JPS62228300A - Dot-blot crossing of genom dna of eucaryote - Google Patents

Abstract

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

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the detection of normal and mutated base sequences or genes in eukaryotic genomic DNA. In particular, the present invention relates to such detection, including nucleic acid hybridization of the sequences of interest using oligonucleotide probes.

Oligonucleotide probes serve as the basis for a powerful method for detecting the presence of specific DNA sequences in mixed DNA populations. Generally, the DNA under test is often nitrocellulose. Immobilized on a sheet of support. Next.

Exposure of the immobilized DNA to labeled oligonucleotides in a hybridization mixture.6 The immobilized phase is then heated such that specific binding is maintained but non-specific binding is destabilized and lost. Washed under strict conditions of buffer and temperature.

This method is called the Southern method.

tting procedure) [Sout
hern), E, M, (1975).

-517]. A simplified version of this procedure eliminates the gel electrophoresis step for separation of the DNA fragments under test. It is not separated, so the so-called "
The DNA tested in the dat-blot J method does not need to be cleaved with restriction endonucleases; the elimination of these two steps provides a positive answer as to whether DNA of a given sequence is present or not. /No' answer is sufficient, significantly shortening and simplifying the above method. In addition to saving time and effort in the dot-plot method, the number of samples that can be tested per medium area of solid support is increased by at least 10 times compared to the Southern method and methods derived therefrom. Become.

Dot-plots and related methods are accepted in prokaryotic systems. Because eukaryotic genomes are 1000 times more complex, restriction/isolation methods have traditionally been necessary for the detection of single copy genes. Thus, until now,
The simple format and ease of handling of the dot-plot method was only possible in prokaryotic systems [
For example, Grunstein.

M., and Hogness, D.

C, (l975), Proceedings of the 1st edition)
[Kandan A, Satue, 3961-3965].

The introduction of synthetic oligonucleotides has revolutionized DNA hybridization and has made it possible to clone genes whose protein sequences are only partially known. In the field of human genetic analysis, it is routine to analyze any length of 30 to include regions of genes whose sequences are known.
The generation of probes up to ~40 nucleotides in size was enabled. Walce and co-workers [Conner, B.J.
, Reyes, A., A., Ma.
rin), C,, Itakura.

Ko, Teplitz, R,L, and Wal 1ace, R,B, (IA, 1st, 278-282) have identified a bell-shaped red cell mutation in the beta globulin gene. investigated and determined that optimal discrimination between normal and bell cell sequences could be made using a 19 nucleotide probe with a mutated base at position 13. However, However, this method has the significant disadvantage of requiring restriction endonuclease digestion and gel electrophoretic separation of the resulting DNA fragment mixture.
have used these elaborate I) NA fractionation and separation techniques to m-111 small DNA fragments for oligonucleotide hybridization. These investigators and others observed high background binding of oligonucleotide probes to high molecular weight DNA, which interfered with the detection of specific hybridizations in unfractionated DNA mixtures.

Wood et al. [Wood.

W, 1. , Gissi? -(Gitschier).

Jl, tasky, l, a, and low y.
(Lawn), R, M, (1985).

585-1588] investigated the use of tetramethylammonium chloride (TMAC) in a rigorous cleaning medium. This salt reduces the sequence dependence of oligonucleotide hybridization, allowing hybridization with mixed sequence probes and stringent washes that discriminate only for sequence pairing criteria. The authors incidentally note that they used oligonucleotide hybridization to detect single base polymorphisms in the factor vM gene. When this method adds one transfer step, the above-mentioned conner
) is the same as the method of et al. This is conne
r) further complicates the method of et al. However, TM
The introduction of AC washing appears to have eliminated the non-4.9 heterogeneous background normally observed in the high molecular weight range. Thus, prior art methods for detecting normal and mutated gene sequences in eukaryotic genomic DNA, particularly human) DNA, by oligonucleotide hybridization, have been shown to be difficult to obtain in order to obtain detectable and specific results. D
Requires extensive and complex NA purification, including restriction digestion of NA and gel electrophoresis.

Many methods of labeling oligonucleotides are currently known in the art. Photochemical labeling is covered by European patent publications.
ion) 131,830'l'j and 156,2
It was described in No. 87. exemplified by double-stranded DNA,
A general method of coupling a nucleic acid to a label, such as a habten or an enzyme, is as follows: Label + Photoreactive Intercalator Labeled Photoreactive Intercalator ↓ Double-stranded DNA Labeled DNA Conventional , there was no disclosure of photochemical labeling using oligonucleotide probes. However, if the oligonucleotide is modified in the hybridization region
It is known that when hybridization is performed, the hybridization parameters may differ from the intact oligonucleotide.

Therefore, background problems involving oligonucleotide hybridization evident from the prior art have severely limited attempts to simplify this method. Thus, in order to make this hybridization analysis practical, for example in clinical laboratories, there is a constantly increasing need to eliminate tedious and time-consuming steps in this technique. have remained constrained by elaborate sample handling and preparation processes. Despite the clear advantages of such techniques, there have been no attempts to perform dot-plot hybridization using oligonucleotide probes to detect sequences in unfractionated eukaryotic genomic DNA. is clear.

It has now been surprisingly discovered that nucleotide sequences in eukaryotic genomic DNA can be detected by dot-plot hybridization of oligonucleotides. This method can be applied to detect iE normal gene sequences and mutated gene sequences, especially sites where point mutations occur. The use of oligonucleotide probes allows this assay to distinguish known n1 base sequences from sequences that differ by few or even one base. This method can be particularly used for genetic analysis of human genomic DNA. This dot block) i'J is the DN of the sample
A fragmentation is not required and eliminates the need for gel electrophoresis to separate fragments containing the sequence of interest from background DNA.

The method of the invention provides a single-stranded form y,! ; involves immobilizing DNA from a test sample in a defined zone or area (Dratau proft) of a sheet of solid support, e.g. nitrocellulose. having a 41-base sequence that is precisely complementary to the base sequence in question, after the unfilled nucleic acid binding sites have been substantially blocked by treatment;
The sheet of support is incubated with a solution of oligonucleotide probes, preferably containing a detectable label. This incubation is accomplished under time conditions that favor hybridization of the probe to the sequence of interest.

After removing the probe solution, the support is washed with sufficient stringency to remove substantially all of the probe that has hybridized to sequences other than the precisely complementary sequence of interest. The presence of hybridized probes in the dot-plot is then detected, and its presence indicates the presence of the sequence of interest in the sample of human genomic DNA. If the probe is labeled, the presence of hybridized probe in the dot-plot is easily determined by detection of the signal or response of the label.

Hybridization conditions are chosen such that after rigorous washing, a level of detectable probe remains in the dot-plot that is easily distinguishable from the background binding of the globe to the surrounding sheet of support.

Because this method is based on dot-plot technology, it eliminates the need for substantial purification and fragmentation of sample DNA as required by prior art techniques. What about the sample? i, for example, by treating with a base and then neutralizing the genomic DNA;
It can then be applied to a sheet of support. An alternative method is 1, for example, by directly extracting sample D from whole cells collected during cancer treatment.
The idea is to immobilize NA on the sheet and then perform in-situ denaturation on the sheet. As a result, the laborious and time-consuming conventional DNA isolation can be reduced to just a few handling sheets of support, and a large number of samples can be analyzed in a unit sheet of two'. .

When analyzing human genomic DNA for the presence of a single gene sequence or a mutated gene sequence of interest,
It is preferable to perform hybridization for each such sequence simultaneously. This is very easily accomplished using the dot-plot technique of the present invention by applying portions of the sample to separate first and second zones on a sheet of support. A pair of oligonucleotide probes is used, one exactly complementary to the normal sequence and the other exactly complementary to the mutated sequence. Before incubating the sheet with the hybridization solution, the sheet of support is cut to form two sections, one containing the first zone and the other containing the second zone.
Contains zones.

Each of the pair of probes is then brought into contact with one of the cut sections, and the method continues as described above for both sections. Analysis of the presence of hybridized probes in two zones cut from the sheet of support will indicate the presence of one or both of the sequences in question in the sample.

The hybridization region of the oligonucleotide is not appreciably altered, whereas the target DNA.

The present inventors have discovered a probe system in which an extra region that is not complementary to the probe specifically carries a label. To achieve this, we discovered a probe system consisting of two synthetic oligonucleotides, one of which has a hybridization region specific for the detection of a particular nucleic acid sequence, Examples are mutations such as point mutations, or frameshifts or deletions and nucleotide residues flanking such hybridization regions. Such nucleotide residues, (are nucleotide residues numbered 5 to 10,000).Nucleotide residues adjacent to the hybridization region do not participate in hybridization.The other oligonucleotide is Complementary to a nucleotide residue.

When these two oligonucleotides are mixed together, they form a double-stranded region and a single-stranded hybridization region in one hybridization molecule. Attach the label to the double-stranded region.

Intercalating drugs that insert double-stranded regions
It can be modified using (drag). This can be carried out using photochemically active insertion compounds such as aminomethyltrioxysalen. The desired label is then attached using an amine. The desired label can be a small molecule, such as biotin or other hapten, such as an immunogen, or a fluorophore, such as fluorescein. It also contains enzymes such as horseradish peroxidase.
peroxidase).

This reaction can be carried out in two ways, namely: (l) With a photoreactive intercalator (intercal
ator), for example, aminomethyltrioxysalen (
aminomethyltrioxs alene)
can be reacted with a label and the final product photochemically reacted with the mixed probe; (2) a photoreactive intercalator, e.g., aminomethyltrioxysalen, is first photochemically coupled to the DNA; , the product can then be reacted with a label.

In order to generate specific and efficient photochemical products, the nucleic acid component and the photoreactive intercalator compound are reacted in a special manner in the dark.

For photochemical coupling to nucleic acids, aminomethylbusoralen, aminomethylangesillin and aminoalkylethidium or methidilam azides are particularly useful compounds. They bind to double-stranded DNA and only the comptex produces a preadduct. Angelicin derivatives are superior to psoralen compounds for the formation of monoadducts. When a single-stranded probe is covalently attached to some extra double-stranded DNA.

It is desirable to use phenanthridium compounds and busoralen compounds because these compounds interact specifically with double-stranded DNA in the dark. The chemistry to synthesize the coupled reagents for modifying nucleic acids for labeling is similar in all cases and is detailed below.

Nucleic acid compounds are DNA or RNA or relatively short oligomers.

The nucleic acid binding ligand of the present invention used to couple a nucleic acid component to a label can be any known nucleic acid binding ligand, in any suitable photoreactive form thereof. Particularly preferred nucleic acid-binding ligands are: intercalator compounds, such as furocoumarins, such as angelicin (imbusoralen) or psoralen or derivatives thereof, which react photochemically with nucleic acids, such as 4' −
Aminomethyl-4,5′-dimethylangelicin, 4′
-aminomethyltrioxysalan (4'-aminomethyl-4,5°, 8-trimethyllopsoralen, 3-carboxy-5- or -8~ or -hydroxyepsoralen), and mono- or bis-azidoamino Alkylmethidiram or ethidium compounds. Various other photoreactive forms of intercalators can also be used and are exemplified in the table below: A. Acridine Dyes Lerman+Journal Enno Molecular Donioyu Two (Stop Yu Mol.

Biol,) 3:18 (l961), Bloom Field (Bloom field) et al. c　Ac1ds) Chapter 7.

Pages 429-476.

Harbor 1 and Ou U, (Harper a) nd ROWe), New -York (l974).

Proflavin, Miller Lysine Orange, Kira, Biopolymer Anacrine Acrifura (Biopolymer Bin 5)
19:2091 (lB, Phenanthridine Bloomfield I” (Class B 1o
omfield et al., 5upra Ethidium Miller, Coralin et al., 5upra Wilson.

n) et al., Journal MaChem, ) 19: 1 Eributicin, Eri Festei (Fest buticin cation, et al., FEBS Leyohy derivative)
Letters 17:321 (l9 71); Kohn et al., Cancer Research 35 (l9 76); LePecq et al., PNAS (USA) 71: 507 8 (1974); Pelabrat et al., Journal 2nd Medicinal Deficiency Studies (Chem.) 23:1330 (1980) C, phenazines Bloomfield (B5-methyl 7. Z-less Ioomfield) cation
et al., 5upraD, Fetchazides Same as above Chlopromacin E, Quinolines Same as above Chlorichinkinin F, Aflatoxin Same as above G, Polycyclic hydrocarbons Same as above - L and their oxirane 1 derivatives 3.4-benzbirene Young (Y a n g) et al.

Benzopyrene Diio-Biochemical Andoyu) 8
2:929 (l Penz Anthrace Amea et al.

Thor5,6-Oki Science
) 176:47 (lH, Actinomycin Bloomfield (Class B)
1oomfield) actinomycin D et al.
5upra ■, Anthracycline Same as above Non-class B-Loudamycin Downamycin J, Thiaxantenone Same as above Miracil K, Anthramycin Same as above, Midomycin Ogawa Uni),
5pec, Publ, 3nia 9 (1977); Akhtar et al., Kana, J. Chem.) 53:2891 M, platinum complex salts Lippard
, Accounts Office, Res, ) 11: N, Polyintercal Warring (Warli Iter)
ng) et al., Nature Echinomysi 7 (Nature) 252: 653 (
1974).

Sea urchin karin (Wake 1in), Biochemika 7: 721 (l976) Kinomycin Lee et al., pi tandem
J.) 173:115 (1978), Hua ng et al., Biochemistry (Bi.

chem, ) 19:55 37 (l980), Viswamitra et al., Nesiacridines LePecq et al., PNA
S (USA) 72:2915 (1975); Carrelakis Ta) 418:277 (1976), Wk e Ifn et al., Biochem, ) 17:5 057 ( 1978); Wke Iin et al., FEBS Letters (Lett.) 104: 261 (1979); Capelle et al., Biochemist IJ- (Bioche Uni) 18:3354 (1979), Wright (1979); Wright et al., Biochem 19:5 825 (1980); Bernier et al., Biochem 199:479 (1981); King et al. 9 people are Chemistry (Biochem,) 21:498 Ethidium dwarf Gaugain
N) et al., Biochemistry (BiOChem,) 17 2 5078 (l97B); Kuhlman et al., Nucleic Acids Research (Bondanto 1, Ac1dS Res,) 5: 2629 (l 978), Markovitz (Marlcovit S) et al., Analytical Hem,) 94:259 (l979), Durban et al. Dez Kushiku Debb
Ar bodies and analogues e) et al., Compt.

Kind: Rend, Ser.

D, 284:81 (t 977); Peraprat (Pelaprat) Stop Med.

Chem,) 23:13 114 Heteroniforms Cain et al.

Journal・Dobu・4 Digital e-chemist (Meh Med.

Chem, ) 21: 65 8 (l978); Gaugain et al., Biochem, ) 17: 5078 (l97);
Chem, Comm, 162 (1983); Atnell et al.

JACS　105:29 0, Norphylin A Loran et al.

JACS 104+32 P, Fluorenes and Bloomfield et al., 5upra Fluorenediamine Witkolasky (Wi
tkowski) et al., WiSs
, Bertr.

-Martin-Luther-Univ, Halle Wittenberg, 11 (18 Q, Furocoumarins Venema Ankerisin et al., MGG, Molecu Genet,) 17
9: 4.5°-Dimethycin di et al., Chem.

Biol, Inter act, 36:275 Psoraren Marciani et al.
nzov) et al., Mutei ZAE Research (M
utat, Res, ) 8 4:11 (1981).

Scott 1, ) 34:63 (l9 5-aminomethyl Hanse-8-
Methoxib n) et al., Tetrahedron psoralen
・Letters (Tet.

Let,) 22:184 4.5.8-triben-/\-(Ben-H methylpunralen ur) et al., Biohimica Acta) 331:
1 4"-Amino Methi Issa 4,5
．． 8-cs), Biochemist Trimethyl Busola
L = (Bioche Len m,) 16:
105B Xanthoxin Fradecma (Hr a d
ecma) et al., 7'y')-e Rologica (Acta Virol, ) (English version) 26: 305 (l98
in) mont) et al., Biohis, A.
cta) 608:1829 (1980) R, penfudipirones Murx et al., Journal of Heterocyclic Chem. 12:417 (1975).

Horte r) et al., Photochemistry and Light Temporary stop (Phtoch) m, Photobi.

±yo) 20: 407 (l S, Monostralfa Juarans
S1 Blue (Mono ranz) et al. Acta 5
tural Fas Histochemistry (Actat)
Blue) Histchem, )70
:130 (198 Such an intercalating agent
A particularly useful photoreactive form of get(s) is the azide intercalator. Their reactive nitrenes are readily generated in long wavelength ultraviolet or visible light, and arylazide nitrenes prefer insertion reactions to their rearrangement products [White et al. Methods in*
s in Enzymology), 64,644
(1977)] Typical azo intercalators are 3-azidoacridine, 9-azidoacridine, ethidium monoazide, ethidium diazide, ethidium monoazide [Mitch et al., JA
C5,104,4265(l982)], 4-azido-
7-chloroquinoline and 2-azidofluorene. Other useful photoreactive intercalators are furocoumarins that form [2+21 cycloadducts with pyridine residues. Alkylating agent 1 For example, bistaroloethylamines and epoxides or aziridines 1 For example, aflatoxins, epoxides of polycyclic hydrocarbons,
Midomycin and norphylin A can also be used.

A label that binds to a nucleic acid component according to the invention can be any chemical () having detectable physical or chemical properties.
or a residue. The label will be a functional chemical that can be chemically bonded to the intercalator compound.

The test sample of the present invention can be applied to the detection of eukaryotic genomic DNA sequences in essentially any specimen or medium, and said specimen or medium can be used directly or A sheet of support for the immobilization of DNA can be processed to produce a test sample suitable for application. Such analyte or medium is
Includes nucleic acids or cells from liquid, solid or solid samples of medical, veterinary, environmental, nutritional or industrial significance. Human and animal specimens and body fluids.

Those containing nucleated cells include, for example, urine, blood (whole blood, serum or plasma), milk, brain, cerebrospinal fluid, tissue sections, and the like.

Genomic DNA of any eukaryote whose base composition is known
The A sequence can be determined according to the method of the invention.

In the case of humans and animals, various genetic diseases such as bell cells, alpha 1-proteinase inhibitor mutations, thalassemias and hemoglobin C can be detected, where normal gene sequences and their or 2 or more''-mutated forms are known. Mutations can be in any form distinguishable from the normal sequence by oligonucleotide hybridization, such as deletions, insertions, inversions, frameshifts, 11! Group changes can be made, especially point mutations in which a single base is changed.

The present invention also provides for restriction fragment length polymorphisms in cases where the sequence in question is known.
fragment length
(RFLP) can be used to simplify analysis. RFLP can be used to diagnose mutations, for example, if restriction nuclease sensitivity is affected by point mutations.

Dot-plots can be used in place of RFLP or in combination with restriction nucleases: First, the DNA
and then use dot-plot hybridization to assay whether the sequence has been cleaved by the enzyme. This procedure is applicable even if there is only a correlation between a given sequence and a given property. In this method,
It is not necessary that the recognition sequence of the oligonucleotide probe be located within or adjacent to the sequence in question; it is only necessary that a correlation can be established.

The following table shows "New Genetics and Clinical Practice (The
New aenetics and ctini
cal Practice) J, D, J.

We thera l (l98)
2) Chopsticks, The Nufffield Pr
ovincial Ho5pitalsTrus
t) describe several gene probes with potential medical importance.

Globin gene cluster α, β,
γ, δ, ε Growth hormone villi 1! ,! Lactation hormone of the anterior pituitary gland Gonatropin of the chorion Prolactin Insulin Interferon α chain of collagen HLA β Histone [several types (severa 1)] Immunoglobulin gene modification, K and light chain) Gastrin Liposomal RNA Melanocytes Stimulating hormones (α, β, γ)corticothrobin β lipoprotein β endorphin actin α1 antitrypsin albumin glucose-6-phosphate dehydrogenase pre-paratyroid hormone β1 microglobulin α-tubulin factor ■ and ■ anti Thrombin 3 Some components of the complement pathway Chromosome 2.3.5.6.11.14.20.22 and X Source: 77 Antonarakis, S
,E., ,Phillips, J.A.
and KaZaZian 7, H, H, (
1982), Miscellaneous Pediatrics, 4 (J, Pedjat
), 100, 845-856.

If it is necessary to positively establish the presence of a normal gene or a specific mutation in a gene, hybridization is performed using separate probes of the test sample using probes complementary to the normal and mutated sequences, respectively. It will be carried out in parts. The method of the present invention provides a convenient means for performing such parallel analyzes and for performing multiple such analyzes on a single batch of test samples. Of course, if a large number of known mutations in the normal gene sequence are of interest, multiple crosses can be performed using the method of the invention, where additional dot-plot zones are applied to the sheet of support. and perform another hybridization using the appropriate respective probes.

In the examples below, purified DNA is used. This was for development purposes as well as for comparison with prior art techniques requiring purified DNA. One of the advantages of the dot-plot technique is that there is considerable leeway in simplifying sample preparation: crude DN
A or even whole cells [e.g. Brandsma (B
r a n dsma), U, and Mill
er).

A) Midan A, 7e, 6851-6855] can be applied to filters for hybridization tests. Crude DNA preparations may be even better than purified DNA because proteins and other cellular components may promote initial adhesion of the test DNA to the sheet of support.

The dot-plot technique can be applied to a sheet of support using any procedure as long as it immobilizes the DNA from the test sample in a single-stranded form in a limited zone of the sheet of support.
You can also create plots. Sample DN of l・min.
It is necessary that A be single-stranded and immobilized on a support so that detectable hybridization with the oligonucleotide probe can occur.

Several procedures for obtaining immobilized single-stranded DNA are preferred. In one such procedure, a test sample is treated in a known manner as a liquid mixture so that the D
The NA is denatured and the resulting solution is applied to a sheet of support. Using a Tingtong don't-plot apparatus, residual liquid is then removed by vacuum through a sheet. Denaturation of the DNA of the sample can be achieved, for example, by adding a base, such as 0.1 to 1.0 mol of sodium hydroxide, and then neutralizing before application to the sheet of support. Heat treatment, melting, and other modifiers may also be used in this process.

Alternatively, a liquid test sample can be applied directly to the support and the DNA denatured in situ. Cao Tong's technology is: a) 0.5 mol NaOH.

It consists of laying the test filter on cancer paper saturated with b) 1.0 molar Tris at neutral PH and then C) 1.0 molar Tris at neutral + 1-3 molar Nacl. However, this procedure limits the sample volume, typically less than 10 μl, preferably 1-2 gl.

The dot-plot procedure can accommodate sample volumes up to several hundred grams. Treatment with strong bases tends to make nitrocellulose paper brittle, which is preferred for dot-plot procedures.

Although Kawatoshi's dot-blotting apparatus can be conveniently used to apply the sample to a sheet of support, any technique that effectively localizes the test sample to a definable area or zone of the support may be used. can be used. Similarly, the shape and size of such zones, generally referred to herein as dot-prongs, are not critical.

Various support materials capable of immobilizing single-stranded DNA are known in the art. Such materials include the following: commercially available nitrocellulose paper, various nylon papers, such as those available from DuPont, Wilmington, Prairie, USA. Gene-screen (Gene-3screen)
and ZETA-PROBE, diazobenzyloxymethyl paper, etc. available from Bio-Rad (California, USA). The choice of support material is not critical; after applying the test sample to the support and performing any additional steps necessary for denaturation, the single-stranded DNA adsorbed to the support is usually further processed, e.g. , heat in vacuum or in alcohol to firmly fix the DNA to the sheet.

Generally, it is necessary to treat the dot-blotted support to block unfilled nucleic acid binding sites in sheet h before hybridizing with specific probes. This can be accomplished by any conventionally known method and will vary somewhat depending on the particular nature of the support used. Typically, the sheet is incubated with a prehybridization solution for a predetermined period of time, generally from about 5 minutes to about 4 hours, preferably from about 5 minutes to about 30 minutes, and the prehybridization solution binds and imparts the desired blocking functionality. Contains exposed chemical reagents. Such solutions usually contain high molecular weight proteins, such as albumin, and non-specific nucleic acids, such as DNA from sources foreign to the test sample (salmon sperm DNA is used for this purpose). will include. Other components of the precrossing solution can be polyvinylpyrrolidone, finyl, sodium dodecyl sulfate. The precrossing solution and the hybridization solution are
If they are not identical, it is analogous to adding the probe to the hybridization mixture. A recent advance is the use of ``Pronto CBlotto'', a skimmed tri-milk preparation [Johnson (Jo
hns.

n),D,A,,cantsch,J,
W, Sportsman, J, R,
and erg-(E 1 d er), J, H,, (l
984), m-gene analysis technology (Gene Anal,
Techn.), 1.

The temperature of the 3-81° precross is generally the same as the hybridization temperature and is usually 10-20° C. lower than the dissociated trait (Td).

The hybridization conditions used in the present invention are suitable for low background from probes that nonspecifically bind to the support or nonspecific DNA in Doh-plots, and for successful detection of specific hybridizations. That's the key. The rate and efficiency of nucleic acid hybridization generally depends on the following key factors: temperature, ionic strength, presence of organic solvent, and concentration of hybridizable nucleic acids. It has been shown in accordance with the present invention that optimal conditions for hybridization allow the use of highly convenient dot-plot techniques in the detection of sequences in unfractionated eukaryotic DNA using oligonucleotide probes. It has been established. Furthermore, the incubation time of the hybridization mixture was found to be particularly important in obtaining easily interpretable results.

Optimal values for temperature, ionic strength and solvent are mostly determined experimentally. In the example below, the specific interaction T
d is approximately 59°C. Therefore, a hybridization temperature of 50°C is used, but this is not critical. Td is high (〉
Formaldehyde is often used to lower the Td when using long (>50 bases) probes (see Wood et al., supra). Crossings are then typically performed in 50% formaldehyde at 68°C.

Regarding the degree of C of the nucleic acids involved in the hybridization, it has been discovered that a relatively lower concentration of probe than is customary for the Sachin method should be used to reduce the background binding of the probe used. . About 1O picomole to about 1
The use of a C degree of the probe in the hybridization solution of 0 nanomolar is an important factor for the successful application of the doy)-plot technique to the hybridization analysis of oligonucleotide probes of eukaryotic DNA sequences. In the specific examples below relating to the detection of calcaneus mutations in human genomic DNA, a concentration of probe of about 0.01 to about 3 nanomolar is preferred, with an optimal concentration of about 0.3 to about 1 nanomolar. . At these concentrations, detectable signals can be obtained from hybridized probes while preventing significant non-specific oligonucleotide binding to the support.

A very significant parameter of the present invention is the incubation time of the hybridization mixture and the support. To limit non-specific binding of the probe to the support, incubation times are usually kept below about 4 hours, with the minimum incubation time required to obtain a detectable signal for the hybridized protein. Governed by sensitivity.

Usually, a cross-breeding period of at least 10 minutes will be necessary, but
Highly sensitive detection methods can reduce this time. The most preferred incubation time was found to be approximately 10 minutes to 1.5 hours.

After removing the hybridization solution from the sheet of support, it is necessary to wash the support under sufficiently stringent conditions such that only the hybrid and exactly complementary base sequences of the probes remain intact. In this way, probes that hybridize to sequences that lack complete homology with the probe sequence are dot-
removed from the plot. The stringent conditions for accomplishing this task are: a) the length of the oligonucleotide, b
) the ionic strength; c) the extent and location of the mismatch or mismatches; and d) the bases containing the mismatch or mismatches.

Ideally, one would be able to calculate the Td of a perfectly paired oligonucleotide in solution and the expected imperfect one. However, in practice the parameters are not known with sufficient precision and Td is determined experimentally.

The preparation of oligonucleotides and their use as probes in DNA hybridization is well known in the art. Such probes have a defined base sequence and can vary from as few as about 10 bases to as many as about 200 bases.

Typically, useful oligonucleotide probes range from about 14 to
It would be 25 bases. The probe sequences can consist of deoxylipo sequences, lipo sequences, and mixed deoxy and lipo sequences. They are prepared by well-known synthetic techniques, such as the phosphite method or the phosphotriester method [Alva
rad-Urbina) et al. (1981), Science (
Science, Vol. 4.270-274].

Hybrid Detection The present invention is not limited to particular methods or reagents for detecting hybridized probes in dot-plots. Any convenient method can be applied. The method of the invention allows for the specific use of labeled probes since conditions have been discovered to limit background binding of the probe to the support.

Various methods exist to determine the presence of hybridized probes. One skilled in the art can choose from conventional means of detecting hybridization between the probe and the sequence to be detected, the presence of which results in immobilized probe, or a decrease in its presence in the reaction mixture.

Generally, the detection step involves the use of a labeled form of the probe;
It may be based on the use of probes that form uniquely detectable hybrids with the sequence in question, or on the use of secondary reactions that can only be carried out when hybridization occurs.

A particularly preferred approach to the detection step involves the use of a labeled form of the probe. This label may be an inherent property of the oligonucleotides made up in the probe, or it may be physically detectable.

It may be a substance with chemical or electrical properties.

When introducing a detectable labeling substance, it can be directly attached to the probe, e.g. by a covalent bond, or indirectly, e.g. by ultimately incorporating the detectable substance and then coupling them to the detection probe. It can be combined by

Labeling substances are well developed in immunoassays;
And in general, most labels useful in such methods can be applied in the present invention. Particularly useful are: enzymatically active groups, such as enzymes [Clinical Chemistry (c1in,
Chem,).

(l976), 2e, 1232. US Reissue Patent No. 31
, 006 and British Patent No. 2,019°408], enzyme substrates (see U.S. Pat. No. 4,492.751), coenzymes (see U.S. Pat. No. 4,230.797 and British Patent No. 4,238) .565) and enzyme inhibitors (see U.S. Pat. No. 4,234,792); fluorescent substances [Clinical Chemistry (cIin, Chem, );
(l979), 25,353]; chromophores; luminescent substances, such as chemiluminescent substances and bioluminescent substances (see U.S. Pat. No. 4,380,580); specifically binding ligands; For example, biotin (European Patent Specification 63,8
(see No. 79) or HABTEN (PCTi line 83-228)
6); and radioactive isotopes, such as 3H23
Labels such as 5S, 3'2p, 125 and 14C may be characterized by their physical properties (e.g. fluorophores, chromophores and radioisotopes) or their reactivity and binding properties (e.g. ligands). , enzyme, substrate, coenzyme and inhibitor). For example, a cofactor labeled species is detected based on the enzyme (or enzymes if a cycling system is used) (the label for said enzyme is detected based on the cofactor and said enzyme). The enzyme can be detected by the addition of one or more 2(K) species labeled with a habten or a ligand (e.g. biotin) or a ligand labeled with a detectable molecule. Detectable molecules can be detected by adding antibodies to proteins (e.g. avidin) or habten that bind to the enzyme.Such detectable molecules can be molecules with measurable physical properties or substances that participate in the enzymatic reaction (e.g. (see list above).

For example, enzymes can be used that act on a substrate to produce a product with measurable properties. Examples of the latter include, but are not limited to, beta-galactosidase, alkaline phosphatase and peroxidase.

Methods for preparing labeled probes for use in preferred embodiments of the invention are readily available from the prior art.

When labeling a probe, use a synthetic approach that is effective for modifying the nucleic acid without substantially interfering with the ability of the labeled probe to participate in hybridization, and that is sufficiently stable under the conditions used for hybridization and that Select a label that allows subsequent detection ~ By way of example, the following approach can be used to label probes. M
I can put it in. Pairs of PI-type brimers can be selected such that one of the pairs can be extended to complete the desired sequence without extending the other.As an example, the following four pairs are templates. would generate a nonadecanucleotide without using one of the same length as: 19 A'5' GGAGACTTCTCG
TC3' vs. 3' GAAGAG
GACTCGTC5' and 19S' 5° GCAGACTTCTGGAC3° vs. GAAGAGGTC; TC
CTC5.

When deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) are used as nucleoside triphosphates (NTPs) for DNA polymerase or inverse transcriptase reactions, only the top button will be extended. . This will produce a 19A'/S' sequence and an unextended bottom strand.

Similarly, complementary heavy chains may be synthesized or labeled by using pyrimidine nucleoside triphosphates in place of dATP and dGTP to generate complementary sequences; the pairs used are: 19S 5' GTCCTGTGGAGAA
GTC3' vs. 3°l: CTCTT
GAGAC: G 5' and 19 A 5' crcGTGAGGAGAA
G 3' vs. 3'
CTCCTCDingTCAGACG 5
'Other useful sets of oligonucleotides that can be labeled by extending only one member of a pair of partially complementary oligonucleotides are those responsible for the common form of antitrypsin deletion. This distinguishes between the mutated gene and the normal alpha1-antitrypsin gene. Deficiency of this protease inhibitor results in emphysema and certain related diseases. The normal allele of this gene is M and the major nucleic acid deletion allele is Z; the nucleotide sequence of the gene near the location of the 7-point mutation [
Kidd, V.J., Wallac
e), R.B., Itakura, N., and Woo, S.

C, C; Detection of alpha-antitrypsin deletions by direct analysis of mutations in the r gene (alph-Antit
rypsin Deficiency Detect
tion By Direct Analysis
Of The Mutation In T
he Gene)J, Nature
), 309, 230-234 (l983)] is such that the non-asymmetric braimer f-tension knowledge can be applied as follows:
GM mold
AGCTGCCTTTchoccc 5'Z pliers
5' TGGTGA (Ic: ATCGACAAGZ template AGCTGTTCTTTCCC5'
Both of these can be labeled by preparing reverse transcriptase or by Tarenow polymerase reaction with the purine deoxyliponucleoside triphosphate precursor alone. The template remains a tetrakaideca nucleotide, while the primer can be extended from 17 in length to 20 by including only dATP in the reaction mixture, or dATP and dGT.
It can be extended to 23 by including both P.

Labeling using these types of pairs creates clusters of nucleotides that act as templates on one strand but not on the other, and sequences that can be identified as having the required closeness for mutation. Limited.

If the shape of the nucleotides in the gene sequence does not allow for the selection of primers and templates for this labeling, synthesize a template whose 3' end is blocked so that it cannot be extended independently of the sequence. is possible.

Such blocking prevents the 2' as the ultimate 3' nucleotide during the chemical synthesis of the template oligonucleotide.
, can be achieved by incorporating 3° dedeoxyliponucleotides.

A key feature of the asymmetric primer extension labeling system described herein is that the length difference between labeled and unlabeled oligonucleotides is determined by electrophoresis or chromatography. This greatly facilitates the separation of labeled oligonucleotides from unlabeled oligonucleotides and unincorporated precursors. Unexpectedly, fractionation of the labeled reaction mixture is not necessary to prepare the hybridization assay! This has also been revealed.

Labeled oligonucleotides are usually purified from unlabeled oligonucleotides. This is because unlabeled oligonucleotides will participate in hybridization and reduce the strength of the signal resulting from the labeled probe binding to the sample DNA.

However, the length difference between the unlabeled PI form and the labeled extension primer is sufficient that under the conditions that can be used for hybridization of labeled probes, the template is too short to form stable hybrids, so probes 72- to the sample DNA of
Do not disturb the ring.

Other common diseases for which sufficient NA sequence information exists to generate synthetic oligonucleotide probes are:
Exists in the family of hemoglobinopathies. Primer extension labeling of alpha and beta thalassemia probes can be accomplished using oligonucleotide pairs as in the following example, without blocking the dideoxyribonucleotides at the 3° end of the template: l, homo Full fur thalassemia of junction 5' ACC: AAGACC ACT ACT CTGGAT
GAAGGGCG 5゜2, Beta+ (IVS-1
) Thalassemia normal 5' GCCTATTGGTG
ATAACCAGATAAAA 5゜suddenly 5' C
TGCCTATTM; TC mutation ATAATC
AGATAAA 5'3, Beta 6 (Beta 3
9) Thalassemia normal 5' CCTTGGACCCAG
ACGTGCGTCTC: CAAGA 5' suddenly 5
°CCTTGGACCTAGA mutation CC:TG
GATCTGCAAGA 5' With a nucleoside triphosphate (NTP) to be added to the desired strand,
It is possible to separate the starting materials (unreacted) by gel electrophoresis or column chromatography. Any type of labeled oligonucleotide can be prepared by selecting the labeled NTP to a suitable J0, e.g. 19
For the A' pair, biotinylated dUTP and dcTP allow the synthesis of a substrate, i.e., a biotinylated oligonucleotide specific for the normal globulin gene; for the fluorescein-labeled NTP, a fluorescent oligonucleotide is prepared. can do. The particular type of label used, whether radioactive or non-radioactive, is not critical, nor is the method of making the labeled probe. Furthermore, dot-
It is clear that any method of detecting the presence of hybridized probes in a plot can be used, whether using labeled probes or other techniques that do not involve them.

The present invention will be illustrated by the following specific examples, but the invention is not limited by these examples.

Dot-plot for detecting the true vine mutation The following solutions are described in the example: Random Brimer w buffer (5x) 0.2 Molar KCl 0.25 Molar Tris-CI pH 8, 110 mmol Dithiothrey 25 mmol MgC12 solution (100x) 2% bovine serum albumin 2% polyvinylpyrrolidone 40 [Calbiochem (ca
lbiochem, Lagiola, Calif., USA] 2% Ficoll [Sigma
), St. Louis, Missouri, USA 12% Sodium Dodecyl Sulfate (S D 5) Ssc (2ox) 175.3 g NaCl 38.2 g Sodium Citrate pH 7.0 to 1 volume with deionized water to 1 Lindl NET ( 20X) 3M NaCl 0.3M Tris-CI pH 7, 520mM EDTA A, oligonucleotide probe preparation and labeling Oligonucleotide probes were labeled with 32-phosphorus using a primer extension reaction. . Radioactive dATP was dried in an Effendorff tube (l, 5 ml) in a Speed-vac apparatus (Savant, Hicksville, NY, USA).
The cooled ingredients are then added to the tube. Normal probe (
For labeling A'), the reaction mixture consisted of the following components: 0.5 mCi of (32-P)-dATP [Amersham, Arlington φ Heights, IL, USA] No. , PB20474.600
0 Ci/mmol lpl (200 ng) of DNA primer (5'-
GCAGACTTCTCCTC)Igl (400ng
) DN14 type (5'-CTCCTGAGGAGAAG
) 2 gl of 0.13 mmol dGTP (7) 3.5 l of 5X Random Brimer Buffer
1.5 pl of deionized water of reverse transcriptase [Molecular
Geneticφ Resources Tampa (Molecul)
ar GeneticsResourcesTa
mpa), 7°Lida, USA, No. MG101] Sequence 5
Use a primer of '-GCAGACTTCTCCAC and a template of 5'-CTCCTGAGGAGAAG.

Gently pump the reaction mixture with your fingers to mix the components and incubate for 90 minutes at 15°C in a water bath. The final probe sequence is: A'
(5'-GCAGACTTCTCCTCAGGA
G) and S゛(5°-GCAGACTTCTCCAC
AGGAG). Under hybridization conditions, the pair of primer-template helices dissociates, eliminating the need for purification.

Excess dATP can result in high background binding, so it is
Calf 1 alkaline phosphatase [Boehringer'--Boehringer-Mannheim
Indianapolis), Indiana, USA
No. 108-146.501'l'/ul] and lysed by incubation for 20 minutes at room temperature.

An alternative method for removing excess dATP that provides excellent hackground reduction is the spinning column method (spu
n column method). Diameter approximately 0
．． Several 5 mm glass beads are suspended above a polypropylene test tube in a yellow Eppendorf tube.
dorf) into the tip of a micropipette. A cleaning device consisting of the upper half of the tip of a blue 1 ml Etzbendorf micropipette is useful in this case. Add TE buffer (1059 mol Tris-C1p) to the yellow tip.
Fill with Sephadex G-25 in H7,5,1 mmil EDTA) so that the settled bed almost reaches the top of the pipette tip.

The test tube containing the mini-column is then spun in a centrifuge for 2
Spin at 00 rpm for 2 minutes. Remove the liquid from the receiving tube. The primer extension reaction is diluted to 40 pl with TE buffer and then applied to the mini-column.

Centrifugation is repeated for 2 minutes at 200 rpm and the eluate is transferred to an Etzbendorf tube for storage. Electrophoresis of the eluent on a polyacrylamide gel shows very little residual dATP, while all of the labeled oligonucleotide is recovered.

B. Trial RDNA? ttal! Polymer state DNA is extracted from peripheral blood lymphocytes or cells present in amniotic fluid, purine (Blin) and staff, u1%.
1c7) (f'fP(Nucl, Ac1ds
Res, ) 3.2303-2308 (l976)
Isolate by force test. To put it simply, 10-20m
Samples of 1 are centrifuged at 3.00 Orpm and the plasma or fluid fraction is removed by aspiration. The remaining amount of plasma was extracted by repeatedly washing the cell pellet with 0.9% NaCl. For extraction of DNA from blood, reticulocytes and old red blood cells were homogenized by addition of 2 cell sediment volumes of sterile water, and lymphocytes were
Collected by centrifugation at 00 rpm for 15 minutes. Lymphocytes and amniotic fluid cells were dissolved in 20 ml of lysis solution (0
, 0 mol Tris pH 7,5, 0,5% sodium dodecyl sulfate (sns), o , i mol NaCl 1,0,
001 mol EDTA, 100 gg/ml proteinase k) and incubated at 37 °C for 4 hours.
Incubation was for 8 hours. After dissolution, the solution was debroitenased by repeated extraction with chloroform-phenol (l:3) and dialyzed overnight at 4°C against 50 mmol Tris p) 18.10 mmol EDTA, 10 mmol NaCl. did. Nucleic acids were isolated by adding 2.5 volumes of ethanol and then centrifuging at 9.00 Orpm for 15 minutes. The precipitate was resuspended in 10 mmol Tris pH 7.5, 1% Le(7) EDTA and 50 ug/ml of heat-treated RNA5e [
RNASe A, Sigma St. Louis, MO, USA] for 30 min at 37°C and extracted with chloroform-phenol. DNA was collected as above by ethanol precipitation and subsequent centrifugation, and approximately 0.2 mg was collected in sterile water.
The solution was redissolved at a concentration of /ml.

Immobilization of sample DNA by C5 dot-blotting Sample DNA was denatured with NaOH and then sampled on nitrocellulose paper for hybridization detection. Since crosses with both the normal probe and the bell cell probe are necessary for the determination of a positive genotype, two dots from each patient must be prepared; the concentration of the DNA test sample is 1.
Determine by diluting 0 pl into 1 ml of TE buffer and reading 0D260 on a Varian 2200 spectrophotometer (Palo Aldo, Calif., USA). 10 micrograms of test DNA to 200
JLl in 0.5 mol (l) NaOH and denatured for 10 minutes at room temperature. after a while,
Prepare the filter. BioRad =
Trocellulose paper (No. 162-0116. Suchsend, Calif., USA) is cut to approximately 12×8 cm and moistened by gradual application to a dish containing 6×sscg buffer. The upper side is then moistened by dipping the paper. Paper that is not properly wetted will not perform consistently. After soaking for approximately 5 minutes, the paper was applied to a PyroRad filter device (BioRad) and
Then, firmly conclude. Then apply vacuum and tighten the screws.

Immediately before application of the sample, the base is neutralized by addition of 34 g 1 2 molar ammonium acetate, pH 5.0. Apply half of the test sample to each of the two wells on the dot-plot device. When the test sample volume is greater than about 50 pl, double all volumes.

After application of the sample, the filter was heated in a vacuum oven at 80°C for 20~
Dry smoke for 60 minutes. It is then dissolved for 50' in the next solution.
Precross in a sealed plastic bag for at least 10 minutes at C: 50 mmol sodium phosphate p
H6, 5X5SC 5X Denhardt's solution 250 pg/ml sonicated salmon sperm DNA (Sigma) The filters were then blotted dry with tear paper and cut into strips for hybridization. (At this point, the filter can be refrigerated for several weeks.) D. Hybridization of Labeled Oligonucleotide with Probe Molecule - For hybridization, prepare the following mixture: 450 JL
750 g of 20X NETu buffer 1507 TL of 20% dextran sulfate 75 L of deionized water 75 L of 100X Denhardt's solution 75 JL of 10% NP-40 detergent (Sigma) To this mixture was added the oligonucleotide prepared in the above section. Add l/10 of probe. The concentration of the final 19-mer probe is approximately 1 nanomolar. Addition of more probe than this was found to increase background. Proportionally less primer, template, and dG to prepare labeled probes for single or several reactions
TP and dATP can be used to scale down the labeling protocols detailed above.

A 0.1× reaction can be carried out in 10 pl and a 0.1× reaction can be carried out in 10 pl;
A 2X reaction can be carried out in 20 IL1... When volume permits, [alpha32P]-α-ATP does not need to be evaporated to dryness.

Place the filter strip in a plastic bag [e.g., Seal-A-Meal and Seal and 5a].
ve), etc.] 4. : Insert with the 1st (7) side open. Up to six different crosses can be carried out in one bag with sealing between the compartments. Several strips can be crossed per compartment; two are routinely performed posterior to posterior. The radiohybridization mixture is added with a Pasteur pipette or syringe with a 21 gauge needle and the last side of the bag is sealed. Crossing periods shorter than 30 minutes result in less than maximum hybridization, and periods longer than 1.5 hours lead to high background.

After hybridization, excess solution is removed with a syringe with a 21 gauge needle. This mixture can be used repeatedly. The strips were collected by excising them with a razor blade and transferred to a large plastic Petri dish (15 cm diameter).
and rinse into the cylinder with 5XSSC. They are then transferred to the bottom of a Petri dish and washed for 15 minutes in 6X SSC with gentle shaking on an orbital shaker.

After rigorous washing, the strips are blotted dry and transferred to a 15 ml prewarmed culture tube containing 14 ml 6XSSC. Wash two strips per tube. Place the tube back into the 57°C circulating water bath for 10 minutes. Pour off the liquid, add another 15ml of prewarmed 6XSSC, and place the tube back into the water bath for another 10 minutes. The tube is inverted intermittently during rigorous cleaning. Blot dry the strips and air dry. Attach them with one strip of double-sided tape to the top of the cancer paper and the other to its bottom.

Cover the foot lip with plastic wrap and coat with Kodak XAR-5 film and Cronex Lig
Autoradiograph at -70°C using a htning-Plus) intensifying screen. - After night exposure, an easily readable signal is obtained.

E. Results Two known samples (XY as a normal control and BG as a bell cell control) and a double-blind sample of IO were analyzed as above. The figures in the accompanying drawings show the results of the hybridization after 36 hours of exposure to X-ray film. Also,
Two non-essential positives, designated Ml 3A and M2B5, were found.
ve) Controls included. These are replicative forms of M13 pillions containing cloned fragments of normal DNA and bell cell DNA, respectively.

As can be seen, the normal signal consists of a strong reaction with the normal (Ao) probe and a weak cross-reactivity with the bell cell (S') probe. The bell red blood cell signal consists of a nonreaction with the normal probe and a strong reaction with the bell red blood cell probe. The heterozygote (sample 5.8.9) is characterized by reaction with both probes, as expected. This reaction is a normal reaction (
Sample xy, 4.6.7.10 and 1) and Ao and S
' can be easily distinguished by the intensity of the dots: the normal sample shows a fairly strong reaction with the A' probe, whereas the heterozygote shows a fairly strong reaction with the S' probe, sample 3 shows a rather strong reaction with the BG or sample 2, i.e. a lower confidence than the other bell cell DNA''A sample; this is due to the reduced amount of DNA in the applied sample (approximately 1-1-5 u- instead of 5 pg). Sample 2 also contained less DNA when analyzed by agarose gel electrophoresis.
shows. These cases demonstrate that correct genotyping can be easily performed despite varying amounts and purity of DNA. Furthermore, since this procedure is rapid, iterative decisions can be made conveniently.

These samples were received by mail and assays were performed. Several individuals have read this film and arrived at the same diagnosis. Next, the genotype determined by Southern analysis (RFLP) in the laboratory where the test sample 1 was purified was sent over the phone! Star. Blind diagnosis was correct in 100% of cases. This was repeated in two cases using 38 unknown samples with identical results.

Example 2 Probes for the detection of mutations in bell-shaped red blood cells For the detection of mutations in bell-shaped red blood cells, the following three probes were prepared using the phosphoramidite method and Applied Biosystem Type 380B DNA synthesis. Synthesized using the equipment: (l) 5 °GATGATGCGATGATCC3'
,,,, (l6L) (2) 5'GGATCATCGCATCATC
GCCCGGCAGACTTCTC CTCAGGAGT3' ,,, (41A'
) (3) 5 'GGATCATCGACATCA
TCATCGCCCGGCA GACTTCTCCACAGGAG T3°,,, (41S') The labeling reaction was performed by injecting 10 pg of small probe 16L into 12.
20 pg long probe dissolved in 1 L buffer,
It was carried out by mixing with 41A' or 41S'. To this was added 60 gg of aminoethyltrioxysaran PEG-biotin compound AMT-PEG-piontin (
(its structure is described below) and the final volume was 160 p l with lO mmol sodium borate pH 8.
It was adjusted to The whole mixture was then divided into four tubes and heated by hand-held UV lamp UVL-
Irradiation was performed for 60 minutes using a 56-type "BLAK-RAM". After irradiation, labeled oligonucleotides were separated from unlabeled material and starting reagents by gel electrophoresis. A 15% polyacrylamide gel containing 7M-urea was run at 250 volts for 15 minutes. The dimensions of this gel were 14 cm long, 19 cm wide and 1.5 mm thick. This gel is U
Three bands were shown under V shadowing (the gel was placed on a fluorescent screen and observed under UV light), the slowest moving band was taken as the cross-linked oligonucleotide, In contrast, the other band migrated with the unlabeled oligonucleotide standard.

Then, three sets of 41″I were cut from the gel and 1.5ml
into an Eppendorf tube and add 1 ml of 20XS
Extracted with SC (20XSSC is 3.0%, llz/N
aC110.3 mol/sodium citrate). The gel mixture was vortexed and incubated for 16 hours at 37°C on a gentle mechanical shaker. To further purify the probe, the gel was removed by centrifugation. The precipitate was washed once with distilled water and the pooled supernatant was applied to a Nensorb column (
DuPont). Washing and elution in methanol/water were performed as described by the manufacturer. Fractions were then run on Savant Speed-Vac.
The mixture was evaporated to dryness in a vacuum (peed-vac) apparatus.

Testing for Biotin The different gel fractions were tested for biotin as follows. A 2 liter sample was diluted in 231 liters of water and the IgI was splatted onto nitrocellulose paper (Bio-Rad). Filters were air dried overnight and dried in a vacuum oven at 80° C. for 30 minutes. Filters were prehybridized for 90 minutes in a blotto at 50°C. The plot was adjusted to 50ml with water2,
5g carnation non-fat dry milk, 15ml 20XSSC and 20ml
of sodium pyrophosphate. Then,
Filters were detected with a BRL kit containing streptavidin and polyalkaline phosphatase according to the manufacturer's instructions. To test the hybridization potential of such probes, M1 containing inserts of either the 1F normal globulin gene or the bell cell globulin gene
Three phage DNAs were spotted and hybridized as described in this example. Crossbreeding and washing at 64°C? '/l, then BRL them (Betheseda Research-Laboratory-1 Betheseda, Maryland, USA)
Detected according to the protocol. The results revealed that the probes thus prepared exhibited both hybridization ability and specificity.

FIG. 2 shows M13 replication forms from viroids containing cloned fragments of normal (A) or bell cell (S) human beta globulin dot-plotted as described. The nanogram amount applied to the spot is shown along the left side. The top four dots contain normal (A) sequences; two sets of four contain sickle cell (S) sequences. C dot is a DNA detection kit)
Positive control consists of 200 pg of biotinylated lambda DNA supplied as part of (BRL). As described herein, the strip on the left was hybridized with a normal specific probe (A') and the strip on the right was hybridized with a sickle cell specific probe (S').

Synthesis of AMT-PEG-Biotin AMT-P which describes the structural formula of AMT-PEG-Biotin
Preparation of EG-biotin required l-amino-17-N-(biotinylamino)-3,6°9.12.15-pentaoxaheptadecane. This was achieved by the following four steps: (l) 3,6.9,12.15-pentaoxaheptadecane (X)-1,17-thiol ditosylate was synthesized.

(2) A 1,17-diphthalimide derivative of (X) was prepared.

(3) A 1,17-diamino derivative of (X) was prepared.

(4) A 1-amino-17-biotinylamide derivative of (X) was prepared.

50 g of hexaethylene glycol (0,177 mol) and 6
To a stirred solution containing 4 ml triethylamine (39,36 g) was added 7
3.91 g of p-toluenesulfonyl chloride (0,
389 mol) was added dropwise over 2.5 hours. The mixture was then filtered, and the filtrate was concentrated in vacuo. The resulting heterogeneous residue was suspended in 500 ml of ethyl acetate and filtered. The filtrate was then concentrated in vacuo to give a yellow oil, which was triturated with eight 250 ml portions of warm hexane to remove unreacted p-toluenesulfonyl chloride. The resulting oil was then concentrated under high vacuum to give 108.12 g of yellow oil (quantitative yield).

Analysis: Calculated for C26H38011S2 Calculated value: C
, 52, 87, H16, 48 actual measurement value: C152, 56,
) (,6,39108 g of 3.6,9,12.15-pentaoxaheptadecane-1,17-thiol ditosylate) (0,183 mol), 74.57 g of potassium fatalimide (0,403 mol) and 700 ml of dimethylacetamide at 160-1
Heated to 70'O for 2 hours, then cooled to room temperature. The precipitate was filtered, rinsed with water and acetone, and 53
．． 05 g of product was obtained as a white powder, which was
It was dried at °C (0°1 mm). Melting point 125-126℃
.

The :jS2 crop of product was obtained by vacuum evaporation from a solution of dimethylacetamide and the resulting precipitate was dissolved in ethyl acetate,
Washed sequentially with water and acetone.

The resulting white powder was dried at 55° C. (0.1 mm) to yield an additional 9.7 g of product 11). Melting point 125
．． 4-126.5°C6 combined yield of products j, Y is 6
2.82 g (68% yield).

Analysis: (1st harvest) Calculated for C28H32N2o9111/2H20 Calculated value: C161.19, H16.05, N, 5.09 Actual value: C161,08; H16,15,N.

5.05 Analysis = (Second Harvest) Calculated value for 20g of C2s Ha 2 N: C1
62, 21, H2S, 97. N, 5.18 Actual value: C161,78, H2S, 15. N, 5.13 60 g of 1,17-phthalimide-3,6°9, t2.
ts-pentaoxaheptadecane (0,118 mol),
A solution containing 14.8 g of hydrazine hydrate (0.296 mol) and 500 ml of z-tanol was heated for 3 h in an oil bath with mechanical stirring.The mixture was then cooled. , then filtered. The filter cake was washed four times with 300 ml portions of ethanol.

The combined solution was concentrated to give 32.35 g of a yellow opaque glassy oil. 150-200℃ (0,0
Evaporative distillation at 1 mm) gave 22.82 g of pale yellow oil (69% yield).

Analysis: Calculation for C12H28N205 ・l/2H20 Direct: C149,80, Hl 10.10°N, 9.68 Actual value: C150,36; H19,58; N, 9.38 [W, Kel 7 (Ke rn), S, Rock wasp (
Iwabachi), H. Sato and V.
, Po-y-(B o h m e r) + 7
(Makrol, Chem,), 1.0,253
9 (l979)].

75 ml C7) A solution containing 7.2 g of 1,17-diami2-3.6,9,12.15-pentaoxaheptadecane (25 mmol) in DMF was mixed under an argon atmosphere with 3.41 g of N- Treatment was by addition of succinylbiotin (10 mmol) in portions over 1.0 h. The resulting solution was stirred at ambient temperature for 4 hours. TLC visualized with spray reagent of dimethylaminocinnamaldehyde (CHC of Si02.70: 10.1
13 CH30HeNHa OH) showed very good conversion to the new product (Rf=0°18).

The resulting mixture was divided in half, each half was absorbed onto 5i02, and 500 g of 5io2-60 (230-
70:10゜1c7) CHC13-CH30H-
Eluted with eNH40H solvent mixture. Fractions containing the product were pooled and condensed to give 2.42 g of a gelatinous waxy solid. The product was precipitated as a solid from impropanol ether, washed with hexane and dried at 55°C (0.1 mm) to yield 1
．． 76 g of white powder was obtained (35% yield).

Analysis: C22Ha 2 N40y S 3/2H2
Calculated value for 0 Ni: C149,51; H18,50; N, 10.4
9 Actual measurement value: C149.59, H18.13, N, 10.3
9 380 mg C7) l-amino-17-N-(biotinylamide)-3,6,9°1 in 3 ml DMF
2. A solution of 15-pentaoxaheptadecane (0.75 mmol) under an argon atmosphere.

It was treated with 146 mg of N,N-carponyldiimitazole (0.9 mmol). TLC(Si20.4
:l of CHCl3 CH30H, visualized with a spray reagent of dimethylaminocinnamaldehyde), biotinylamine (Rf=0°1) is imidazourea (Rf=
0.5), indicating complete conversion. The reaction mixture was then mixed with 193 mg of 4°-aminomethyl-4,5
',8-trimethyl-bussoralen (commercially available from HRI, California, USA) (0,75
mmol) and 2.7 μl triethylamine (l,
The resulting mixture was then heated to 60° C. overnight.

TLC (5302, 4:1 CHC]3-CH30H)
showed that the imidazolide was converted to a new product (Rf=0°52), which was Uv fluorescent and tested positive with the dimethylaminocinnamaldehyde spray reagent. Removal of the solvent in vacuo yielded a gelatinous oil, which was dissolved in CH30H and 5i
02. The impregnated solid was then flash-chromatographed on 60 g of 5i02 60 (230,400 mesh) with 9:1 CHC13CH.
Eluted with a 30H solvent mixture. The fractions containing the partially purified product were poured into 60 g of 5i02
chromatography and eluted with the same solvent system.

Melting point: Slow decomposition, 129.5°C-149.50C
0 Analysis: C3s Hs s Ns Ot IS * H
Calculate total 3 for 20 = [I1Ili: C156,49,H,7,1
1,N, 8.67 Found: C156,58, H17,16,N, 8.53 The above examples and description provide representative embodiments of the invention. Obviously, many modifications may be made without departing from the inventive characteristics of the invention.

4.14 Brief explanation of fi'i7 Figure 1 shows the normal form of the human beta globulin gene jE.
FIG. 2 is a photograph of a dot-plot obtained from the hybridization of a radiolabeled probe according to the invention applied to the detection of mutated forms of calcaneus and calcaneus cells. FIG. 2 is a photograph of a dot-plot according to Example 2.

(2) Men's Palpitation 5/No change in content) A'S'A'S'MI3A
@ ・ ・ 6M13S Association ・
Cram school 7 86 taste -・ 9 2 ・ ・ , #1゜4 Fist 9
・ ・ 1 ・Go to 6 1' Continued 111) Official document (Method) April 1988 241J Commissioner of the Patent Office Kuro 11 Akio Tono Crossing Act 3, relationship with 11 cases involving arrest Patent applicant name Name Molecular Thai Agnostics Incorporated 6, Object of Odori-dome 7, Contents of Yu-dome As shown in the appendix (l) On the last line of page 100 of the specification, there is a photo of ``J'' in the photo.
In the diagram, correct it as l.

(2) On page 101, line 3, change the text ``with one photo'' to ``with illustration r''.

(3) Figures 1 and 2 of the drawings will be corrected as shown in the attached sheet.

that's all

Claims (1)

[Claims] 1. The following steps: (a) immobilizing DNA in single-stranded form from a sample of eukaryotic genomic DNA in a defined zone of a sheet of a solid support; (b) ) treating the sheet of support to substantially block unfilled binding sites thereon; (c) incubating the support with a solution containing probes of oligonucleotides under hybridization conditions; said probe has a base sequence that is precisely complementary to the base sequence of interest in the sample; (d) separating a solution of excess probe from said support; (f) washing the support with sufficient stringency to remove substantially all of the probes that have hybridized to sequences other than the sequence; and (f) removing the presence of hybridized probes in the zone of the sheet of the support. Detecting eukaryotic genomic DNA comprising:
A method of detecting a specific base sequence in a sample by nucleic acid hybridization. 2. The method of claim 1, wherein the support on which single-stranded sample DNA is immobilized is incubated with the oligonucleotide probe for a period of less than about 4 hours. 3. The concentration of the probe in the solution incubated with the support on which single-stranded sample DNA is immobilized is about 10 pM to 10 nM.
The method described in section. 4. The sample is a liquid mixture that is treated to denature the DNA therein and applied to a sheet of solid support, and the residual liquid or sample removed by vacuum is transferred to the sheet of solid support. 4. A method according to any one of claims 1 to 3, wherein the method is applied to and denatures the DNA therein in situ. 5. The probe comprises a detectable label, said label being detected in said zone of the sheet of support to detect the presence of hybridized probes.
The method described in any of the paragraphs. 6. The steps of: (a) obtaining a sample containing nucleated cells from a human patient; (b) isolating essentially unfractionated genomic DNA from the cells contained in said sample; and (c) ) denaturing an isolate of unfractionated genomic DNA by treatment with a base to form a single-stranded form of genomic DNA; (d) neutralizing the liquid isolate treated with said base; e) applying said neutralized liquid isolate in two separate defined zones to a sheet of solid support, said sheet of support capable of immobilizing single-stranded DNA; and applying a vacuum to the opposite side of the sheet of support to remove any remaining liquid; (f) contacting the sheet of support with a precrossing solution for a predetermined period of time, the precrossing solution being applied onto the sheet of support; (g) cutting said sheet of support into two sections, one of said sections containing the first zone and the other containing the second zone; (h) contacting one of the sections of the sheet of said support with a hybridization solution containing an oligonucleotide probe having a base sequence exactly complementary to the normal sequence of the gene in question; The other section of the body sheet is contacted with a hybridization solution containing probes of oligonucleotides having base sequences exactly complementary to the mutated sequence, each of said probes containing a detectable label; ) contacting each said support section with said hybridization solution and incubating for less than about 4 hours at a temperature suitable for hybridization between said probes and their complementary sequences; (j) said hybridization solution; (k) removing substantially all of the probes that have hybridized to sequences other than precisely complementary sequences of the sheet of support with sufficient stringency to remove substantially all of the probes that have hybridized to sequences other than exactly complementary sequences; washing the sections; and (l) detecting the presence of probe labels in the first and second zones of the cut sheet of support. A method for detecting normal or mutated genes by dot-plot hybridization. 7. The method of claim 6, wherein the normal gene in question is the human beta globulin gene, and the mutated form thereof is one that causes human sickle cell anemia. 8. Components: (a) a first synthetic oligonucleotide, said first oligonucleotide comprising: (i) a hybridization region specific for the detection of a particular nucleic acid sequence; and (ii) located adjacent to said hybridization region. a plurality of nucleotide residues, and (b) a second synthetic oligonucleotide, when the first oligonucleotide and the second oligonucleotide are combined, form a double-stranded region and a single-stranded hybridizable region. and (c) a label, said label being attached to a double-stranded region. 9. The probe according to claim 8, further comprising a photoreactive intercalator attached to the double-stranded region. 10. The probe according to claim 9, wherein a label is attached to the intercalator. 11. Claims 8 to 11, wherein the label is biotin.
The probe according to any one of Item 10. 12. The probe according to any one of claims 8 to 11, which includes [there is a gene sequence] or [there is a gene sequence] or [there is a gene sequence].