 ACGTCACGAT GCGGATCGCG TTTGTGGATT CCAGTTCCAT CCGGAATCCA TGCAGTGCTA CGCCTAGCGC AAACACCTAA GGTCAAGGTA GGCCTTAGGT 610 620 630 640 650 TTCTCACCAC CCAGGGCGCT CGCCTGCTGG AACAAACGCT GGCCTGGGCG AAGAGTGGTG GGTCCCGCGA GCGGACGACC TTGTTTGCGA CCGGACCCGC 660 670 680 CAGCATAAAC TAGAGCCAGC CAACACGCTG CAA CTCCTATTTG ATCTCGGTCG GTTGTGCGAC GTT

The 683 base pair Salmonella typhimurium trp D gene is shown below.

[Table 14] 10 20 30 40 50 GAAGCCGATG AAACCCGTAA TAAAGCGCGC GCCGTATTGC GTGCTATCGC CTTCGGCTAC TTTGGGCATT ATTTCGCGCG CGGCATAACG CACGATAGCG 60 70 80 90 100 CACCGCGCAT CATGCACAGG AGACCTTCTG ATGGCTGATA TTCTGCTGCT GTGGCGCGTA GTACGTGTCC TCTGGAAGAC TACCGACTAT AAGACGACGA 110 120 130 140 150 CGATAACATC GACTCGTTCA CTTGGAACCT GGCAGATCAG CTACGAACCA GCTATTGTAG CTGAGCAAGT GAACCTTGGA CCGTCTAGTC GATGCTTGGT 160 170 180 190 200 ACGGTCATAA CGTGGTGATT TACCGTAACC ATATTCCGGC GCAGACGCTT TGCCAGTATT GCACCACTAA ATGGCATTGG TATAAGGCCG CGTCTGCGAA 210 220 230 240 250 ATCGATCGCC TGGCGACAAT GAAAAATCCT GTGCTAATGC TCTCCCCCGG TAGCTAGCGG ACCGCTGTTA CTTTTTAGGA CACGATTACG AGAGGGGGCC 260 270 280 290 300 CCCGGGTGTT CCCAGCGAGG CAGGTTGTAT GCCGGAGCTG CTGACCCGAC GGGCCCACAA GGGTCGCTCC GTCCAACATA CGGCCTCGAC GACTGGGCTG 310 320 330 340 350 TACGCGGCAA GTTACCGATC ATCGGCATTT GTCTGGGGCA TCAGGCGATT ATGCGCCGTT CAATGGCTAG TAGCCGTAAA CAGACCCCGT AGTCCGCTAA 360 370 380 390 400 GTCGAAGCTT ACGGCGGTTA CGTCGGTCAG GCGGGAGAAA TCCTGCATGG CAGCTTCG AA TGCCGCCAAT GCAGCCAGTC CGCCCTCTTT AGGACGTACC 410 420 430 440 450 CAAAGCCTCC AGCATTGAGC ATGACGGTCA GGCGATGTTC GCCGGGCTCG GTTTCGGAGG TCGTAACTCG TACTGCCAGT CCGCTACAAG CGGCCCGAGC 460 470 480 490 500 CGAATCCGCT ACCGGTCGCG CGTTATCATT CGCTGGTCGG CAGTAATGTT GCTTAGGCGA TGGCCAGCGC GCAATAGTAA GCGACCAGCC GTCATTACAA 510 520 530 540 550 CCTGCCGGGC TGACCATTAA CGCCCATTTC AACGGCATGG TGATGGCGGT GGACGGCCCG ACTGGTAATT GCGGGTAAAG TTGCCGTACC ACTACCGCCA 560 570 580 590 600 ACGTCATGAT GCGGATCGCG TTTGCGGTTT TCAATTTCAT CCCGAGTCCA TGCAGTACTA CGCCTAGCGC AAACGCCAAA AGTTAAAGTA GGGCTCAGGT 610 620 630 640 650 TCCTGACGAC ACAGGGCGCG CGTCTACTGG AGCAAACATT AGCCTGGGCG AGGACTGCTG TGTCCCGCGC GCAGATGACC TCGTTTGTAA TCGGACCCGC 660 670 680 CTGGCGAAGC TGGAACCGAC CAACACCCTA CAG GACCGCTTCG ACCTTGGCTG GTTGTGGGAT GTC

The homologous region between both sequences is shown below. here,
The upper sequence is E. coli and the lower sequence is Salmonella typhimurium.

[Table 15] area 1 ** * * * * ** * * * ** 452 AAACCCGCTGCCGGTGGCGCGTTATCACTCGCTGGTTGGCAGTAACATTCC-GGCCGG-T 453 AA-TCCGCTACCGGTCGCGCGTTATCATTCGCTGGTCGGCAGTAATGTTCCTGCC-GGGC * * * * * * * TTAACCATCAACGCCCATTTTAATGGCATGGTGATGGCAGTACGTCACGATGCGGATCGC TGA-CCATTAACGCCCATTTCAACGGCATGGTGATGGCGGTACGTCATGATGCGGATCGC * ** * * * * * * * * * * * * GTTTGTGG-ATTCCAGTTCCATCCGGAATCCATTCTCACCACCCAGGGCGCTCGGCTGCT GTTTGCGGTTTTC-AATTTCATCCCGAGTCCATCCTGACGACACAGGGCGCGCGTCTACT * ** * ** ** * * * * * * * * GGAACAAACGCTGGCCTGGGCGC-AGCATAAACTAGAGCC-AGCCAACACGCTGCA 682 GGAGCAAACATTAGCCTGGGCGCTGGC-GAAGCTGGAACCGACC-AACACCCTACA 682% = 78.390 P (236, 185) =. 000E + 00 E =.
000

[0138]

[Table 16] Area II * ** ** * ** * * * 1 GAAGCCGACGAAACCCGTAA-CAA-AGCTCGCGCCGTACTGCGCGCTATTGCCACCGCGC 1 GAAGCCGATGAAACCCGTAATACGGC * * * * * ATCATGCACAGGAGAC-TTTCTGTCCGA- * TC * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ** -CGACAATGA-AAAATCC * * * * * * * * ** * * ** * * GGTGCTGATGCT-TTCTCCTGGCCCCGG-TGT-GCC-GAGCGAAGCCGGTTGTATGCCGG TGTGCTAATGCTCTCC-CCCGGCCC-GGGTGTTCCCAGCG--AGGCAGGTTGTATGCCGG * * * * * * * * ** * AACTCCTCACCCG-CT-TGCGTGGCAAGCTGCCCATTATTGGCATTTGCCT-CGGACATC AGCTGCTGACCCGACTACGCG--GCAAGTTACCGATCATCGGCATTTGTCTGGGG-CATC * ** * * * * * AGGCGATTGTCGAAGCTTACGG-GGGCTATGTCGGTCAGGCAGGTCGAAATTCT TTACGGCGG-TTACGTCGGTCAGGCGGGAGAAATCCTGCATGGC * * * * TAAAGCCTCCAGCATTGAACATGACGGTCAGGCGATGTTTGCCGG 445 AAA-GCCTCCAGCATTGAGCATGACGGTCAGGCGATGTTCGCCGG% = 80.215 P (465,373) =. 000E + 00 E =.
000

The restriction sites for both sequences are shown below.

Table 17 E. coli HpaII (CCGG) 187 229 254 272 283 443 443 463 502 506 592 HphI (GGTGA) 177 552 HphI (TCACC) 285 597 MboI (GATC) 135 564

[0140]

[Table 18] S. typhimurium HpaII (CCGG) 187 248 253 283 443 463 506 HphI (GGTGA) 177 552 MboI (GATC) 135 204 317 564 Mnll4 (CCTC)

The E. coli sequence has Mbo I at 135 and 564.
It has a (GATC) site. There are 429 base pair fragments that can be detected with the probes in regions 1 and 2. The same enzymes are used in Salmonella typhimurium 135, 204,
It is also at position 317 and 564. The two homologous region probes detect 69, 113 and 247 base pair fragments. Thus, the identified genetic characterization of E. coli by this probe and enzyme is 429 and that of Salmonella typhimurium is 69, 113, 247.

Example 3 Use of the α-Fetoprotein Gene as a Probe This example illustrates the use of the homologous region (endonuclease MnI I in the human and rat α-fetoprotein gene sequences.
It shows the use of (GAGG).

Human α-fetoprotein message c
The DNA (1578 base pairs) is as follows.

[Table 19] 10 20 30 40 50 AGCTTGGCAT AGCTACCATC ACCTTTACCC AGTTTGTTCC GGAAGCCACC TCGAACCGTA TCGATGGTAG TGGAAATGGG TCAAACAAGG CCTTCGGTGG 60 70 80 90 100 GAGGAGGAAG TGAACAAAAT GACTAGCGAT GTGTTGGCTG CAATGAAGAA CTCCTCCTTC ACTTGTTTTA CTGATCGCTA CACAACCGAC GTTACTTCTT 110 120 130 140 150 AAACTCTGGC GATGGGTGTT TAGAAAGCCA GCTATCTGTG TTTCTGGATG TTTGAGACCG CTACCCACAA ATCTTTCGGT CGATAGACAC AAAGACCTAC 160 170 180 190 200 AAATTTGCCA TGAGACGGAA CTCTCTAACA AGTATGGACT CTCAGGCTGC TTTAAACGGT ACTCTGCCTT GAGAGATTGT TCATACCTGA GAGTCCGACG 210 220 230 240 250 TGCAGCCAAA GTGGAGTGGA AAGACATCAG TGTCTGCTGG CACGCAAGAA ACGTCGGTTT CACCTCACCT TTCTGTAGTC ACAGACGACC GTGCGTTCTT 260 270 280 290 300 GACTGCTCCG GCCTCTGTCC CACCCTTCCA GTTTCCAGAA CCTGCCGAGA CTGACGAGGC CGGAGACAGG GTGGGAAGGT CAAAGGTCTT GGACGGCTCT 310 320 330 340 350 GTTGCAAAGC ACATGAAGAA AACAGGGCAG TGTTCATGAA CAGGTTCATC CAACGTTTCG TGTACTTCTT TTGTCCCGTC ACAAGTACTT GTCCAAGTAG 360 370 380 390 400 TATGAAGTGT CAAGGAGGAA CCCCTTCATG TATGCCCCAG CCATTCTGTC ATACTTCA CA GTTCCTCCTT GGGGAAGTAC ATACGGGGTC GGTAAGACAG 410 420 430 440 450 CTTGGCTGCT CAGTACGACA AGGTCGTTCT GGCATGCTGC AAAGCTGACA GAACCGACGA GTCATGCTGT TCCAGCAAGA CCGTACGACG TTTCGACTGT 460 470 480 490 500 ACAAGGAGGA GTGCTTCCAG ACAAAGAGAG CATCCATTGC AAAGGAATTA TGTTCCTCCT CACGAAGGTC TGTTTCTCTC GTAGGTAACG TTTCCTTAAT 510 520 530 540 550 AGAGAAGGAA GCATGTTAAA TGAGCATGTA TGTTCAGTGA TAAGAAAATT TCTCTTCCTT CGTACAATTT ACTCGTACAT ACAAGTCACT ATTCTTTTAA 560 570 580 590 600 TGGATCCCGA AACCTCCAGG CAACAACCAT TATTAAGCTA AGTCAAAAGT ACCTAGGGCT TTGGAGGTCC GTTGTTGGTA ATAATTCGAT TCAGTTTTCA 610 620 630 640 650 TAACTGAAGC AAATTTTACT GAGATTCAGA AGCTGGCCCT GGATGTGGCT ATTGACTTCG TTTAAAATGA CTCTAAGTCT TCGACCGGGA CCTACACCGA 660 670 680 690 700 CACATCCACG AGGAGTGTTG CCAAGGAAAC TCGCTGGAGT GTCTGCAGGA GTGTAGGTGC TCCTCACAAC GGTTCCTTTG AGCGACCTCA CAGACGTCCT 710 720 730 740 750 TGGGGAAAAA GTCATGACAT ATATATGTTC TCAACAAAAT ATTCTGTCAA ACCCCTTTTT CAGTACTGTA TATATACAAG AGTTGTTTTA TAAGACAGTT 760 770 780 790 800 GCAAAATAGC AGAGTGCTGC A AATTACCCA TGATCCAACT AGGCTTCTGC CGTTTTATCG TCTCACGACG TTTAATGGGT ACTAGGTTGA TCCGAAGACG 810 820 830 840 850 ATAATTCACG CAGAGAATGG CGTCAAACCT GAAGGCTTAT CTCTAAATCC TATTAAGTGC GTCTCTTACC GCAGTTTGGA CTTCCGAATA GAGATTTAGG 860 870 880 890 900 AAGCCAGTTT TTGGGAGACA GAAATTTTGC CCAATTTTCT TCAGAGGAAA TTCGGTCAAA AACCCTCTGT CTTTAAAACG GGTTAAAAGA AGTCTCCTTT 910 920 930 940 950 AAATCATGTT CATGGCAAGC TTTCTTCATG AATACTCAAG AACTCACCCC TTTAGTACAA GTACCGTTCG AAAGAAGTAC TTATGAGTTC TTGAGTGGGG 960 970 980 990 1000 AACCTTCCTG TCTCAGTCAT TCTAAGAATT GCTAAAACGT ACCAGGAAAT TTGGAAGGAC AGAGTCAGTA AGATTCTTAA CGATTTTGCA TGGTCCTTTA 1010 1020 1030 1040 1050 ATTGGAGAAG TGTTCCCAGT CTGGAAATCT ACCTGGATGT CAGGACAATC TAACCTCTTC ACAAGGGTCA GACCTTTAGA TGGACCTACA GTCCTGTTAG 1060 1070 1080 1090 1100 TGGAAGAAGA ATTGCAGAAA GACATCGAGG AGAGCCAGGC ACTGTCCAAG ACCTTCTTCT TAACGTCTTT GTGTAGCTCC TCTCGGTCCG TGACAGGTTC 1110 1120 1130 1140 1150 CAAAGCTGCG CTCTCTACCA GACCTTAGGA GACTACAAAT TACAAAATCT GTTTCGACGC GAGAGATGGT CTGGAATCCT CTGATGTTT A ATGTTTTAGA 1160 1170 1180 1190 1200 GTTCCTTATT GGTTACACGA GGAAAGCCCC TCAGCTGACC TCAGCAGAGC CAAGGAATAA CCAATGTGCT CCTTTCGGGG AGTCGACTGG AGTCGTCTCG 1210 1220 1230 1240 1250 TGATCGACCT CACCGGGAAG ATGGTGAGCA TTGCCTCCAC GTGCTGCCAG ACTAGCTGGA GTGGCCCTTC TACCACTCGT AACGGAGGTG CACGACGGTC 1260 1270 1280 1290 1300 CTCAGCGAGG AGAAATGGTC CGGCTGTGGT GAGGGAATGG CCGACATTTT GAGTCGCTCC TCTTTACCAG GCCGACACCA CTCCCTTACC GGCTGTAAAA 1310 1320 1330 1340 1350 CATTGGACAT TTGTGTATAA GGAATGAAGC AAGCCCTGTG AACTCTGGTA GTAACCTGTA AACACATATT CCTTACTTCG TTCGGGACAC TTGAGACCAT 1360 1370 1380 1390 1400 TCAGCCACTG CTGCAACTCT TCGTATTCCA ACAGGAGGCT ATGCATCACC AGTCGGTGAC GACGTTGAGA AGCATAAGGT TGTCCTCCGA TACGTAGTGG 1410 1420 1430 1440 1450 AGTTTTCTGA GGGATGAAAC CTATGCCCCT CCCCCATTCT CTGAGGATAA TCAAAAGACT CCCTACTTTG GATACGGGGA GGGGGTAAGA GACTCCTATT 1460 1470 1480 1490 1500 ATTCATCTTC CACAAGGATC GTGCCAAGCT CGGCAAAGCC CTACAGACCA TAAGTAGAAG GTGTTCCTAG CACGGTTCGA GCCGTTTCGG GATGTCTGGT 1510 1520 1530 1540 1550 TGAAACAAGA GCTTCT CATT AACCTGGTGA AGCAAAAGCC TGAACTGACA ACTTTGTTCT CGAAGAGTAA TTGGACCACT TCGTTTTCGG ACTTGACTGT 1560 1570 GAGGAGCAGC TGGCGGCTGT CACTGCAG CTCCTCGTCG ACCGCCGACA GTGACGTC

The rat α-fetoprotein 3'endo cDNA is as follows (540 base pairs).

[Table 20] 10 20 30 40 50 GAGGGACTGG CCGACATTTA CATTGGACAC TTGTGTTTAA GACATGAGGC CTCCCTGACC GGCTGTAAAT GTAACCTGTG AACACAAATT CTGTACTCCG 60 70 80 90 100 AAACCCTGTG AACTCCGGTA TCAACCACTG CTGCAGTTCC TCGTATTCCA TTTGGGACAC TTGAGGCCAT AGTTGGTGAC GACGTCAAGG AGCATAAGGT 110 120 130 140 150 ACAGGAGGCT CTGCATCACC AGCTTTCTGA GGGACGAAAC CTACGTCCCT TGTCCTCCGA GACGTAGTGG TCGAAAGACT CCCTGCTTTG GATGCAGGGA 160 170 180 190 200 CTACCATTCT CTGCGACAAA TTCATCTTCC ACAAGGAATC TGTGCCAAGC GATGGTAAGA GACGCTGTTT AAGTAGAAGG TGTTCCTTAG ACACGGTTCG 210 220 230 240 250 TCAGGGCCGA GCACCACAGA CCATGAAACA AGAGCTTCTC ATTAACCTAG AGTCCCGGCT CGTGGTGTCT GGTACTTTGT TCTCGAAGAG TAATTGGATC 260 270 280 290 300 TGAAACAAAA GCCTGAAATG ACAGAGGAGC AGCACGCGGC TGTCACTGCT ACTTTGTTTT CGGACTTTAC TGTCTCCTCG TCGTGCGCCG ACAGTGACGA 310 320 330 340 350 GATTTCTCTG GCCTCTTGGA GAAGTGCTGC AAAGACCAGG ATCAGGAAGC CTAAAGAGAC CGGAGAACCT CTTCACGACG TTTCTGGTCC TAGTCCTTCG 360 370 380 390 400 CTGTTTCGCA AAAGAGGTCC AAGTTGATTT CCAAACTCGT GAGGCTTTGG GACAAAGC GT TTTCTCCAGG TTCAACTAAA GGTTTGAGCA CTCCGAAACC 410 420 430 440 450 GGGTTTAAAC ATCTCCAAGA GGAAGAAAGG ACAAAAAAAT GTGTCGACGC CCCAAATTTG TAGAGGTTCT CCTTCTTTCC TGTTTTTTTA CACAGCTGCG 460 470 480 490 500 TTTGGTGTGA GCTTTTCGGT TTGATGGTAA CTGGTGGAGA CTTCCATGTG AAACCACACT CGAAAAGCCA AACTACCATT GACCACCTCT GAAGGTACAC 510 520 530 540 GGATTTCTAT GCCTAAGGAA TAAAGACTTT TCAACTGTTA CCTAAAGATA CGGATTCCTT ATTTCTGAAA AGTTGACAAT

The regions of homology between the two are as follows (human: upper row, rat: lower row).

【Table 21】 *** * * * * * 1367 CTCTTCGTATTCCAACAGGAGGCTATGCATCACCAG-TTTTCTGAGGGATGAAACCTATG 90 CTC --- GTATTCCAACAGGAGGCTCTGCATCACCAGCTTT-CTGAGGGACGAAACCTACGAA ** TAC ** GACGATCCATCCATCCATCCATCCATCCATCCATCCATCCATCCATCGACCATCCATCCATCCATCCATCCATCGATCCATCGACCATCCATCGACCAGTCATCCATCGACCAGTCATCCATCCATCGACCAGTCATCCATCGACCAGTCATCCATCGACCAGTCATCCATCCATCGACCATCCATCGACCAGTCATCCATCCATCGACCATCCATCGACCATCCATCGACCAGTCATCCATCAGCATCCATCGACCATCCATCAGCA-CAACACATCATCTC-CCCCATTCTCTGAGTC-CAACACATCATC-CCCCATTCTCTGAGTC-CAACACATCATC-TCCCATTCTCTGAGTC-CAACACATCATC-CCCCATTCTCTGAGTC-CAACACATCATC-CCCCATTCTCTGAGC TTCATCTTCCACAAGGAATCTGTGCCAAGCTC * ** * * * * * * -GG--CAAAGC-CCTACAGACCATGAAACAAGAGCTTCTCATTAACCTGGTGAAGCAAAA AGGGCCGAGC = GCCTGAGCGCGCGCGCGCGCGCGCGCGCGCGC 000E + 00 E =.
000

Restriction sites between both human and rat are as follows.

[Table 22] Human MboII (GAAGA) 108 261 328 1066 1069 1230 MboII (TCTTC) 881 916 1361 1449 MnlI (CCTC) 273 574 1190 1200 1219 1245 1439 MnlI (GAGG) 44 47 358 449 653 887 1070 1162 1250 1274 1378 1402 1436 1544

[0147]

Table 23 Rat MboII (GAAGA) 435 MboII (TCTC) 168 MnlI (CCTC) 100 159 323 MnlI (GAGG) 39 98 122 267 357 384 412

Fragments containing conserved sequence portions (24, 34, 1
It can be calculated that (04 and 108 base pairs) describe human DNA. Also, fragments 24, 59, 9
0 and 145 base pairs describe rat DNA. On the other hand, both sequences contain a 24 base pair fragment, which is an important fragment set (a taxonomic feature).

[Brief description of drawings]

Figure 1: Pseudomonas aeruginosa (Pseud)
2 shows EcoRI restriction endonuclease digestion of DNA isolated from a strain of O. monas aeruginosa. E. coli 16S and 23S as probes
CDNA for type ribosomal RNA (rRNA) was used.

Figure 2: Pseudomonas aeruginosa (P. aer
Pst I of DNA isolated from S. uginosa)
Shows restriction endonuclease digestion. CDNA for 16S and 23S rRNA of E. coli as a probe
A was used.

FIG. 3 shows EcoRI restriction endonuclease digestion of DNA isolated from glucose-nonfermentative Gram-negative rod species. Escherichia coli 16S type and 2 as probes
The cDNA for 3S type rRNA was used.

FIG. 4 shows Pst I restriction endonuclease digestion of DNA isolated from glucose-nonfermentative Gram-negative rod species. Escherichia coli 16S type and 2 as probes
The cDNA for 3S type rRNA was used.

FIG. 5: Various Bacillus subtilis
S. subtilis) Ec of DNA isolated from the strain
3 shows oRI restriction endonuclease digestion. CDNAs for Escherichia coli 16S type and 23S type rRNA were used as probes.

FIG. 6 shows Pst I data obtained with the same probe for the same strains as in FIG.

FIG. 7 shows BglII data obtained with the same probe for the same strains as FIGS. 5 and 6.

FIG. 8 shows Sac I data obtained with the same probe for the same strains as FIGS.

FIG. 9: B. Subtilis and B. Polymica (B.po
2 shows EcoRI restriction endonuclease digestion of DNA isolated from lymyxa). CDNA for 16S and 23S rRNA from E. coli as a probe
A was used.

FIG. 10 shows Pst I data obtained with the same probe for the same strains as in FIG.

FIG. 11 shows BglII and SacI data obtained with the same probe for the same strains as FIGS. 9 and 10.

FIG. 12: Streptococcus pneumoniae (Streptococcus pneumoniae) in EcoRI digested DNA from infected mouse tissues using cDNA for 16S and 23S rRNA from E. coli as probe.
neumoniae) detection.

FIG. 13 Mus Musculous Domesticus (Mu
Figure 3 shows the identification of mouse species by comparing Pst I digestion of DNA isolated from mammalian tissue using cDNA for 18S and 28S rRNAs from cytoplasmic ribosomes of S. musculus domesticus (mouse).

FIG. 14 Mus Musculius domesticus 28S
2 shows EcoR I digested DNA from mouse and cat tissues that hybridized with type rRNA, a cDNA probe.

FIG. 15 Mus Musculius Domesticus 18S
And 28S rRNA, Sac I digested DNA from mammalian tissue hybridized with cDNA probe
Indicates.

FIG. 16 Mus Musculius domesticus 18S
Eco from mammalian tissues and cell cultures hybridized with p-type and 28S-type rRNA, cDNA probes
RI digested DNA is shown.

Claims (31)

[Claims] 1. A kit comprising a carrier partitioned to tightly contain one or more containers, the first one containing probe organisms, derived probe organisms, or a matching sequence. Conserved gene material sequence information-containing nucleic acid (not ribosomal RNA information containing nucleic acid)
Or a kit containing a catalog with hybridized or repaired chromatographic band patterns of at least two different known organic species,
Alternatively, the kit comprises at least two known organism species or genetic material derived therefrom. 2. The method according to claim 1, wherein the genetic material is DNA. 3. A kit according to claim 1 or 2 which also comprises a second container containing one or more restriction endonuclease enzymes. 4. The method according to claim 2, wherein the stored DNA sequence information-containing nucleic acid probe is detectably labeled. 5. The kit according to claim 1 or 3, wherein the stored DNA sequence information-containing nucleic acid probe is RNA. 6. The conserved DNA sequence information-containing nucleic acid probe is DNA complementary to RNA.
Item Kit. 7. The kit of claim 5 which also comprises a container of one or more detectably labeled deoxynucleoside triphosphates. 8. The kit according to claim 1, wherein the probe organism is a pronuclear organism. 9. The kit according to claim 1, wherein the probe organism is a eukaryotic organism. 10. The method according to claim 8, wherein the prokaryote is a bacterium.
Item Kit. 11. The kit according to claim 9, wherein the eukaryotic nucleic acid probe is derived from its organelle. 12. The kit according to claim 7, wherein the cDNA is labeled with ³² P, ¹⁴ C or ³ H. 13. The kit of claim 7 in which the cDNA is a faithful copy of the rRNA from which it was derived. 14. The kit according to claim 1, which also comprises one or more containers of the viral nucleic acid probe. 15. The kit according to claim 1 or 3, wherein the catalog is a book, a computer tape, a computer disk or a computer memory. 16. The kit according to claim 1 or 3, wherein the catalog also contains a virus chromatographic pattern. 17. The kit according to claim 1, wherein the catalog also includes a taxonomic pattern of subspecies or less. 18. A kit comprising a carrier partitioned to contain exactly one or more containers therein to detectably detect probe organisms, derived probe organisms, or matching sequences. A kit comprising a first container containing a conserved DNA sequence information-containing nucleic acid containing labeled nucleic acid (not a ribosomal RNA information containing nucleic acid) and a second container containing one or more restriction endonuclease enzymes. 19. The kit according to claim 18, wherein the stored DNA sequence information-containing nucleic acid probe is RNA. 20. The kit according to claim 18, wherein the stored DNA sequence information-containing nucleic acid probe is DNA complementary to RNA. 21. The kit of claim 19, further comprising a container of one or more detectably labeled deoxynucleoside triphosphates. 22. The kit of claim 18, wherein the probe organism is a pronuclear organism. 23. The kit of claim 18, wherein the probe organism is a eukaryotic organism. 24. The method according to claim 2, wherein the prokaryote is a bacterium.
Item 2 kit. 25. The kit according to claim 23, wherein the eukaryotic nucleic acid probe is derived from its organelle. 26. The kit according to claim 20, wherein the cDNA is labeled with ³² P, ¹⁴ C or ³ H. 27. The kit of claim 20, wherein the cDNA is a faithful copy of the rRNA from which it was derived. 28. The kit of claim 18, which also comprises one or more containers of the viral nucleic acid probe. 29. The kit according to claim 18, wherein the catalog is a book, a computer tape, a computer disk or a computer memory. 30. The kit according to claim 18, wherein the catalog also contains a virus chromatographic pattern. 31. The kit according to claim 19, wherein the catalog also contains taxonomic patterns of subspecies or less.