JP2729457B2 - Kit for identification of organisms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] The present invention relates to bacteria, plants and animals.
Organisms including prokaryotic and eukaryotic organisms such as
How to quickly and accurately characterize and fix
It is. [0002] BACKGROUND OF THE INVENTION The classification of living organisms has traditionally been
Performed more or less arbitrarily, along some artificial lines
Have been. For example, the living world is divided into two worlds.
: Plants and animals (Animal)
ia). This classification applies to commonly known organisms
Is convenient, but organisms such as unicellular organisms (eg,
Green flagellates, bacteria and cyanobacteria). What
These are basically "plants" and "animals"
Is different. [0003] Organisms are simplified by the internal structure of their cells.
It was proposed to divide it. In this way, all
Cell organisms are prokaryotic or eukaryotic
(Eukaryotic). Prokaryotes
Prokaryotes are eukaryotes
yotes) are not as complex, they include unit membrane tissue
There is no internal partition by and lacks a clear nucleus. Previous
The genetic information of the nucleus is expressed in the cytoplasm on double-stranded circular DNA.
Transported to No other DNA is present in the cells (F
Germs, bacterial viruses, and autonomous replication
Except when a circular DNA plasmid is present). other
On the other hand, eukaryotes have a wide variety of unit membrane tissues.
Separates many functional components into specialized and isolated areas
Have a role to do. For example, genetic information (DNA)
Nucleus, a small organ found in a clearly partitioned nucleus
Body (mitochondria) and (in photosynthetic organisms) leaf green
It is also found throughout the body. Eukaryotic genome replication, transcription and
And translation are performed in two or three distinct sites in the cell,
Occurs in the cytoplasm, glomeruli and chloroplasts. However, the difference between prokaryotes and eukaryotes
Shows that when comparing glomeruli and chloroplasts in prokaryotic cells,
Shattered. These organelles are nowadays free
Derived from free-living prokaryotes
The free-living prokaryotes are believed to be
Endogenous eukaryotes and endosymbioti
c) Entering a relationship and ultimately tightly integrated with the host cell structure
That independent existence is no longer possible (see
For example, Fox, G .; E. FIG. 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, Riboso
Obtained from mouse L cell filaments carrying the RNA gene region
DNA was used for Escherichia col
i) show significant sequence homology with ribosomal RNA
And strongly support the endosymbiotic model (Van
  Etten, R .; A. et al, “Cell” 2
2: 157-170 (1980)). Also, corn
23S-type ribosome D of Zea mays chloroplast
The nucleotide sequence of NA is the 23S ribosome of E. coli.
It has also been shown to have 71% homology with DNA
(Edwards, K. & Kossel, H., "N.
ucleic Acids Research "9: 2
853-2869 (1981);
(Bonen, L. & Gray, MW, ibid. 8:
319-335 (1980)). Further support the general concept
I have [0005] In this model, eukaryotic cells are viewed from the nature
Phylogeny, with organelle parts clearly prokaryotic
Target "chimera". The prokaryotic-eukaryotic dichotomy is also
Even broad classification methods have drawbacks. Classification of organisms
Crossing and breeding (b)
attempt to identify plants or animals for the purpose of reading
And so-called "higher" organisms or
Accurate and reliable identification of microorganisms that may infect other media
This is the case where the identification is to be performed with certainty. For example, planting
Breeders, livestock breeders or fish breeders should
You will want to have a quick and reliable way to specify.
Veterinarians, physicians or horticulturists should be able to
Infectious organisms (parasites, fungi, bacteria, etc.) and whey
You will want to accurately identify Ruth. These organisms
Identification of virus and virus species is of particular importance.
You. This problem explains the identification of bacteria.
I understand best. Bacterial species names identify many strains.
Usually forgotten, one strain is derived from one cell
Considered a group. Bacterial species are typically found in species strains.
By describing the degree of homogeneity and diversity of attributes
Is generally defined. Express accurate definitions of bacterial species
To establish the boundaries of the species,
Difficult because it is necessary to define boundaries (Buch
anan, R .; E. FIG. , "International
Bulletin of Bacteriological
al Nomenclature and Taxon
omy ", 15: 25-32 (1965)). Species definition
Of phenotypes for the practical application of DNA to the identification of unknown bacterial strains
Appropriate processes, such as substrate and conditions for detecting attributes
Selection of lobes and labeling with radioactive material from the same species
DNA is required. Due to the diversity of bacterial species,
Leaning is a classic, progressive method for strain identification
Is the first tool used in screening
Where are the results of the method where the laboratory methods and reagents define the strain
It is used to predict whether it is appropriate for global identification. same
After all, a constant between the unidentified strain and the characterized species
Based on similarities between phenotype and genotype. Challenging
Is to define the boundaries of the species,
Using standard probes that give species-specific information
Is to do this. By doing so, the species is defined
It is directly and equally applicable to the identification of unknown injuries. Barge's Manual of Determine
Active Bacteriology (Buchanan, R .;
E. FIG. and Gibbons, N.W. E. FIG. Editing, 197
4, Eighth Edition, Williams & Wilkins
(Baltimore) is the easiest to understand bacterial taxonomy, especially
The name method, basic strain, related literature, etc. are shown. I
However, this is only a starting point for species identification.
is there. Because this is especially outdated,
Species are too easy to describe
It is. (Reference example; Brenner DJ (Brenner)
  D. J) "Manual of Clinical"
Microbiology, 3rd edition, American Society for Microbiology,
Washington D. C. , 1980, pp. 1-6). When used in bacteria, the term "species"
With different types of organic matter with distinguishable characteristics
And also in essential organic tissue characteristics, generally
Has been defined as a group of organisms with similarities to each other
Was. The problem with these definitions is that they are subjective
(Brenner, supra, p. 2).
Also, the species is simply a matter of host range, pathogenicity, and fermentation of certain sugars.
Whether it produces gas or not, and whether the sugar fermentation is fast or slow.
It was sometimes defined based on such criteria. In the 1960's, numerical bacterial classification (compilation)
(Also known as computer or genetic phenotyping)
Has become widely used. Numerical taxonomy is organism
As many potentials as possible
It is based on testing. Classification based on many characteristics
By doing so, strains with a certain degree of similarity
Can form seeds, and these can be considered seeds
it can. However, to characterize one species
A valid test may not always be useful for the next species
This method of defining species is straightforward and practical for identifying unknown strains.
Not applicable to Even though this seems to be species specific
By selecting the attributes that will be
Used for strain identification and partially overcome
However, this type of definition is only applied indirectly.
(Brenner, supra, p. 2-6). Moreover
The general method is to use this as the sole basis for
If you use it, there are some problems,
Include the number and nature of the tests to be used and
Should be assessed to determine how relevant
And how much similarity to choose
Whether similarity criteria can be applied to all groups, etc.
You. Hugh R. H (Hugh, RH) and Gillier
G. L (Gilliardi, GL), "Manua
l of Clinical Microbiology
y "Second Edition, American Microbial Association, Washington, D.C. C. ,
1974, p. 250-269 include genomic fragments
As a means of determining bacterial species using
The characteristics of the phenotype are listed. Many from one species
By studying randomly selected strain samples
Is the most highly stored or very large number of bacteria
You can select attributes that are common to strains and define species.
Wear. The use of least characteristics is an advance.
Starting with a screening method to tentatively identify strains.
So that an appropriate additional medium can be selected. Then
Expect the strain to have most of the minimum properties
While examining known attributes stored in the species. Most
Some of the minor characteristics are found in all strains of the species.
Not necessarily. The related thinking is that
Species type, neotype or identified control strain of
There are comparative studies. Media and methods vary by laboratory
This is necessary because of the differences, and the strain is the species standard.
(Standard), not a method. A molecular approach to bacterial classification is
DNA-DNA repairing (reassoc) of the two genomes
iation). Species inheritance
The tentative definition is that the species strain is more than 70% related
Is included. The strain can be identified by DNA-DNA recombination.
The reason is that the radiolabeled DNA probe and the unknown DNA are the same.
Only if they are of the same species. But this 70% species
-Practical application of the definition requires the selection of appropriate probes.
Limited by what must be done. This is a rematch group
Select phenotype attributes that appear to be interrelated with
May be partially overcome, but these are simply
When used alone, species-specific DNA-DNA repairing
The definition still applies only indirectly. [0011] Brenner, supra, page 3, describes bacterial species.
The ideal way to confirm is to isolate the gene and immediately
The nucleic acid sequence in the strain can be replaced by any known species (speci
es-something) standard pattern
Is similar to mass spectrophotometry
Su ". [0012] However, Brenner is an isolated genetic
Restriction endonuclease for determining the general sequence of offspring
We can do the analysis, but we say,
Box, especially suitable for use in clinical laboratories
Black boxes are unlikely to be available. "
You. His words apply equally to all species of organisms
It is. From this brief review of the prior art, the unknown details
Identify fungi and other organisms and quickly classify them
In particular pathogenic organisms or available biochemical reactions.
Quick, accurate and reliable for identifying living organisms
We come to the conclusion that a law is needed now. The method is clinical research
Must be universally and easily accessible in the ultimate room
Must be performed, the number of trials performed, and the clinician's subjective bias.
And must not rely on past, accidental or inevitable
Trial and error method
od). Furthermore, any living
To identify and differentiate the genera and species of organisms
Medical, plant grower, toxicologist, animal breeder, entomologist
And in other relevant areas where such identification is needed
Must be easy and reliable
It is. [0014] Accordingly, the object of the present invention is to
Organisms, especially but not limited to microorganisms,
Provides a fast, accurate and reliable way to objectively identify
It is to be. [0015] Another object of the present invention is the presence of bacteria such as bacteria.
Provides a method to identify the body using the genome of the organism
It is to be. Another object of the present invention is to provide an animal or plant
To be able to characterize and identify the cause of the disease
Characterize pathogenic organism species and genera in floor laboratories
To provide a method for identification. [0017] Another object of the present invention.
Provides a variety of products that are useful for the taxonomy described above.
Is Rukoto. These and other objects of the invention are:
The following methods are provided, as will be more readily apparent below.
Was achieved by offering. That is, an unknown organism
Method to characterize the species of
From or to a known probe organism
Origin of ribosomal RNA information-containing nucleic acid and hybrid
Restricted endonuclease-digested D
Chromatography of NA-by determining the pattern
Restriction method in genetic material of unknown organisms)
The unknown organic site corresponding to the known position of the clease cleavage site
Some or all of the evolutionary conserved sequences in the genetic material of the body
Position, thereby identifying a site for identification of the unknown organism.
Genetic characterization and identification of the conserved sequence
At least two sets of genetic characterizations for
Information from each of the pairs defining the fuselage and the features
This is a method that is based on comparison with the attachment. Another object of the present invention provides the following method.
That was achieved. Pathogenic organic in certain samples
A method for diagnosing systemic infection, including
A method comprising identifying by the method described above. The present invention is directed to the event of species
If it is a collection of individually isolated strains to be associated
Objectively define species boundaries, despite decentralization
There should be similarities shared by the strains and strains of the species
Provides structural information to help them find their common roots.
Based on the inventor's perception that they should have
You. The past history of organisms is the most semantic (se
remains in DNA and RNA
(Zuckerkndle, E. and Paul
ing, L .; "Journal of Theoret
Ial Biology, Vol. 8: 357-366
(1965)). European Patent Application (EP-A-) No. 0
In 076123 (added here as a reference),
The inventor believes that the ribosomal RNA (rRNA)
The definition and characterization of species of organisms that use information
Described the system. Ribosomal RNA for protein synthesis
It has a structural and functional role (Schup, "Jo
urnal of Theoretic Biol
ogy "Vol. 70: 215-224 (1978))
Obtained from rRNA-DNA hybridization studies
The general conclusion is that the basic sequence of the ribosomal RNA gene
Have undergone changes during evolution compared to most other genes.
Harder to be preserved (Mo
ore, R .; L. Author, Current Topics
  InMicrobiology and Immun
obiology ", 64 volumes, 105-128 (197
4), Spring-Verlag, New York
H). For example, the 16S type rR obtained from a number of bacterial species
Primary structure of NA deduced from oligonucleotide analysis
(Fox, GE et al, "Inter
national Journal of System
"Matic Bacteriology", Volume 27:
44-57 (1977)). 16S type of several strains of E. coli
There is a negligible difference in the oligomer catalog (Uc
hida T .; et al; “Journal of
  Molecular Evolution, Volume 3:
63-77 (1974)).
It can be used to create a phylogenetic classification table (Fo
x, G. E. FIG. "Science" Volume 209: 457-4
63 (1980)). Different strains of a bacterial species
The restriction maps are not necessarily the same, and different Ec
oRI site in rRNA gene of two strains of E. coli
(Boros, IA et.
al, “Nucleic Acids Research
ch ", Volume 6: 1817-1830 (1979)). Fine
Bacteria appear to share conserved rRNA gene sequences
And other sequences can vary (Fox, 197).
7, ibid.). Thus, the present inventor has identified the restriction endonuclease of DNA.
Nuclease digests may be used in certain organisms (eg, bacteria)
Are similar in other strains, but different in strains of other species of organisms.
Having a combination of fragments containing conserved sequences
That is, despite the mutation of the strain, it has a high frequency of occurrence,
Specific set of enzymes of restriction fragments with minimal genotype characteristics
It was found that the combination determines the species. This is EP-
A-0076123 is the essence of the invention, wherein
It is also the essence of the invention described. The present invention is disclosed in EP-A-0076123.
It is an extension of the evolved concept,
In addition to sequences, there are highly conserved sequences through evolution.
Was found to be
It may be as useful as rRNA sequences. Okay
Thus, the present invention is not a rRNA, but a conserved probe.
By using the same method as in EP-A-0076123.
To provide a way to implement
You. The present invention is also used in the identification process.
Additional embodiments of the method that may be provided are also provided. The inventor has
Are the same or different in taxonomy (classical)
Prokaryotic and eukaryotic regardless of
Conserved nuclei from organisms other than the organism to be determined
Applicable to both prokaryotic and eukaryotic DNA using acid probes
It was also found to be general in that it could be cut. The present invention
Is located at a known location, such as a restriction endonuclease site.
Based on the conserved sequence of the corresponding DNA or other genetic material
To provide an objective way to define an organism. Storage
For detection of restriction fragments containing sequences, DNA sections were probed
Hybridization with nucleic acids containing conserved sequence information from the body or
This is done by re-pairing. [0024] The present invention is characterized by the process of the present invention.
Airframe "(this term is interpreted to include" identify ")
By definition, DNA or DNA in its genome by definition
Means virtually any organism, including RNA. this
Refer to the traditional taxonomy for information on points
Is useful. Monera kingdom, plant (Pla)
ntae) and animals (Animalia)
Includes all organisms. For example, Monera
a) In the world, mycobacterium (myxobacter)
ia), spirochetes, yu
-Bacteria (eubacteria), rickettcher
(Rickettsiae) fission products (bacteria) of the class and
Cyanobacteria: cyanophytha
Can be mentioned. Euglenoids in the plant kingdom
(Euglenophtha), green algae: chlorophyta
(Chlorophyta), Chlorophysae (Chl)
orophyceae and Charophyse (Char)
ophysea, rope, chrysotha
phta) xanthophysae (xanthophyc)
eae), chrysophysea
e), bacillaryophies
yceae); pyrophyta
a) The class Dinoflagella
tes), brown algae: phaeophyta
ta); Red algae: Rhodophyta
a); Slime molds: Mycomycophyta
phyta), mycomyceta (myxomycete)
s), acrasiaes, plasmodes
Plasmodiophore, la
Lebrinthlieae class;
-Mycophyta: fungi (Eumycophyt)
a), phycomycetes, a
Zucomicetes (ascomycetes), bacidomycetes
(Basidomycetes) class; bryophytes, hepati
D (hepaticae), anthellot (antho)
cerotae), Musci class; Vascular planting
Things Tracheophyta,
Psilopsida, lycosida (l
ycosyda), Sphenopsida (sphenops)
ida), pteropsida,
Permopsida subfamily, saika
De (cycadae), zinc goe (gingkoa)
e), conifere, gunete (gn)
eteae) and angiospermae (angios)
permae), decotyledone
donee), monocotiroedone (monocoty)
loedoneae) are described. In the animal kingdom,
Protozoa, Protozoa
a) Plasmodroma subplasma
Gate, flagellata, sarcode
Sarcodina and sporozoa (spor)
ozoa class; ciliophor
a) Subphylum, class Ciliata;
Kingdom, polyhera (sponge porifera), kalka
Calcarea, Hexachinerida (hex)
actinellida) and desmospogee (d
esmospongiae); Mesozoa, Mesozoic
Phylum: metazoan subdivision Radiata
Gate, Coelenterata Gate, H
Drozoa (hydrozoa), sifozoa (scyp
hozoa), Utozoa rope,
Nofora (ctenophora) gate, tentakrata
(Tentaculata) and Nuda
Rope; protostoma section
Platyhelminte
s), tubellana, tremato
Trematoda and cesto
da) rope; nemertina gate, red
Acanthocephala gate;
Aschelmintles Gate, Loti
Spatula (rotifera), Gastrotrica (gast)
rotricha) and kinorhync (kinorhync)
ha), pre-applida, nema
Nematoda and nematomorpha (n
genus Ematomorpha; entproctor (ent)
oecta); ectoproctor (ectopro)
cta) Gate, gymnolemat
a) and phyllactola
emata) rope; phoronida
Gate; Braciopoda Gate, Inner
Inarticulata and artik
Class of Articulata; molska mollusc
(Mollusca) gate, amfinula (amph)
ineura), monoplacophora (monoplac)
ophora), gastropod
a), Scaphopoda, Pele
Psycypoda and Cephalopo
Cephalopoda; Sipunculida (s)
ipunculida); echiuida
Rida) gate, annelida gate, poly
Caterers (polychaeta), oligocaters (ol
igochaeta and hirudinea (hirud)
inea) rope; onychophor
a) Gate; tardigrada gate;
Pentastoimida
Gate; Arthropoda Gate; Bird
Trilobita Gate, Keli Selata (c
helicerata submonkey, Kifuosura (xipho)
sura), arachimida, pic
Pycnogomida rope, mandebrata
(Mandibulata) Amon, crustae (cru
staea), chiropoda, de
Dipropoda, polopoda (p
auropoda), symphyla
Rope, colembola, protura
(Protura), diplura, dip
Sanura, efumerida (eph)
emerida), odonata, ol
Topoptera (orthoptera), dermaptera (d
ermaptera), embinai (embiani)
a), plecoptera, zorap
Terra (zoraptera), Condentier (cor)
rodentia), mallophaga (mallophaga)
ga), anoplura, chisasnop
Tera (thysasoptera), hemi-ptera (h
emiptera), neuroptera (neuropte)
ra), Coleoptera, Hime
Noptera (hymenoptera), Mecoptera (m
ecoptera), siphonaptera (siphonap)
utera), diptera, trichop
Trichotera and Lepidoptera
(Lepidoptera) insects of the order;
Keto, an animal in the Mia (Denterostoma) category
The gatenata (chaetognatha), Echinoderma
Echinodamata Gate, Clinoidea
(Crinoidea), astrodea (astero)
dea), ophiuroid
a), echinoidea and e
Rotouloidea, Pogono
Pogonophora Gate, Hemicolodata
(Hemichordata) Gate, Enteronoista
(Enteropneusta) and Pterobranchi
(Pterobranchia) class; cordata (c)
hordata) gate, urocor data (urochor)
data) subphylum, ascidiacia
e), thaliaceae, larvae
Cephalocor data (c)
ephalochdata) Amon, Vertebrata
(Vertebrata) Amon, Agnat
ha), chondrichthye
s), ostechthys, sa
Scocopteiygii subclass, black
Crossopterygii and de
The order of dipnoi, amphibia (amph)
ibia), repitilia, ave
(Aves) and mammaria (mammalia)
Class, prototheria, class
Theria subclass, Marsapiaris (mars)
upialis), insectibora (insecti)
vora), dermoptera,
Chiroptera, primatis (p
rimates), edentata,
Phoridota, lagomorpha (l
agomorpha, rodentia
a), cetaceae, carnibola (c)
arnivora), tubulentata (tubuli)
dentata), probosicd
ea), Hiracoidea, Shire
Nearia (silenia), perisactyla (peris)
sodactyla and arti-dachila (arti)
odactyla). Under my eyes
Still have family, tribe, genus, species and subspecies,
It is known that there are taxa, strains or individuals. That
Above, cultured cells (plants or animals) are the same as viruses.
Can be specified. These classifications are
Used for descriptive purposes only and used in a limited manner
Not something. Identify organisms known or unknown
But may be the most commonly unknown. Functionally
Means that all organisms are eukaryotic cells for the purposes of the present invention.
It is convenient to separate them into groups and pronuclear cell groups. Pronuclear organisms
When identifying, DNA can be found in cells or
Are present in chromosomes that do not exist. Eukaryotic organisms
If specified, nuclear DNA or organelle DNA (granulosum)
DNA or chloroplast DNA). Briefly, conserved sequences (and possibly species
Classification below the level or subclassification below the subspecies (infras
ubsspecific subdivision)
Sequence that can be used to retrieve
High molecular weight DNA and / or small circular DNA
It is separated from and identified. DNA is available to those skilled in the art.
It is extracted by a known method. DNA is: 1) The presence and location of conserved sequences and
And 2) both the positions with respect to the endonuclease restriction sites.
It is analyzed to see who is. Existence of saved sequence
The easiest way to analyze the location is to use the conserved DNA sequence
Use polynucleotide probes that can be hybridized with
It is to use. However, due to chemical sequencing and analysis
Direct sequence information as obtained is also used. EP-
The probe utilized in A-0076123 was r
Although the probe was an RNA information-containing probe,
Any other probe with a sequence can be used.
You. In a similar manner, an endonuclease restriction site
The easiest way to find a given set of
Is to cut the DNA with restriction enzymes (in fact, this is
Method taught and practiced in EP-A-0076123
Is). However, sequences linked to known restriction site sequences
Column information or other methods such as truncation and partial alignment
Can also be used. Most commonly, DNA is controlled at specific sites.
Cleavage into fragments by restriction endonucleases.
You. The fragments are measured by size in a chromatographic system.
Is separated. In EP-A-0076123, gel
Chromatography is a useful chromatographic system
Used as an example. However, high pressure liquid chromatography
Or capillary zone electrophoresis or other separations
Other systems, such as technology, can also be used. Gel black
When using chromatography, the fragments are separated and
The gel is stained using known methods,
Using the standard curve created, standardize the fragment size
I do. Then, the separated fragments were collected in Southern
rn) spot method (Southern, EM "J.
ownnal of Molecular Biolo
gy ", 38: 503-517 (1975), see here.
Nitrogen cellulose cellulose paper)
And covalently bonded thereto by heating. Save the sequence
The fragment containing the nucleic acid probe then contains the conserved sequence information
The position is determined by the ability to hybridize.
Also, whether hybridization occurs after digestion and before separation
Or restriction fragments are generated after hybridization, and
It may be separated. The nucleic acid probe is labeled with a non-radioactive substance.
Or preferably labeled with a radioactive substance.
When labeling with a radioactive substance, the probe may be RNA.
Can be synthesized by reverse transcription, or
Signed by a nick translation
Complementary to RNA contained on cloned fragments that can be
DNA (cDNA) may be used. In addition, synthetic oligodeo
Xyribonucleotides are synthesized from labeled nucleotides.
It may be. A well-defined probe will be seen later
Obtained or matched from any chosen organism as
May be obtained from the sequence. Once hybridized
Once this occurs, the hybridized fragment is a double-stranded nucleus.
Whether to detect by selectively detecting acid (non-radioactive
Labeled probes) or, for example, radioautograph methods
Therefore, make it visible (radiolabeled probe
B). The size of each hybridized fragment is limited
It is related to the part and the travel distance is calculated using the standard curve described above.
It is decided from separation. Hybridization amount, hybridization
The size of patterns and hybrids
Organic compounds individually or in combination
Used to identify the body. Emerging from this technology
Genetic characterization is at least two, otherwise numerous
Equivalent characteristics from known standard organisms, genera or species of
Easy to compare with Preliminary broad classifications (for example,
After already done (using classical classification),
Inspection and matching with appropriate chromatographic patterns
Hybrid (as in EP-A-0076123)
By comparing the size of the digested restriction fragments, the band intensity
(Hybridization amount) or a combination of these
By doing so, a comparison can be made. Ideally
Is a point-of-sale
e) One-dimensional computers, such as those used for processing
It is better to compare by a pattern recognition device. When using the above method, the inventor
Organisms have very similar genetic characterizations,
The major difference is the difference between subspecies due to changes in strains,
Between and between genera (and at a higher level of classification
Is very large. Enzyme-specific between certain strains
By utilizing the fact that the fragment is mutated, for various purposes, for example,
In the case of bacteria, strains should be typed for epidemiological purposes.
Can be. In fact, restriction enzymes distinguish strains within a species
You can choose what you can do. "Probe organism" used in this study
(And the resulting nucleic acid probes) are listed above.
Any eukaryotic or prokaryotic organism
May be. The only restriction is that the conserved sequence-containing probe
Must hybridize maximally with DNA of unknown organisms
Must be given by the fact that Conserved sequence information-four types of contained probes
1) pronuclear probes (especially obtained from bacteria), 2)
3) Eukaryotic chloroplast probe 4) True
Nuclear non-organelle probes. DNA (digested with endonuclease)
There are also four types of sources: 1) prokaryotic DNA, 2) true
Nucleoplast DNA, 3) eukaryotic chloroplast DNA, 4) eukaryotic cells
Nuclear DNA. Thus, the following hybridization table was created:
(Table 1). [0037] [Table 1] The table shows which probes are generally unknown organisms.
Can hybridize maximally with other DNA
Is shown. For example, to identify a certain eukaryotic organism
To extract the species-specific glomerular or chloroplast DNA,
This is digested with endonuclease, and the digest
Eukaryotic probes and hives extracted from lobes or organelles
What is necessary is just to make a lid. Similarly, identify prokaryotic organisms
To do this, the species-specific cellular DNA is extracted and endonucleated.
Digest it with creatase and digest the digest with a pronuclear probe or
Hybridization with eukaryotic probes extracted from organelles
I just need. Extracting and digesting the species-specific nuclear DNA,
Can be hybridized with a non-organelle eukaryotic probe.
Also, eukaryotic organisms can be identified. true
Nuclear cells are nuclei, glomeruli, or, in some cases, chloroplasts.
May be determined by one or some combination of
did it. These cross-hybridizations are due to eukaryotic organelles.
Broad homology of an incoming nucleic acid with an evolutionarily conserved sequence from a prokaryotic nucleic acid
Nucleus-extracted eukaryotic DNA and prokaryotic DNA
The homology is generally so broadly distributed between
Not based on the fact that. The DNA to be digested and the associated
The choice of lobe pair is arbitrary and depends on the organism to be identified.
Will depend. That would depend on the question being asked.
For example, to detect and identify infectious agents, eukaryotic cells
Pronuclei present in or together with (eg animals or plants)
When detecting cell species (eg, bacteria), use a pronuclear probe.
And DNA from organelles is not extracted or minimal
What is necessary is just to process under the condition of extracting a limited amount. Use this technique
If present, interference between organelle-derived DNA and pronuclear DNA is minimal.
You can be confident that it is small. Eukaryote (it is prokaryotic
(Not infected by some cells) with a pronuclear probe
For example, when organelles are separated from the nucleus and then
By extracting only DNA,
It is best to maximize the concentration of DNA. Of prokaryotic organisms
If you want to identify infected eukaryotic organisms,
It is best to use probes derived from public, non-prokaryotic cells.
You. Because this probe combines DNA from prokaryotic cells
Does not generally form sufficient hybrids. The same world, or the same sub-world, or the same division, or
Is the same gate, or the same subphylum, or the same class, or the same subclass,
Or from the same eye, or from the same family, or from the same race, or from the same genus
It is preferable to use one pair (DNA and probe)
No. Before hybridizing with prokaryotic DNA,
Using nuclear cell probes (eg bacterial probes)
Particularly preferred. In this way, the genus, species and species of the prokaryotic organism
Strains can be detected, quantified and identified.
Would. One of the most preferred pronuclear probes is obtained from bacteria.
That are particularly easy to use and obtain
In this respect, E. coli is preferred. E. coli
Robes can be used on any organism, especially all prokaryotes
Can be used to identify
Most preferred for the determination. Other, particularly preferred embodiments
Uses eukaryotic probes from a given family
To identify eukaryotic organisms of the same family (eg,
For example, use mammalian probes to identify mammalian organisms
There). Most preferred are the same subfamily and / or eyes and
And / or DNA obtained from a family of organisms
That is, for example, to identify certain mouse species.
It is better to use a mouse-derived probe). The most sensitive and effective paired system is the probe output.
Between the source and the source of the unknown DNA
It is a system that has less or less variance. In the present invention, the term “evolutionary conserved remains”
The phrase “transgenic material sequence” refers to plant, animal or microbial
Show homology between at least two different species
Used to represent the sequence of genetic material, for example DNA
You. The homology between two conserved sequences is
Such that one of the sequences of the DNA molecule is detectable.
If known, two single-stranded DNA molecules or fragments
High enough when they are placed under hybrid conditions with each other
Bridging or annealing can occur,
By standard methods (ie, radiolabeling, enzyme labeling, etc.)
The result is a duplex that is sufficiently stable to be detectable. Examples are given in EP-A-0076123.
The conserved evolutionary conserved sequences are those of the ribosomal RNA gene.
Was. This is still a highly desirable gene sequence
is there. However, it is not well preserved over evolutionary distances.
That other useful gene sequences exist
Was. Examples of such additional sequences can be found in references
Dayhoff's "Atlas o
f Protein Sequence and St
structure ", Volume 5, Supplem
ent 3, 1978, NBR, 1979, pages
  9-24, Superfamily (Superfamily)
(Family), preferably the subfamily (Subfamily)
ly) or as belonging to the same Entry (Entry)
Encoding the indicated transfer RNA or protein
The sequence of the gene or part. The protein family is
With no more than 50% of the amino acid residues in these sequences
Are also distinguishable from each other. Protein subfamily
Because less than 20% of the amino acid residues in the sequence
Both two proteins are distinguishable. The gate is
No more than 5% of the amino acid residues in any of the two
White is distinguished. Gene sequences that can be used or appropriate parts thereof
A special example is the cytochrome C-related gene, cytochrome
Room C_ThreeRelated gene, cytochrome C₁Related, cytochrome
B_FiveRelated, ferrodoxin related, libredoxin related
Ren, flavodoxin-related, alcohol dehydrogenase
Related, peroxidase related, adenylate kinase related
Ream, phospholipase A_TwoRelated, tryptophan opero
Related, carboxypeptidase related, subtilisin related
, Penicillinase-related, protease inhibitor-related
Ren, Somatotropin related, Corticotropin related, Lipo
Tropine-related, glucagon-related, snake venom toxin (snak
even toxin), plant toxin (pla
nt toxin-related, antimicrobial toxin (antiba)
terial toxin) -related, immunoglobulin
(Immunoglobulin) related gene, ribosomal
Ribosome-related genes other than RNA, heme carrier
-(Heme carrier) gene, chromosomal protein
(Chromosomal protein) gene,
Fibrous protein gene
And so on. Some of these additional DNA sequences
Preservation of the thing is ribosomal RNA (rRNA) inheritance
Spread through the animal, plant or microbial world as much as the offspring
It is not something. (Thus, the use of rRNA is still
preferable). However, this may be more restrictive or
Additional within the sub-world to identify or characterize organisms
May be available, so use
It does not constitute a major obstacle. For example, trp
  D Generate microbial probes and then within the microbial world
To use this probe for testing in microbes
It is possible to utilize the trpD sequence from. Thing
In fact, the same species, family or genus
The world (for example, Enterobacter)
teriaeae) or Bacillus
s) test for the presence of
It is possible to use. Therefore, the additional probe sequence
Some applications are as wide as rRNA probes
Not so, but its adaptability is extremely effective in a narrow space
Would be a target. The probe containing the conserved DNA sequence information is E
Contains rRNA information exemplified in PA-0076123
It is manufactured by the same operation as the method of manufacturing the probe. Thus
The lobe may be RNA, DNA or cDNA, etc.
No. The individual steps involved in the technique are
Mentions both nuclear and prokaryotic cells (where applicable)
Or if there is any technical difference,
Will be described separately for each type of cell.
You. The first step is the extraction of DNA from unknown organisms
It is. Nuclear DNA from eukaryotic cells is a standard known to those skilled in the art.
Can be selectively extracted by quasi-methods (examples and
By Drohan, W et al, "Bioche
m. Biophys. Acta ", 521 (197
8), 1-15, here inserted as reference
). Because organelle DNA is small and circular,
The spooling method is used to transform a non-cyclic nuclear DNA into a circular form.
Helps to separate from DNA from organs and organelles. result
The unspooled material is derived from organelles
Of DNA, which are individually isolated by density gradient centrifugation.
Can be separated. Alternatively, thread granules
(Or chloroplasts) from a mixture of crushed cells
And refined fractions of the thread (or chloroplast)
Nuclear fraction purified and used for preparation of government-derived DNA
Is used for the preparation of nuclear DNA. (For example, Bonen
L. and Gray M.S. W "Nucleic Ac
ids Research "8: 319-335 (19
80)). Pronuclear DNA extraction is also well known to those skilled in the art.
I have. For example, industrial fermentation suspensions, agar media, plants or
Unknowns present in media such as animal tissues or samples, etc.
Bacteria were set to extract high molecular weight DNA
Treat under known conditions. For example, cells of an unknown organism
Is suspended in the extraction buffer, lysozyme is added thereto, and the
Incubate the suspension. Cell destruction is achieved by adding detergent.
Can be further promoted by heating and / or temperature rise.
Wear. Chloroform / phenol extraction after protease digestion
Extraction and ethanol precipitation to complete the DNA extraction.
You. Much faster than phenol / chloroform extraction
One extraction method uses ethanol precipitation to speed up DNA.
It is a method of separating crab. This method directly coats DNA.
Knee, or used to separate from small volumes of liquid media.
Will be This method is described in Davis, R .; W et al
"A Manual for Genetic Eng
inering, Advanced Bacteri
al Genetics ”(hereafter referred to as“ Davis ”)
), Cold Spring Harbor research
Place, Cold Spring Harbor, New York
See, 1980, p. 120-121 (here references)
). DNA (prokaryotic or eukaryotic (nuclear or
Non-nuclear)) dissolved in physiological buffer for the next step
It is. Following the isolation of the desired DNA,
Tep is diverse. One of these steps
One is endonuclease digestion. Digestion of the extracted DNA is restricted to restriction endonucleases.
Perform with the enzyme lease. Any restriction endonuclease yeast
Element can also be used. Same as what you are trying to check
More preferably, it is not obtained from the same organism. so
Otherwise, the DNA will remain completely intact. (This
That would eventually identify the organism.
Because the enzyme was obtained from a species of its own origin
Because it does not seem to cut the DNA). Characteristic
Since the organism species to be shaken will not be known,
It takes a minimum amount of trial and error to get, but this is
Can be routinely performed by those skilled in the art without undue experimentation
It is a job of a degree. Possible restriction endonucleus
Examples of the enzyme are Bgl I, BamH I, 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 here for reference)
In) One or more mixtures of endonucleases
Can be used for Usually, DNA and endonucleus
The reases are put together in a suitable buffer for a suitable time (1 to
Within 48 hours, the temperature range is 25 ° C-65 ° C, preferably
Or 37 ° C). Genetic Characterization for Identification Resulting
Is the type of one or more endonucleases used.
And is specific for endonucleases
there will be. Therefore, which enzymes (one or
It is necessary to pay attention to whether Because
If the comparative characterization used in the catalog is the same enzyme
Or it must be made with enzymes
is there. Another step is to digest the desired DNA molecule.
For example, sequencing and restriction site libraries
Reference to the desired DNA molecule.
To clarify the nuclease site. clear
In addition, digestion is not a more effective way to indicate the site.
But should not be limited to this. Essence of invention
Is the DN associated with the location of the endonuclease restriction site
The location of the conserved sequence along A is a characteristic set for each species.
It lies in the discovery that it constitutes a set. Follow
The desired information (the relationship between the location of the site and the location of the gene).
All of the techniques for obtaining
Would. Further, the position of the conserved sequence along the DNA molecule
By using a hybridization probe.
Best shown. This probe controls unknown DNA.
Allow the fragments to anneal. But like arraying
Other methods for determining conserved DNA sequences are also useful.
You. When using hybridized probes,
Thus, the DNA fragments are first digested and separated, and then separated.
Preferably, the separated fragments are hybridized. I
But first digested, with excess moles of probe and / or
Did the probe anneal the DNA with a complementary sequence?
Thus, the mixture can be separated. For example, the unknown D
NA is digested with restriction endonucleases, denatured,
A small detectable labeled DNA fragment in liquid, or
Complementary synthetic orientations for some or all of the conserved sequences of interest
One or more of the godeoxyribonucleotides
It can be hybridized in molar excess. Most restriction enzymes
Cuts very rarely in DNA,
If one or more double-stranded regions of the hybrid are
It will be small compared to the size of the fragment. hybrid
In the reaction, only oligodeoxyribonucleotides
It is performed under conditions that cause bridging. Single chain DN
Unreacted material such as fragment A and hybridized
DNA fragments containing Godeoxy nucleotides are
Separated by chromatographic techniques. Labeled
DNA fragments appear in fractions sized as expected.
Will be. First, the DNA is
Anneal and then digest, then separate the mixture
It is also possible. Solution in short time or low Cot
When incubating, the restriction sites are hybridized or
Will be limited to the duplex region. Prolong solution or high
When incubating with fresh Cot, unknown DNA
Neal and prone to restriction endonuclease cleavage
This will result in a labeled duplex. Single chain end without recombination
The ends are removed by a nuclease such as S1.
Unpaired bases are DNA polymerase I or T4
Filled with polymerase. In addition, stored sequence information (for example, 20-, 30-
-Or 50) in the storage selected as the probe.
Subsequences that are more conserved than the rest of the
There is. These "short" sequences can be synthesized as desired
Or labeled enzymatically
You may put in. Single chain from unknown, predigested D
NA is an integral part of these short, highly conserved fragments.
It can be cubated and hybridized to it. It
Separation from partially labeled probes for short labeled probes.
Performed on a digestion mixture containing the quenched fragments.
(Therefore, separation occurs after hybridization). Everything
The digestion mixture is essentially single-chain for all practical purposes.
Behaves like a mixture of fragments of
Performed by matography. As pointed out, the preferred method is to first erase
And then separate and then hybridize
is there. Therefore, after digestion with endonuclease,
The incubation mixture containing the fragments
Preferably, they are separated by a chromatography method. C
If hybridization is the last step, nucleic acid digest
Can be separated by size, followed by nucleic acid
What is the method that enables hybridization with a probe?
However, it can be used. For example, gel electrophoresis, high pressure
Liquid chromatography or capillary zone swimming
Motion can be used. (Jorgenson, J. et al.
W. , J .; of HRC and CC, 4: 230-
231 (1981)). Currently preferred is gel electrophoresis
An agarose-gel electric method is particularly preferred.
It is an electrophoresis method. In this device, the DNA digest is usually
After electrophoresis in a suitable buffer, the gel is usually
A standard marker immersed in an ethidium bromide solution and possibly added
Put in UV-light-box to make car fragments visible
It is. Use labeled standard marker fragments for detection
You can also. After separation and visualization, the D
The NA fragments were prepared by the Southern method using nitrocellulose filter paper or
Is a charge-modified nylon membrane (charge-modify
dnylon membranes) ("Jou
rnal of Molecular Bilog
y "38: 503-517 (1975)). This transfer
Can be performed after the denaturation and neutralization steps, usually
Take a long time (about 10-20 hours) or apply electric power
And transferred from the gel to filter paper. Transfer from gel to filter paper
Equipment that facilitates movement is commercially available. Nitro received
Cellulose filter paper is then high to bind DNA to the filter paper.
Heat for several hours at a temperature (60-80 ° C). Also, see Purrello, M .; (Ana
l. Biochem. , 128: 393-397 (19
83)) using recent methods for direct hybridization
By doing so, metastases are avoided. Hybridization of DNA digestion fragment bound to filter paper
The probe used for the kit is a nucleic acid probe,
A given well-known organism or known base
Preferably, it is obtained from the sequence. The probe sequence has a natural counterpart (c
It is preferable that the material does not have an outerpart). Sand
In other words, the probe sequence is heterogeneous, and many residues of the same sequence
In each of the most commonly occurring parts, the base is identical.
May be a column. The same sequence, no matter how naturally
A more stable hybrid than one of the offending sequences
Can be generally formed. Probes can be added
Made by covalently linking various nucleotides
Even synthetic oligodeoxyribonucleotide molecules
Good. Synthetic molecules, for example,
(Alvardo-Orbina et a)
1, Science 214: 270-274 (198
Prepared in 1)). Probe molecules of any size
It is useful, and only one or more sequences need be present in the probe solution.
For example, several 20-base sequences are highly expressed in rRNA genes.
Used to detect several stored parts. It
Is detectably labeled or labeled
Are not marked, but are marked so that they can be detected.
It is desirable to be informed. In this case, the nucleic acid
Lobes are detectable labeled RNA, preferably notched
Translation labeled DNA, cloned DNA or probe organism
Detectable DNA (cDN) that is complementary to RNA from
A) is a highly conserved DNA sequence information
Everything that includes information. Synthetic oligodeoxynucleotide
Otides are prepared with detectable labeled nucleotides
The molecule incorporates labeled nucleotide residues
Labeling. Depending on the choice of combination, professional
Can be either prokaryotic or eukaryotic (cytoplasmic,
Or from organelles). Most preferred
Indicates that the detectable label is a radioactive label such as radioactive phosphorus.
(E.g.,³²P,^ThreeH or¹⁴C) or bio
Based on tin / avicin (biotin / avidin
-Based) system. Nucleic acid probe is a metal source
It may be labeled with a child. For example, uridine and sisi
Nucleotides can form covalent mercury derivatives
it can. Nucleoside triphosphate bound to mercury is reverse-transcribed
Good substrate for many nucleic acid polymerases, including enzymes
(Dale et al, "Proceedings
of the National Academy o
f Sciences "70: 2238-2242, 1
973). Mercurization by direct covalent bonding of natural nucleic acids reported
Was told. (Dale et al, "Biochem
Istry "14: 2447-2457). Mercury polymerization
The rear annealing of the body is
Similar to that of the corresponding mercury-free polymer (Dale
  and Ward, "Biochemistry" 1
4: 2458-2469). Metal-labeled probes are examples
For example, light-hearing sound spectroscopy, X-ray spectroscopy
Peaks, eg X-ray fluorescence, X-ray absorption or photon spectroscopy
It can be detected by copying. Isolation of Probes Containing the Desired Conserved DNA Sequence
And preparation are within the skill of the art. For example, eukaryotic cells and
Isolation of rRNA from prokaryotic and pronuclear cells is well known to those skilled in the art.
Have been. Thus, the eukaryotic cytoplasmic ribosome converts rRN
In order to prepare A, RNA must be prepared in whole cells or ribosomes.
From sucrose and separated by sucrose gradient centrifugation
And the 18S and 28S fractions have molecular weight
Can be collected using known markers (eg, P
erry, R .; P. And Kelly, D.M. E. FIG. , Author
"Low production of 28S and 18S ribosomal RNA
5S RNA when inhibited by actinomycin D
Sustainable Synthesis of " Cell. Physiol. , 7
2: 235-246 (1968), herein incorporated by reference.
Marked). As a corollary, the organelle-derived rR
NA is separated from organelle fractions in a similar manner.
And purified (eg, Van Etten RAE)
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
If this is the case,
Grown in a nutrient or culture medium containing such compounds
Alternatively, it is separated from the cultured probe organism. Plow
If the DNA is complementary DNA (cDNA)
Converts RNA isolated from probe organisms to radionuclides
Reoside triphosphate (for example,³²P-nucleoside or
^Three(H-nucleoside) by reverse transcription.
Made The labeled probe may also be a notched translation DNA
Molecules, especially those obtained from all-circular DNA from organelles
It may be. Specifically, the chloroplast or filamentous ring
DNA is incised and translated in the presence of a radiolabel
Thus, a labeled DNA probe is obtained. Chloroplast signs
Probe best hybridizes with chloroplast DNA
However, the nucleolar-labeled probe is best associated with nucleolar DNA.
Will hybridize. Chloroplast (or filament)
The incision translation-labeling probe is a globule (or chloroplast)
It will hybridize the second best with DNA. So
Although this is generally less preferred, whole plants
Will also hybridize with the DNA of the product (or animal)
U. This method is a good choice for practical use.
Cut even from eukaryotic cell nuclear DNA
May be obtained by embedded translation. Achieve this
A more useful approach is to eukaryotic cells from nuclear DNA.
Cut the conserved gene (by restriction enzyme) and separate the fragments.
To identify the gene sequence (by hybridization)
Then, the gene sequence is separated (by electrophoresis).
T) that is. Then the separated sequence is a plasmid or
Is religated into another vector and transformed into a suitable host.
After (transformation)³²In P-containing medium
The cloning may be carried out. Another method is the trait
Propagate the transformed host, then isolate the DNA, cut it and invert it.
Label it by translation or isolate the DNA,
The sequence is cut out and then labeled. The obtained riboso
Probe hybridizes in the same state as the cDNA
(See below). Preferred nucleic acid probes are probe organisms
Is radiolabeled DNA that is complementary to RNA from E. coli.
The RNA is usually a messenger that decodes a conserved gene.
Jar RNA, substantially carrying DNA (tRNA)
Or (unless rRNA is used) ribosome R
It does not include other RNAs such as NA (rRNA). if
If rRNA is used, prokaryotic rRNA is usually 3
Includes sub-types. So-called 5S, 16S,
And 23S-fragment. Three types of reverse transcription to cDNA
Performed on all mixtures or otherwise 16S and
And a mixture of 23S fragments. These rRNA components
Performing reverse transcription with only one of them is possible under certain conditions
However, it is not very desirable. Eukaryotic rRNA
Usually has two subgroups, 18S and 28
S is included. And reverse transcription to cDNA is 18S and 2
Performed by a mixture of 8S fragments or each of these
You. Pure, almost free of other types of RNA
That can reverse transcribe rRNA into cDNA
Incubate with transcriptase. Preferred in this case
Primers such as calf thyroid DNA hydrolyzate
In the presence of avian myeloblastosis virus (AM
V) by incubating with reverse transcriptase
You. The mixture contains the appropriate deoxynucleoside triphosphate
And at least one of the nucleosides
One is, for example,³²P is radioactively labeled
You. For example, deoxycytidine 5 '-(³²P), deoki
Cythymidine 5 '-(³²P), deoxyadenine 5 '-(
³²P) or deoxyguanidine 5 '-(³²P) · Mirin
Acids are used as radionucleosides. 30 minutes to 5
And incubated at 25 ° C-40 ° C for
And phenol extraction and centrifugation
After chromatography, combine radiolabeled fractions
And use it as a cDNA probe. Substantially pure type protection
Radiolabeled cDNA probe containing DNA information
CD complementary to other types of RNA without unlabeled molecules
No NA, no proteinaceous material, cell formation of membranes, organelles, etc.
Unclear radiolabeled cDNA probes are also one of the present invention.
Make up the surface. A preferred probe is labeled c for prokaryotic cells
DNA, most preferably bacterially labeled cDNA.
You. The species of the probe is a bacterial microorganism such as Enterova
Enterobacteria (Enterobacteriacea)
e), Brucella, Bacillus (Ba)
Cillus), Pseudomonas (Pseudomon)
as), Lactobacillus (Lactobacillus)
s), Haemophilus,
Microbacterium
m), Vibrio, Neisseria (Nei)
sseria), Bactroides
s) species included in the family, and other anaerobic groups, such as
Legionella or the like is used. Book
Examples of prokaryotic cells in the application are bacterial prokaryotic probes
Although limited to the use of E. coli as an organism,
It is by no means limited to this microorganism. With probe
The use of radiolabeled forms of cDNA
Due to the greater stability during bridging, the radiolabeled R
Preference is given to using NA. When the labeled cDNA probe is
Must be a copy, i.e.
All nucleotide sequences of the template RNA are transcribed
It is important to recognize that this has been done. Plastic
The use of immers is essential in this regard. cDNA is faithful
Being a copy means that after hybridization
Prove by the fact that it has two properties
You can: 1. cDNA is 100% ribonucleic acid of labeled rRNA.
Must protect against ase digestion; and 2. The labeled cDNA shows resistance to S1 nuclease
Anneals specifically to the rRNA,
There must be. Belgian ski M. M. et al., “CR Acad Sc
  Paris "t286, Series D. p. 1825-
1828 (1978), derived from E. coli rRNA^Three
H-radiolabeled cDNA has been described. This
The cDNA in the study of
Not prepared with reverse transcriptase in the presence of
Lease U_TwoRRN previously divided using
A prepared as a template with DNA polymerase I
It is. The rRNA digestion product of Beljansky et al. (R
NA se U_TwoDiffers from the first rRNA
Base ratios, bases and / or short fragments are lost
It shows that. CDNA obtained in this manner
Is not a faithful copy. And Belgian skis
The use of DNA polymerase I
Stimulate the superiority of homopolymerization transfer over polymerization transfer
(See, Sarin PS et.
al, "Biochem, Biophys, Res. C.
omm. 59: 202-214 (1974)). To summarize these, the probe is a) an example
Genomic DNA containing conserved sequences, such as genes
By cloning and / or incision translation,
b) from RNA itself or c) from cDNA
A is obtained by reverse transcription. Typically, the next step in the process of the present invention is
Unlabeled DNA digests isolated from known organisms
Or (preferably) radiolabeled RNA or DN
Hybridization with the A probe. Hybridization
Is used to remove covalently labeled DNA from unknown organisms.
Of the hybridized paper containing the probe
This is done by contact with the compound. Incubation
The heating is performed over a long period of time under heating (50-70 ° C).
Afterwards, wash the filter paper to remove unbound radioactivity (necessary
If not, then air dry and prepare for detection. Already
One, much faster than the above method, very preferred
A new hybrid is described in Corn D. E (Kohne,
D. E) et al., "Biochemistry" 16:53.
29-5341 (1977).
Room temperature phenol emulsion repair method. After hybridization, the method is appropriately
Selective detection of hybridized fragments is required. This
Detection indicates that the hybridized fragment has a double-stranded
And the selective method (unlabeled pro
Radiograph or computer
It may or may not be
A suitable radiation scanner method that will increase exit speed
(In the case of a labeled probe).
These methods are well known to those skilled in the art,
I won't go any further in this regard. The end product of this method is that various
Intensity peaks and valleys, or preferably bright and
Chromatographic band pattern with dark areas
Regular gene characterization. These positions are EcoR
Of a marker such as I digested lambda bacteriophage DNA
By subjecting the separation method to a specific fragment size (kg
Base pair) can be easily matched. like this
Easy relative position of bands and absolute size of each band
Can be confirmed. Genetic features for identification of unknown organisms
Compare the signature with the characterization in the catalog or library
Compare. At least 2 for catalogs or libraries
Of almost as many as infinite
Consists of books that list the genus and species characteristics of organisms
May be. For example, the pathological consequences of human disease
The number of bacteria involved is estimated to be about 100, and
The standard catalog of fungi has 50-150 such characterizations
It is estimated to contain. For epidemiological judgment system
A catalog of bacterial strain types may also be included. Characterization is the chosen endonuclease enzyme
Depending on the type or types of
Specific source used as the source of the probe (probe organism)
Storage used in organisms and also for probe preparation
DNA sequence information The composition of the nucleic acid (eg, 5S, 1
6S or 23S type subtype or 16S and 23S type
Only or the matching sequence). Thus,
The catalog contains the recorded band size and
Includes various enzyme-specific characterizations with relative strength
Maybe. The concentration of bound DNA bound to the filter paper decreases.
Only the strongest band becomes visible,
Alternatively, the species can be identified by the size of these bands. The above changes or permutations, of course, are all live
Used for rallies. Moreover, for eukaryotic organisms,
The library is a type of DNA, or organelle,
And / or the use of each-DNA combination
May contain various patterns. Of each DNA digest
The turn will depend on the probe composition. The catalog is
If one or more strains or species are present in the extracted sample
And if detected by the probe, then
It is organized so that it can interpret the cunning characterization. You
The user can use the resulting characterization, for example,
Can be compared to
One-dimensional computerized digital scanner
Can also be compared. These computers
Tarscanner is a time-of-sale (time-sale)
of-sale) processing (a commonly used "superma
Checkout barcode or pattern reading
It is well known to those skilled in the art of As an ideal
Is the library or catalog relative to multiple organisms
The characterization and the absolute value of the molecular weight or size of the fragment
Should be in pewter memory. so
If possible, the catalog comparison should include one or both of the storage information elements.
(Relative characterization and / or absolute size factor)
This means that unknown characterizations are
This is done by matching one of the Compare with standard
The intensity of each band at the time of
The extent of the organism,
For example, to estimate the presence of prokaryotic cells in eukaryotic cells
Can be used. If the user is given a property of an organism
If you want to confirm the
User can transform an unknown organism into a second different endonucleus
Digested with ase, the resulting characterization was chosen second
Catalog characterization of organisms for endonucleases
Compare with. This process is for accurate identification
Can be repeated as many times as necessary. But
Usually, one analysis with a single probe is usually sufficient
Is enough. The invention and its variants are infinitely applicable.
You. The present invention allows plant growers or animal breeders to
May be used to confirm
Present in any medium, including eukaryotic cells, in biology laboratories
May be used to identify bacteria, parasites or fungi that
No. In this latter case, the method is compatible with standard microbial assays.
Used as Because of the isolation and growth of microorganisms
It is not necessary. In vitro
Growth and characterization are currently
Plastic (Mycobacterium leprae)
For some microorganisms such as (Leprosy pathogens)
Possible and inevitable intracellular bacteria (such as ricketters,
Some microorganisms (such as gramida) on standard media
Impossible or very dangerous if not impossible
(Eg, B. anthracis)
(Anthrax pathogens). The method is based on the isolation of nucleic acids.
Does it perform conventional bacterial isolation and characterization?
These problems can be eliminated. This method is
To detect microorganisms that have not been formally described before
Seems to be able to. In addition, the method uses different strains
Which can be identified, for example, by epidemiology in bacteriology.
Useful for typology. This method is used in criminal investigations for plants or animals.
Forensic practices should be used to correctly and clearly identify tissue
Can be used in the laboratory. Also check the nature of crop damage
Entomologists should be able to identify insect species quickly.
Is also used. Further, the method is applied to taxon groups other than subspecies (for example
Plant root nitrogen enzyme gene; see: Hennecke H
291 "Nature" 354 (1981))
By linking, this methodology has left individual strains
It can be used to investigate and identify genotypes. [0078] The method of the present invention can be used to detect microorganisms.
It is preferably used in any place for the identification of microorganisms. This
These microorganisms can be found in both physiological and non-physiological
Will be found. They are industrial growth media, cultured meat
Found in juice, etc., and concentrated, for example, by centrifugation
Will be made. Microorganisms are found in physiological media
Is preferred and it is found in infected animal sources
Is most preferred. In this latter example, the method applies to animals,
Preferably used to diagnose bacterial infections in humans
Can be Detection and detection of bacterial DNA with pronuclear probes
Is highly selective, and the presence of animal (eg, mammalian) DNA
Can be done without obstacles even in the presence. If pronuclear pro
If hybrids are used, hybridization with the nucleolar DNA
Choose the condition that minimizes the
Can be deducted. In this way, this technology is
For use in floor laboratories, fungal depositors, industrial fermentation laboratories, etc.
Can be. Of particular interest are the species of infectious microorganism and
In addition to strain identification, some special genetics
The possibility of discovering the existence of a child sequence. An example
For example, on the transmissible plasmid R factor that mediates drug resistance
Can detect the presence of antibiotic resistance sequences found in
it can. Labeled factor R DNA or clone labeled antibiotic
By adding the resistance sequence to the hybridization mixture
To determine whether the organism is resistant to antibiotics.
(One or several bands that are not regular appear.
). In addition, the added antibiotic resistance sequence probe (1 or several
Re-hybridizes the filter paper once hybridized in the presence of
It may be lidded. Alternatively, unknown DNA
Into aliquots and identify the first aliquot for identification
Next, a second aliquot was added for the presence of the drug resistant sequence,
A third aliquot is used for the toxin gene
Can also be tested. Alternatively, one radiation
Nuclides (for example,³²Includes conserved genetic information labeled in P)
Probes can be connected to different^ThreeH or¹⁴C)
Hybridization mixing with intelligent R-factor probe
It could be used in things. After hybridization,
The presence of R-factor DNA in unknown DNA is determined by two types of scans.
It can be tested by scanning with a channer. One is a seed
And for strain identification (eg,³²P), the other one
Is for drug resistance etc. (for example,^ThreeH or
¹⁴C). This method allows the experimenter to isolate and characterize the microorganism.
Identify genera and species without straining and strain strains
Classification, drug resistance, toxin production or other properties, or
Is the species level detectable by the labeled nucleic acid sequence or probe
All of the following taxon tests can be performed in a single experiment.
Can be. The R-factor is universal and intersects species boundaries.
Identification in any bacterial genus or species
This can be done with the same R-factor probe (see: To
mkins, L .; S. et al. J. Inf. Di
s. , 141: 625-636 (1981)). Further, in eukaryotic cells or prokaryotic cells,
The presence of virus or virus-related sequences may also affect the method of the invention.
It can be bound and detected and identified. "Manu
alof Clinical Microbiology
y "Third Edition (Publisher Lenette, EH Ame
r. Soc. Microb. , 1980, 774-77
Identify all viruses listed in 8)
Can be. For example, Picornaviride
aviridae), caliciviride
iridae), reovirida
e), togaviridae, ol
Orthomyxovirida
e), paramyxoviri
dae), rhabdovirida
e), retroviridae,
Arenaviridee, corona
Coronaviridae, Buniavi
Bunyaviridae, pub-o-vilide
(Parvoviridae), papovaviride (pa
povaviridae), adenoviride (adeno)
viridae), herpes vide (herpesv)
iridae), vidovirid
ae) and poxviridae
etc. A) The viral genome is integrated into the host DNA
(For example, DNA viruses such as Papo Villide member)
ー, RNA viruses, for example, RetroVide members)
Extracts high molecular weight DNA from tissue and
To digest. The overall procedure is the same as for bacteria. C
The choice of an eelsogenic probe is again required.
"Probe viruses" and detection
Depends on the degree of homology between the virus-related sequences to be done
You. To obtain convenient sequence homology, use probes and
And the organization of the tissue relates to the same family or species of virus.
It is necessary to be. In addition to the degree of stored sequences
The virus probe is related to the virus of the host DNA.
Hybridization with the sequence is determined by hybridization.
Conditions, such as stringent conditions
It will depend on whether it is a Lux condition. Hybrid
The result of the hybridization was the virus incorporated into the host DNA.
A single band or band pattern indicating the presence of a sexual arrangement
right. This information helps predict carcinogenesis. Cloning
Any of labeled complementary nucleic acid probes containing viral sequences
These can also be used as probes. RNA viruses
For example, reverse transcriptase using viral RNA
DNA can be made from DNA, and in the case of DNA viruses
For example, wheels labeled by incision translation
DNA can be used. Again multiple pros
Probes, especially with differently labeled probes.
Wear. The same general features apply to DNA and RNA
Equally applies to Rouss. Virus genome is relative
Small, and sedimented nucleic acids are preferably collected by centrifugation.
Good. All operations may be performed on all nucleic acids,
Each operation may be performed separately. Before centrifugation,
By removing cell DNA by spooling,
It is believed that viral nucleic acids can be concentrated. this
Controls whether the virus genome is integrated.
Can be used to read. Hybridizing a virus probe
To do so, the probe must be of the same family, genus or
It must be a seed and at least most preferred. reaction
Conditions, stringent or relaxed, given
Hybridization between probe and unrelated genome
Decide whether to do it. Probes are labeled claw
May be a virus sequence or a complete genome
Or may be part of it. The method described in Southern described above is
DNA fragments (about 0.5 kilobases or more)
Useful for transfer to nitrocellulose paper after denaturation
is there. This method is useful for DNA viruses,
Maybe useless in the case of RNA viruses
No. Activate RNA on cellulose paper (diazobenzyloxy)
(Methyl methyl paper) and covalently bonded, and
Can be used for RNA viruses. Sau
Thomas' modified version of Zahn's method (Thomas, P.,
"Proc. Nat. Acad. Sci" USA 7
7: 5201-5205 (1980))
Small DNA fragments for hybridization.
Can be used to transfer efficiently to low cellulose paper
You. RNA and small DNA fragments are
Denatured with dimethylsulfoxide and agarose gel.
And electrophoresis. By this operation, 100-2000nu
DNA fragments and RNA that are nucleotides are efficient
To nitrocellulose paper during hybridization
I do. This is true even for small ribosomal DNA fragments.
Useful. Therefore, it is most preferable to use restriction enzymes.
The digested sample is divided and some of its nucleic acids are
It is to denature with Saar. Southern and Toma
The operating procedure will give the maximum amount of information. B) In the case of DNA viruses, the double strand (D
S) Existence by performing restriction analysis on viral DNA
Existing viruses can be identified. Single chain (S
S) DNA viruses have genomes of different lengths
right. Probes that form hybrids (sequence information is
DS-DNA), forming a hybrid
Fragment pattern and / or one or more sizes
Can be used to identify viruses. Again, complementarity
There are a number of ways to obtain nucleic acid probes. For example, DS-
In the case of DNA, incision translation can be used.
In the case of S-DNA, the DNA polymerase is cDNA
Used for synthesis. C) In the case of RNA viruses, RNA is controlled
Not digested by restriction endonuclease (sequence information
May have been converted to DS-DNA). Different RN
The A virus genome has different sizes and some R
There are more than one molecule in the genome of NA viruses.
This allows certain probes or union probes to
RNA viruses along the detected nucleotide sequence
Can be specified. One example of a probe is the Virus R
This is a cDNA synthesized using NA. When searching for infectious pathogens in a specimen,
By extracting nucleic acids from the book, or
Increase the number of pathogens by culturing in cells, or use centrifugation.
Use an enrichment process or try all approaches
You can search directly by looking. From the present invention, it is necessary to carry out the method.
It is easy to create a “kit” containing the essential elements.
You. Such kits may include test tubes or vials
One or more containers can be packed tightly in it
It would consist of partitioned carriers. The above container
One is an unlabeled nucleic acid probe or, for example, an organism probe.
Like radiolabeled cDNA for RNA from a probe
Detectable labeled nucleic acid probe (to identify bacteria
Prokaryotic cDNA is more preferred for
) May be included. Labeled nucleic acid probe is frozen
In dry form or, if necessary, in a suitable buffer
Would. One or more containers contain DNA from unknown organisms
One or more endonucleases used for digestion
Will contain the enzyme. These enzymes alone or
The mixture can be in lyophilized form or in a suitable buffer solution.
Exists. Enzymes used in the kit are
Ideally an enzyme whose catalog is prepared
It is a target. But users compare themselves during the experiment
Nothing prevents you from creating a standard, so if you
That the unknown is actually a given genus or species
If you suspect that a person is
Create an identified characterization and identify it with the characteristics of the unknown
Compare it with the sign. In this way, the kit
Include all the elements necessary to perform the
No. These elements are one or more known organisms (such as bacteria).
Or DNA isolated from known organisms. So
In addition to the kit, the kit may be widely used as a booklet,
Catalog or computer tape defined as
Or disk or computer access number
Including plant species, mammalian species, bacterial species,
Certain groups such as pathologically important bacteria, insect species, etc.
Incorporates the identified characterization of various organisms. this
In this way, the user prepares the characterization of the unknown organism,
This can be visually (or computer-
Just compare them. The kit also comes with one
A probe RNA for probe synthesis is already contained in the vessel.
Radioactively labeled deoxyribonucleoside in one container
And triphosphoric acid, and a primer in another container
May be included. In this way, the user
You can make a probe cDNA. Finally, the kit includes a buffer, a growth medium,
Element, pipette, plate, nucleic acid, nucleoside / triphosphorus
Acid, filter paper, gel material, transfer material, autoradiograph
Required to perform the technique of the present invention, such as fee replenishment
All of the additional elements may be included. It is also
Antibiotic resistance sequence probe, virus probe or its
Probes with other specific properties may also be included. While the invention has been described generally above,
The present invention has been described with reference to certain reference experiments and examples.
Will be better understood. Note that the embodiments are merely described.
It is described only for the purpose of and is not specified
There is no intention to limit the present invention as far as it is possible. [0091]Materials and methods A. Bacteria Extraction of high molecular weight DNA The bacterial broth culture was spun down and the cells were washed with cold saline. So
Gram weight of packed cells
Approximately 10 times the volume of the extraction buffer (0.15 M
Sodium, 0.1M EDTA, 0.03M Tris
  pH 8.5). Lysozyme 10mg / ml
It was added to a final concentration of 0.5 mg / ml. 37 suspension
Incubated at 30 ° C for 30 minutes. Cell destruction is 25
%, SDS to a final concentration of 2.5%,
For 10 minutes to 60 ° C. water
After cooling in the bath, the mercaptoethanol is brought to a final concentration of 1%.
Added to be. Pronase^TM20mg / ml 0.02M
Predigestion of Tris buffer (pH 7.4) at 37 ° C for 2 hours
And then added to a final concentration of 1 mg / ml. So
Was incubated at 37 ° C. for 18 hours. Pheno
1 l of redistilled phenol, double distilled water
5 l, 270 ml of saturated tris base, mercaptoethanol
12 ml and final concentration 10^-3The amount of EDT to be M
A and mixing at 4 ° C. to separate the mixture.
Prepared. The phenol was washed with a washing buffer (10^-1M
Sodium chloride, 10^-3M EDTA, 10 mM Tris pH
8.5). Then add an equal volume of fresh buffer
Was. Mercaptoethanol to a final concentration of 0.1%
Added. The solutions were mixed and stored at 4 ° C. Prepared
Lyse cell solution with half volume of phenol and half volume of chloroform
Added. Shake this for about 10 minutes and at 3400 × g
Centrifuged for 15 minutes. Remove aqueous phase with 25mg glass pipette
did. This extraction procedure is performed until there is almost no sediment at the boundary.
I repeated the work. 1/9 volume of 2N sodium acetate (pH
5.5) was added to the aqueous phase. Double volume of -20 ° C 95%
Slowly pour chilled alcohol along the wall of the flask
No. Melt the tip of the Pasteur pipette, close and settle
Used to spool DNA. High molecular weight DNA
Dissolved in buffer (10^-3M EDTA, 10^-2M to
Squirrel pH 7.4). DNA concentration is the absorbance as a conversion factor
Absorbance at 260 nm, using 30 μg per unit
It was measured in degrees. [0092]Restriction endonuclease digestion of DNA The EcoR I restriction endonuclease reaction was 0.1 M
Tris-HCl pH 7.5, 0.05 M NaCl,
0.005M MgCl_TwoAnd 100 μg / ml calf blood
Made in Qing albumin. The EcoR I reaction mixture is
Contains 5 units of enzyme per DNA / μg,
Incubated at 37 ° C. for 4 hours. PSTI restriction
The nuclease reaction is 0.006 M Tris-HCl pH
7.4, 0.05M sodium chloride, 0.006M chloride
Magnesium, 0.006M 2-mercaptoethanol
And 100 μg / ml calf serum albumin
Was. The PSTI reaction mixture was duplicated per DNA / μg.
Incubate at 37 ° C for 4 hours
did. Normally, 10 μg of DNA is consumed in a final volume of 40 μl.
Was A 10-fold concentration of buffer was added. Sterile distilled water
Added depending on DNA volume. λ-bacteriophage D
NA creates marker bands for fragment sizing
Therefore, it was restricted by EcoRI. Usually 2 μg λ DNA is
Digested with 20 units of EcoRI to a final volume of 20 μl
Became. [0093]Gel electrophoresis and DNA transfer Add up to about 20% glycerol and broth to the DNA digest.
Mophenol blue dye was added. λ DNA digest
In this case, 20 μl of 1 × EcoRI buffer was added to each 20 μl.
  Added to the reaction mixture. Normally 75% glycerol 15
4 μl and 5 μl of 0.5% bromophenol dye
Added to 0 μl reaction mixture. Digested bacterial DNA 10μ
g and 2 μg of digested λ DNA
r well), and cover the surface with agarose
U. Digests 0.02M sodium acetate, 0.002M
  EDTA, 0.018 M Tris base, and 0.02
0.8% agarose containing 8M Tris HCl pH 8.05
Medium, 35V, electrophoresis until dye migrates 13-16cm
Movement. Afterwards the gel is ethidium bromide
(0.005 mg / ml) so that the λ fragment can be seen.
Placed in a UV light box. DNA from Southern
And transferred to nitrocellulose filter paper. Gel vibrates
Denaturing solution (1.5 M sodium chloride,
0.5M sodium hydroxide). Medium denaturing solution
Solution (3.0 M sodium chloride, 0.5 Tris HCl pH
7.5), and after 40 minutes, check the gel with pH paper.
I checked. After neutralization, the gel was washed with 6 × SSC buffer (SSC =
0.15 M sodium chloride, 0.015 M sodium citrate
(Lium) for 10 minutes. Gel and nitrocellulose
6 x SSC with a bundle of paper towels
The DNA fragment is removed from the gel
Transferred to Lurose paper. Between two 3mm chromatograph papers
Put the filter, and with the aluminum wheel, shine the outside
And dried in a vacuum oven at 80 ° C. for 4 hours. [0094]Synthesis of ³² P ribosomal RNA complementary DNA
( ³² P-rRNA and DNA) E. coli R-13 23S and 16S ribosome RN
Complementary to A³²P-labeled DNA was used for avian myeloblastosis
Synthesized using reverse transcriptase from Lus (AMV). Anti
The reaction mixture contained 5 μl of 0.2 M dithiothreitol,
25 μl 1 M Tris pH 8.0, 8.3 μl 3 M chloride
Potassium, 40 μl of 0.1 M magnesium chloride, 7
0 μg actinomycin, 14 μl 0.04M d
ATP, 14 μl of 0.04 M dGDP, 14 μl
Contains 0.04M dTTP, and 96.7 μl of water
Was out. The following were added to the plastic tube:
137.5 μl of reaction mixture, 15 μl of calf thorax
Immer (10 mg / ml), 7 μl H20, 3 μl rR
NA (40 μg / OD Using unit concentration 2.76
μg / μl), 40 μl of deoxycytidine 5 ′
− (³²P) Triphosphoric acid (10 mCi / ml), and 13 μl
AMV polymerase (6,900 units / μl). Enzyme anti
The reaction was performed at 37 ° C. for 1.5 hours. Then the solution
Extract with 5 ml of chloroform and prepared phenol
Was. After centrifugation (JS13,600 RPM), the aqueous phase is
Contact Sephadex^TMG-50 column (1.5 × 22cm)
Laminated on top. Empty plastic 10 ml pipette
Used as a system. Place one small glass bead at the tip
And a rubber tube with pinch fittings
Degassed G-50 swelled by immersion in 5% SDS was added. water
The phase is poured directly along the wall into the G-50, then
Eluted with 05% SDS. 20 ml of 0.5 ml each
Were collected in plastic vials. Peak fract
The tube containing the^ThreeEach sample using H-discriminator
Count for 0.1 minutes each time, and record the total count.
Discovered by Nkov measurement. Combine peak fractions
Was. Aliquot aquesol^TM(Available commercially)
Well, per 1ml³²Cinch of P's CPM (counting every minute)
It was measured by a ration counter. [0095]Hybridization and autoradiograph
Eee Filter the fragment containing the ribosomal RNA gene sequence into a filter
The above DNA³²Hybridization to P-rRNA cDNA
After that, detection was performed by autoradiography. H
The filter was mixed with the hybridized mixture (3 × SSC, 0.
1% SDS, 100 μg / ml denatured and sonicated
Dog DNA and Dynehardt solution (0.2% each)
Calf serum albumin, ficoll,
And polyvinylpyrrolidine)) at 68 ° C for 1 hour
Soaked.³²4 × 10 PrRNA cDNA⁶CPM / ml
And the hybridization reaction solution is allowed to stand at 68 ° C. for 48 hours.
Incubated. Then filter 3xSSC
And then with 0.1% SDS at 15 minute intervals for 2 hours or
Is about 3,000 cpm of washing solution³²To contain P / ml
I washed it. Air-dry the filter and remove the plastic
Wrap in plastic wrap and use Kodak X-OMATR film for -7
Autoradiography was performed at 0 ° C. for about 1 hour. [0096]B. Mammal experiment RR of Mus musculus domesticus (mouse)
NA probe is 18S and 28S type and 28S type
Was synthesized from fresh rRNA. Nucleic acid extracted from mouse liver
Settled. High molecular weight DNA was spooled and removed.
The remaining nucleic acid was collected by centrifugation, and 50 mM MgCl_TwoAnd
And 100 mM Tris pH 7.4 buffer. DNA
se (excluding RNAse) to a concentration of 50 μg / ml
Added. Incubate the mixture at 37 ° C for 30 minutes
did. The resulting RNA is re-extracted, ethanol precipitated,
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. Add the sample and
Gradient at 35K RPM for 7 hours with SW40 rotor
I let it. Fractions were collected by optical density. well-known
Compared with molecular weight markers, 18S and 28S
Action. Relax in all mammalian experiments
(Relaxed) hybridisation conditions were used. 54
° C. The washing process is performed at 54 ° C, and 0.05% SDS is added.
Washed three times for 15 minutes each in 3 × SSC. In Reference Experiments 1 to 8, the rRNA information-containing
The experiments performed in the lab are described. In Examples 1 to 3,
Histone gene information containing probe, tryptophan operation
Ron trpD gene information containing probe and α-fe
Compile using probe containing gene information
Computer simulations are described below. [0098]Reference experiment 1 The bacterial species is a restriction endonuclease in the ribosomal RNA gene.
Determined by Lease analysis. Used in this experiment
P. Several strains of Aeruginosa have been
Has few phenotypic characteristics (Hugh RH, et.
al. "Manual of Clinical Mi
crobiology "2nd edition, ASM, 1974, p.
p, 250-269) (Table 2). Three other pseudomos
Eggplant and two Acinetobacter (Acineto
Bacter strains were selected and the species and genera were compared (Table
3). [0099] [Table 2] Pseudomonas and Acinetobacter species
The strains used for the comparison are listed in Table 3. [0101] [Table 3] To compare Acinetobacter species with genera
I chose because they have certain attributes
This is because they are shared with other species. The size of the fragment in the EcoRI digest (K
(Base pair) P. stutzer
i) 16.0, 12.0, 9.4; Fluoressen
(P. fluorescens) 16.0,10.
0, 8.6, 7.8, 7.0; Putida (P. put
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 smallest three fragments are
Not calculated); 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 (do not calculate the size of the three smallest fragments)
Was). Fragment size in PSTI digest
(Kilobase pairs) are shown below; Stowzeri6.
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; Anitratas36.
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. Obtained from 7 strains of Aeruginosa
Comparing the hybridized restriction fragments, this species
Is based on 10.1, 9.4, 7.6 and 5.9 kilobases
Ec of fragment containing rRNA gene sequence of pair (KBP)
oR I-defined by a specific combination
(Fig. 1). The 7.6 KBP EcoRI fragment was obtained from
In the sample, it appears in 4 out of 7 strains. A similar situation is the seed
It also occurs among some phenotypic traits of the strains. 7 strains
EcoRI combination of fragments from
The fact that it is used to separate Aergi
The assumption that there are two species with the minimal phenotypic characteristics of Noza
To argue. Experimental results of digesting DNA with PSTI
(FIG. 2) shows that the EcoRI 7.6 KBP fragment
Conclude that mutations in strains indicated in
Lead the argument. Because one conserved set of PSTI fragments
Combined, 9.4, 7.1, 6.6 and 6.4 KBP
For there is a seed. 9.
The PSTI fragments of 4 and 6.6 KBP were obtained from P. Ael
Appears in 6 out of 7 strains of Guinosa; 7.1 and
The 6.4 KBP PSTI fragment was used for all strains
Appears. Mutation of the PSTI fragment was performed by EcoR
Occurs on strains that do not contain the I7.6KBP fragment; RH15
1 has the 10.1 and 8.2 KBP fragments and RH80
9 does not contain the 9.4 KBP fragment, but the 6.0 KBP fragment
Have. And RH815 of the type strain is 6.6.
Contains no KBP fragment. Of the hybridized fragment
The pattern is determined by the conserved combination specific to the enzyme.
Support the conclusion that it can be used for One species of fungus
Strain probably has a large number of fragments as conserved combinations
Will be. Even if some strains have a fragment mutation,
Do not interfere with identification and may be useful for epidemiological studies.
It can be said that it proves. P. EcoR in Aeruginosa strains
  Mutation of the I7.6KBP fragment was
Hybridization Ec found in eggplant type strains
Perspective by examining oRI fragments
It may be (Figure 3). P. Stotueri, P.S. Fluo
Lessens and P.L. The type strain of Putida is 7.6KB.
An EcoRI fragment of the same size without the P fragment
Have in common; Aeruginosa and p. S
Eucalyptus has a 9.4 KBP fragment and P. Stotsuye
And P. Fluorescens has 16KBP fragment
And P. Fluorescens and p. Petida is 10KB
It has a P fragment. Generally, the size of a fragment is four sh
Unique to each type of Domonas species;
Each type of strain has a different size range
With fragments. These general descriptions are given in PSTI digestion.
The same is true for objects (Fig. 4). Four Pseudomonas species and two reeds
Pattern of each fragment of Netobacter bacterium or its I bacterium
When comparing strains, species of each genus are similar, but genus
Can be concluded. Two Acinetobacter
-The species is hybrid compared to the four Pseudomonas species
A larger range of fragments size. Escherichia coli, Bacillus thuringinensis (B
acillus thuringiensis) and
B. Can be obtained from B. subtilis
Without the help of a restriction enzyme map where
Do you cut the NA gene or the number of copies per genome?
Non-homologous furan between multiple genes or heterogeneous genes
There are Kings (flanking regions)
It is impossible to predict. Escherichia coli rRNA
The cRNA probe has some restrictions including rRNA gene sequence.
May not form hybrids with restriction fragments,
If so, this proceeds between the test organism and E. coli.
It reflects the presence of chemical distance or variance. rRNA
Arguing that this is not the case using the conserved nature of
Can be However these are any unknown
The advantage of having standard probes that are equally applicable to species
This is a small problem compared to. [0109]Reference Experiment 2 Ratio between restriction analysis and DNA-DNA liquid hybridization
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.
Was. Labeling DNA of RH3021 and RH2990
Using liquid DNA-DNA hybridization data
Collected. Table 6 shows the results. [0113] [Table 6] This data has two hybrid groups.
Indicates that Similar data can be found in About Subtilis
(Seki et al, "I
international Journal of S
systematic Bacteriology "2
5: 258-270 (1975)). RH of these two groups
To be represented by 3021 and RH2990
it can. Restriction endonuclease of ribosomal RNA gene
When the enzyme analysis is performed, the EcoRI digest (FIG. 5)
Could be divided into groups. Represented by RH3021
Group has two strongly hybridizing fragments
(2.15 and 2.1 KBP). By RH2990
Group represented by two strongly hybridizing fragments
(2.6 and 2.5 KBP). EcoR I
B. using data. Subtilis strain is transformed into DNA-DNA
It can be placed in the appropriate place in the hybridized group. DNA
-According to the DNA hybridisation 70% rule, S
Butyris is actually two species. However, P
Considering the STI data (FIG. 6),
To two common ancestors or speciation events.
It can be considered a dispersed population. B. Subtilis
The conclusion that one species is correlated with phenotypic data
You. The strains listed in Table 5 are Gordon R. "T
he Genus Bacillus "Agricult
ure Handbook No 427 (U.S.A.
Ministry of Agriculture, Agricultural Research Services Washington, D.C. C. ) P
36-41. Subtilis has been identified. Limit
Analysis is comparable to DNA-DNA hybridization data
Data can be prepared and the appropriate enzyme can be selected.
By analysis, restriction analysis provides enough seeds despite variance.
Can be determined in minutes. RH3061 is a PSTI
Lost a kit. However, EcoRI data does not
Is a strain of B. Suggests that it is subtilis. Same thing
And BglII data (FIG. 7) and SacI data
(FIG. 8). [0115]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)
Bgl II data (Fig. 11 left) and Sac I data
(Right in FIG. 12). PST
Due to the large difference in the I-band pattern,
We can conclude that mosquitoes are in the wrong genus
You. Both species produce spores, but they have a phenotype
Not similar in nature. Culture of both ATCC and NRRL
Collection B. Polymica type strains have the same band pattern
It's true that they have But no important data
That is, spore mutants can be identified. if
Bacillus species if they cannot make spores
Identification is very difficult and probably impossible. [0118]Reference Experiment 4 Identify bacterial species in mouse tissues without isolation Swiss mouse, Mus musculus domesticus
(Mus musculus domesticus)
(Inbred strain) to Streptococcus pneumoniae
(Streptococcus pneumonia
e) Cloudy suspension of RH3077 (ATCC6303)
0.5 ml was inoculated intraperitoneally. The mouse is moribund
At that time, my heart, lungs, and liver were removed. High molecular weight DNA
These organizations, S.M. Pneumonia RH3077 and Switzerland
Eco isolated from mouse organ and digested DNA
Using RI, the restriction endonuclease of the rRNA gene
The operation of the enzyme analysis was performed. Wash the filter with 3 × SSC
0.3 × SSC and 0.05 × 2 × 15 min.
Washed with% SDS. 48 hours autoradiography
went. The data (FIG. 12) was 7 new moniers
Is determined by the hybridizing fragment of
(17.0, 8.0, 6.0, 4.0, 3.3, 2.
6 and 1.8 KBP). CDNA probe for this bacterium
Represents two mouse DNA fragments (14.0 and 6.8 KB).
Does not hybridize much with P). Hybrid type
The adult fragments are S. cerevisiae in infected tissues Know the existence of Nyumonie
It is what makes you. All 7 bands in heart DNA extract
Can be seen. Their strength is higher in liver DNA extracts
Small, but you can see everything by autoradiography
Can be. Only 6.0KBP band in lung DNA extract
Appears. The low number of bacteria in the lungs
Explain that you have sepsis rather than pneumonia
Can be. Lungs did not show solidification on culture. This
To determine the sensitivity of the assay, bacterial DNA was
Diluted with A and subjected to electrophoresis. 0.1 μg bacterial DNA
To see all seven bands on the autoradiograph.
I was able to. 10^-3For μg bacterial DNA, 17.
The 0, 8.0 and 6.0 KBP bands were seen. 10
⁶S. 5 × 10 per pneumoniae cell^-3μg DNA
If you use the numbers (Biochem Biophys
  Acta 26:68), 10^-1μg is 2 × 10⁷Fine
Equivalent to a vesicle. This method can be used to diagnose infectious diseases at this sensitivity level.
It is useful. In this reference experiment, the bacterial probe was
Hybridizing with specific EcoRI fragments
(See FIG. 9, 14.0 and 6.8 KBP).
Fragments). These fragments were derived from mouse 18S and 28S
Eco detected by a ribosomal RNA probe
Corresponding to the RI fragment (FIG. 14, said fragment was 6.8 KBP).
2 shows that the fragment contains a 28S rRNA sequence. ) Bacteria
The lobes are distinct from the mammalian ribosomal RNA gene sequence.
Belts are less intense because they do not hybridize well
Bacterial probe and nuclear mammalian DNA systems are sensitive.
For DNA of smaller, infected prokaryotic cells
The sex appears clearly. Bacterial probe was run in lane (la
ne) hybridized to 10 μg digested bacterial DNA
In the experiment, the bacterial band was clearly visible in one lane.
Per 10 μg digested human or mouse DNA
No lid formation was found. [0120]Reference Experiment 5-8 Mammal experiment These reference experiments are based on rRNA restriction analysis.
The concept of confirming that
Explain that it applies successfully to the aircraft.
You. FIG. 13 shows that the genus of mammals is Mus musculus.
・ Dometics 18S and 28S rRNA probes
And some species of Mus are distinguished
Show what you can do. In this figure, the enzyme is PSTI,
The objects and the respective bands are as follows. 1. Mus Musculus Merosinas
(Mus musculus melosinus)
(Mouse) 14.5, 13.5, 2.6 2. Mus musculus domesticus (mouse) 1
3.5, 2.6 3. Canis famil
iaris) (dog) 12.0 4. Cavia porcells
us) (guinea pig) 17.0, 14.0, 13.0,
One band of 8.8, 5.7, 4.7 and 3.0 or less 5. Crisetulus 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 norvegic
icus) (rat) 12.5 9. Mus musculus domesticus (mouse) 1
3.5, 2.6 10. Mus Servicoreau Servicoreau (Musc
servicecolor (mau)
S) 14.0, 2.7 11. Mus servicolope Papeus (Mus ser
vicolor papeus (mouse) 13.5,
2.6 12. Mus pahari (Mau
S) 13.0, 3.7 13. Mus cookie (Mus cookie)
Mouse) 13.5, 2.6 FIG. 14 shows that the DNA of mouse and cat was 28S.
Type rRNA cDNA alone, and
Pattern of hybridized and hybridized bands
It depends on the composition. In FIG. 14, the enzyme is EcoR
At I, the objects and bands are as follows. 1. Mus musculus domesticus (mouse)
6.8KBP 2. Ferris Catus (cat) 8.3KBP In FIG. 15, the enzymes are Sac I, the target and
The obi is as follows. 1. Erythrosebu Pasta
s patas) (Patus monkey) 8.5, 3.7, <3.
0 2. Ratus norvegicus (rat) 25.0, 9.
5, 3.6, <3.0 3. Mus Musculus Domesticus (Mouse)
6.8, <3.0 4. Ferris Katas (cat) 9.5, 5.3, 4.0,
<3.0, <3.0 5. Homo sapiens (human) 10.5, <3.0 6. Macaca mulatt
a) (Larus monkey) 9.8, <3.0 FIG. 5 (Sac I digestion) is compared with that of other mammals.
Hybridization pattern is specific to the enzyme
You can see that. FIG. 16 shows the behavior of primates.
Indicates that objects can be distinguished. Cultured cells are
The cells have a common band with the tissue.
Can be distinguished by crossing or chipping
You. In this figure, the enzyme is EcoRI and the target and
The bands are as follows. 1. Erythro Sebas Patas (Patus monkey)> 22.0,
11.0, 7.6, 2.6 2. 10. Makaka Muratta (Laser Monkey) 22.0,
5, 7.6 3. Homo sapiens (human)> 22.0, 22.0,
16.0, 8.1, 6.6 4. M241 / 88 (Langer monkey cultured cells)
0, 7.2, 5.7 5. HeLa (human cultured 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 [0126]Example 1 Use of H4 histone gene probe Identification and characterization of two animal species (sea urchin and mouse)
Computer simulation using H4 histone gene
This was performed using a conserved DNA sequence obtained from Sea urchin (Ps
ammechinus milaris)
The Ston H4 gene sequence is shown below. Where A, T,
C and G are represented by known nucleotides, and N is currently known.
Represents a site that does not exist (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 GAATAACG 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 GGTCTGCTCA 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 CGACACAGGGGG ACACAAAGGT        960 ATGAGTGGCT GTGCTTGA TACTCACCGA CACGAACT The regions homologous to the above-mentioned sequences are shown below.
Shown below. Where the asterisk is not a homologous site
Is displayed. In the indicated region, the first 118 base pairs are
It has a homology of 80.5%.
Base portion of sea urchin (upper) used as row probe
Is the 449th to 567th, and the base part of the mouse (lower) is
257-375). [Table 10] % = 84.503 F (342,289) =. 000E + 00 E =. 00
0 The restriction endonuclease cleavage sites were
Determined from the sequence. Cuts in the sea urchin mouse sequence
The list of places is shown below. If there is no site name in parentheses,
The number indicates the 5 'side of the cleavage site, and only the recognition site is known.
Indicates that there is. [0131] [Table 11]Sea urchin         Arrangement 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 (GCCGTG) 491 HgiCI (GGQPCC) 494 HgiJII (GPGCQC) 483 HhaI (GCGC) 125 253 295 497 HindIII (AGGCTT) 145 HinfI (GANTC) 200 431 561 HpaII (CCGG) 275 376 HphI (GGTGA) 368 HphI (TCACC) 195 365 532 MboI (GATC) 324 MnII (CCTC) 267 342 MnII (GAGG) 4 42 54 280 299                                 311 372 463 484 NarI (GGCGCC) 495 NspBII (GCMGC) 256 PvuI (CGATCG) 327 ScrFI (CCNGG) 276 377 533 SfaNI (GCATC) 409 527 TaQI (TCGA) 117 327 [0132] [Table 12]mouse         Arrangement 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 [Ecopl] (AGACC) 418 496 882 [Ecopl] (GGTCT) 285 771 [Ecop15] (CAGCAG) 765 EcoRI (GAATTC) 2 EcoRI * (PPATQQ) 4 790 845 EcoRII (CCLGG) 338 368 443 536 Fnu4HI (GCNGC) 366 543 574 577 FnuDII (CGCG) 162 164 380 461 682 FokI (GGATG) 526 905 910 FokI (CATCC) 111 322 346 442 587                                 711 748 HaeII (PGCGCQ) 305 312 537 HaeIII (GGCC) 404 542 612 624 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) 220 339 462 495 MboI (GATC) 188 MboII (GAAGA) 105 124 MboII (TCTTC) 629 MnII (CCTC) 202 227 236 415 559 MnI (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 (GAANNNNTC) 439 The sequence of the sea urchin mouse was Hha I (GC
GC) and the probe sequences described above. Sea urchin array
Creates a 202 base pair (bp) fragment with 295
It has a cleavage site at position 497,
Hybridizes with the probe sequence. Hh of mouse sequence
a I (GCGC) sites (166, 304, 311 and
And 380 sites), the fragments 69 and 138
Indicates that the column can be detected. In this way, sea urchin
The genetic characterization is 202 and 69 for mice.
138. [0134]Example 2 Trp D remains of tryptophan operon as probe
Use of a gene Using the trp D gene as a probe,
Computer simulation was performed similarly. this
E. coli and Salmonella typhimuri
Umm (Salmonella typhimuriu)
m) can be identified by restriction fragments containing conserved sequences.
Give argument. A 684 base pair (bp) E. coli (E.co.
li) The 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 CAGCTTCGAA 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 683 bp Salmonella typhimurium
The mutrp 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 CAGCTTCGAA 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 regions of homology between the two sequences are shown below. here,
The upper sequence is E. coli, and the lower sequence is salmonate
Ifimurium. [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 GAAGCCGATGAAACCCGTAATAAAGCGCGCGC--CGTATTGCGTGCTATCGCCACCGCGC                     * * * * *     ATCATGCACAGGAGAC-TTTCTGATGGCTGACATTCTGCTGCTCGATAATATCGACTC-T     ATCATGCACAGGAGACCTT-CTGATGGCTGATATTCTGCTGCTCGATAACATCGACTCGT      ** ** * * * * * * * * * ** *     TTTAC--GTACAACCTGGCAGATCAGTTGCGCAGC-AATGGG-CATAACGTGGTGATTTA     T-CACTTGGA-ACC-TGGCAGATCAGCTACG-AACCAA-CGGTCATAACGTGGTGATTTA        * * * * * * * * * * * ** *     CCGCAACCATATACCGGCGCAAAC-CTTAATTGAACGC-TTGGCGACCATGAGTAA-TCC     CCGTAACCATATTCCGGCGCAGACGCTTA-TCGATCGCCTGG-CGACAATGA-AAAATCC     * * * * * * * * ** * * ** * *     GGTGCTGATGCT-TTCTCCTGGCCCCGG-TGT-GCC-GAGCGAAGCCGGTTGTATGCCGG     TGTGCTAATGCTCTCC-CCCGGCCC-GGGTGTTCCCAGCG--AGGCAGGTTGTATGCCGG      * * * * ** ** * * * * * * ** *     AACTCCTCACCCG-CT-TGCGTGGCAAGCTGCCCATTATTGGCATTTGCCT-CGGACATC     AGCTGCTGACCCGACTACGCG--GCAAGTTACCGATCATCGGCATTTGTCTGGGG-CATC                           * ** * * * * * *     AGGCGATTGTCGAAGCTTACGG-GGGCTATGTCGGTCAGGCGGGCGAAATTCTCCACGG-     AGGCGATTGTCGAAGCTTACGGCGG-TTACGTCGGTCAGGCGGGAGAAATCCTGCATGGC     * * * *     TAAAGCCTCCAGCATTGAACATGACGGTCAGGCGATGTTTGCCGG 445     AAA-GCCTCCAGCATTGAGCATGACGGTCAGGCGATGTTCGCCGG % = 80.215 P (465,373) =. 000E + 00 E =.
000 Restriction sites for both sequences are shown below. [Table 17]E. coli HpaII (CCGG) 187 229 254 272 283                                 443 463 502 506 592 HphI (GGTGA) 177 552 HphI (TCACC) 285 597 MboI (GATC) 135 564 [0140] [Table 18]Salmonella typhimurium HpaII (CCGG) 187 248 253 283 443                                 463 506 HphI (GGTGA) 177 552 MboI (GATC) 135 204 317 564 MnII (CCTC) 417 The Escherichia coli sequence contains Mbo I at 135 and 564.
(GATC) site. Probes for regions 1 and 2
There is a 429 base pair fragment that can be detected at Same enzyme
Are Salmonella typhimurium 135, 204,
It is also at positions 317 and 564. Two homologous regions
Probe detects 69, 113 and 247 base pair fragments
I do. Thus, this probe and the enzyme
The identified genetic characterization was 429, with Salmonella
It is 69, 113, 247 for fimurium. [0142]Example 3 Use of α-fetoprotein gene as a probe This example demonstrates human and rat α-fetoprotein genetics.
Homologous regions in the codon sequence (endonuclease MnI
(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 ATACTTCACA 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 AAATTACCCA 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 CTGATGTTTA 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 GCTTCTCATT AACCTGGTGA AGCAAAAGCC TGAACTGACA ACTTTGTTCT CGAAGAGTAA TTGGACCACT TCGTTTTCGG ACTTGACTGT       1560 1570 GAGGAGCAGC TGGCGGCTGT CACTGCAG CTCCTCGTCG ACCGCCGACA GTGACGTC Rat α-fetoprotein 3 'endo
The 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 GACAAAGCGT 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 ATTTCTGAAAAGTTGACAAT The regions of homology between the two are as follows:
(Human: upper row, rat: lower row). [Table 21]         *** * * * * * 1367 CTCTTCGTATTCCAACAGGAGGCTATGCATCACCAG-TTTTCTGAGGGATGAAACCTATG   90 CTC --- GTATTCCAACAGGAGGCTCTGCATCACCAGCTTT-CTGAGGGACGAAACCTACG      * ** * ** ** * * *      CCCCTC-CCCCATTCTCTGAGGA-TAAATTCATCTTCCACAAGGA-TC-GTGCCAAGCTC      TCCCTCTACC-ATTCTCTG-CGACAAA-TTCATCTTCCACAAGGAATCTGTGCCAAGCTC      * ** * * * * * *      -GG--CAAAGC-CCTACAGACCATGAAACAAGAGCTTCTCATTAACCTGGTGAAGCAAAA      AGGGCCGA-GCACC-ACAGACCATGAAACAAGAGCTTCTCATTAACCTAGTGAAACAAAA             * **      GCCTGAACTGACAGAGGAGCAGCTGGCGGCTGTCACTGC 1576      GCCTGAAATGACAGAGGAGCAGCACGCGGCTGTCACTGC 299 % = 85.845 P (219,188) =. 000E + 00 E =.
000 The restriction sites between human and rat are as follows:
It is. [Table 22]Human MboII (GAAGA) 108 261 328 1066                             1069 1230 MboII (TCTTC) 881 916 1361 1449 MnII (CCTC) 273 574 1190 1200                             1219 1245 1439 MnI (GAGG) 44 47 358 449                               653 887 1070 1162                             1250 1274 1378 1402                             1436 1544 [0147] [Table 23]Rat MboII (GAAGA) 435 MboII (TCTTC) 168 MnI (CCTC) 100 159 323 MnII (GAGG) 39 98 122 267 357                               384 412 Fragments containing conserved sequence portions (24, 34, 1
04 and 108 base pairs) describe human DNA.
It can be calculated that Also, fragments 24, 59, 9
0 and 145 base pairs describe rat DNA. one
On the other hand, both sequences contain a 24-base pair fragment, which is an important fragment.
Set of pieces (a taxonomic feature).

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 Pseudomonas aeruginosa (Pseud
1 shows the EcoRI restriction endonuclease digestion of DNA isolated from a strain of S. omonas aeruginosa). Escherichia coli 16S and 23S as probes
CDNA for the type ribosomal RNA (rRNA) was used. Fig. 2 Pseudomonas aeruginosa (P. aer
uginosa) Pst I of DNA isolated from strain
Figure 5 shows restriction endonuclease digestion. CDN against 16S and 23S rRNA of Escherichia coli as a probe
A was used. FIG. 3 shows EcoRI restriction endonuclease digestion of DNA isolated from glucose-non-fermentative Gram-negative bacillus species. Escherichia coli 16S type and 2
CDNA for 3S-type rRNA was used. FIG. 4 shows Pst I restriction endonuclease digestion of DNA isolated from glucose-non-fermentative Gram-negative bacillus species. Escherichia coli 16S type and 2
CDNA for 3S-type rRNA was used. FIG. 5: Various Bacillus subtilis (Bacillu)
Escherichia coli DNA isolated from S. subtilis strain
1 shows oRI restriction endonuclease digestion. CDNA for Escherichia coli 16S and 23S rRNA was used as a probe. FIG. 6 shows Pst I data obtained using the same probe for the same strain as FIG. FIG. 7 shows BglII data obtained using the same probe for the same strains as FIGS. 5 and 6. FIG. 8 shows Sac I data obtained using the same probe for the same strains as in FIGS. FIG. Subtilis and B. Polymica (B.po
1 shows the EcoRI restriction endonuclease digestion of DNA isolated from lymyxa). CDN for 16S and 23S rRNA from E. coli as probe
A was used. FIG. 10 shows Pst I data obtained using the same probe for the same strain as FIG. FIG. 11 shows BglII and SacI data obtained using the same probes for the same strains as FIGS. 9 and 10. FIG. 12: Streptococcus pneumoniae in EcoRI digested DNA from infected mouse tissues using cDNA for 16S and 23S rRNA from E. coli as probe.
(Neumania). FIG. 13: Mus Musculus Domesticus (Mu
2 shows the identification of mouse species by using cDNAs for 18S and 28S rRNA from cytoplasmic ribosomes of S. musculus domesticus (mouse) and comparing Pst I digestion of DNA isolated from mammalian tissues. FIG. 14: Mus Musculus Domesticus 28S
Figure 2 shows EcoRI digested DNA from mouse and cat tissues hybridized with type rRNA and cDNA probes. FIG. 15: Mus Musculus Domesticus 18S
I digested DNA from mammalian tissues hybridized with native and 28S rRNA, cDNA probes
Is shown. FIG. 16: Mus Musculus Domesticus 18S
And 28S rRNA, Eco from mammalian tissue and cell culture hybridized with cDNA probes
3 shows RI digested DNA.

   ────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Webstar, Jyon A. , Jiuni               Ya               United States 01821 Masachi Youse               Tutu, Billerica, Apartment 21,               Building 5, Kenmar Drive                 5                (56) References "Molecular and Bi               chemical Parasito               logys, 3 (1981). p. 47-56 "

Claims (1)

(57) [Claims] A kit for use in a method of identifying a species of an unknown organism, the method comprising: 1) Evolutionary conserved sequences in DNA of unknown organisms (ribosomal RNA information-evolutionary conserved sequences appearing in nucleic acids containing nucleic acids) ) And conserved DNA sequence information capable of hybridizing to the evolutionary conserved sequence of the unknown organism-a probe containing a nucleic acid containing the nucleic acid; 2) restriction endonuclease cleavage in DNA in the unknown organism. Determining the location of the conserved sequence in the unknown organism corresponding to the site, thereby obtaining a genetic characterization of the unknown organism; 3) the characterization obtained in step 2); A sufficient number of additional genetic characterizations to determine the species, including the evolutionary conserved sequences in the DNA of known organisms (ribosomal RNA information-evolutionary conserved sequences appearing in nucleic acids containing And a probe containing conserved DNA sequence information-containing nucleic acid capable of hybridizing to the evolutionarily conserved sequence of the known organism, followed by restriction endonuclease cleavage in DNA in the known organism. Comparing the position of the conserved sequence in the known organism corresponding to the site with the additional genetic characterization obtained by determining the position of the unknown organism; and 4) identifying the species of the unknown organism; The kit comprises: (a) one or more containers that are partitioned so that they can be neatly placed, wherein the first container has an evolutionarily conserved genetic material sequence of or derived from the probe organism. An information-containing nucleic acid (excluding ribosomal RNA information-containing nucleic acid) and a carrier containing a detectably labeled nucleic acid; and (b) a restriction endonuclease. A combination of at least two of the chromatographic patterns of the degraded fragments, wherein each combination of the chromatographic patterns comprises a catalog having a combination defining a known different species of organism; A kit by which the species of the unknown organism can be identified by comparing the chromatographic pattern of the restriction endonuclease degraded fragment of the body with the species-specific chromatographic pattern in the catalog. 2. The kit of claims 1 or 2, further comprising a second container containing one or more restriction endonuclease enzymes. 3. 3. The kit according to claim 1, wherein the conserved DNA sequence information-containing nucleic acid probe is RNA. 4. 3. The kit according to claim 1, wherein the stored DNA sequence information-containing nucleic acid probe is a DNA complementary to RNA. 5. The kit of claim 3 further comprising a container of one or more detectably labeled deoxynucleoside triphosphates. 6. Claim 1 wherein the probe organism is a prokaryotic organism
Or the kit of item 2. 7. Claim 1. The probe organism is a eukaryotic organism.
Or the kit according to any one of the above items 2. 8. 7. The kit according to claim 6, wherein the prokaryote is a bacterium. 9. 8. The kit according to claim 7, wherein the eukaryotic nucleic acid probe is derived from the organelle. 10. The kit according to claim 4, wherein the cDNA is labeled with ³² P, ¹⁴ C or ³ H. 11. 5. The kit of claim 4, wherein the cDNA is a faithful copy of the rRNA from which it is derived. 12. 3. The kit according to claim 1, further comprising one or more containers of viral nucleic acid probes. 13. 3. The kit according to claim 1, wherein the catalog is a book, a computer tape, a computer disk, or a computer memory. 14． 3. The kit according to claim 1, wherein the catalog also includes a wheel chromatographic pattern. 15. 3. The kit according to claim 1 or 2, wherein the catalog also contains categorized patterns of subspecies or less.