JPH0665320B2 - Novel gene detection method - Google Patents

Description

FIELD OF THE INVENTION This invention relates to assays or techniques for diagnostic detection in genetic material such as eukaryotic, prokaryotic or viral genes.

Prior Art, Problems to be Solved by the Invention A human gene using a genomic Southern blot with any restriction endonuclease polymorphism such as sickle cell anemia and some point mutations in the ras oncogene For the detection of existing mutations, currently available analyzes are Geever, RF, Wilson, LB, Nallaseth, FS, Mil.
ner, PF, Bi-ttner, M and wilson, JT (1981), "Direct identification of sickle cell anemia by blot hybridization", Proc. Natl. Acad. Sci. US .78 , 5081 , 5085 and San
tos E., Martin-Zanca, D., Reddy, EP, Pier-otti, MA, D
ella., Porta, G., and Barbacid, M. (1984), “Kr occurring in tumor cells in non-normal tissues of patients with scaly cell lung cancer.
Sc mutations important for malignant transformation of as oncogene "Sc
ience, 223 , 661-664, and is a synthetic oligonucleotide as a radioactive probe such as the mutated ras oncogene β-thalassemia and most of the alpha 1 antitrypsin is Guerr- ero, I., Villasant
e, A., Corces. V. and Pellicer, A. (1984), "Activation of CK-ras oncogene by somatic mutation in gamma-radiation-induced mouse lymphoma," Science, 225 , 11
59,1162 ， Kidd, VJ, Wallace, RB, Itakura, k.and Woo
d, SLC (1983), Narure , 304 , 230-234, and Woo, SL
C., Kidd, VJPam, ZK., Wallace, BR, and Itakura, k. (1
983), "α-1-antitrypsin deficiency and emphysema; identification of reverse homozygosity by direct analysis of mutation site of chromosomal gene". Cold Spring Harbor Labor for application of recombinant DNA to human diseases
atory, Vol. 14 , page 105-112 Bambury Report (Bam
bury Report). On the other hand, the recently developed DNA denaturing acrylamide gradient gel has a characteristic rearrangement-based analysis of a DNA heteroduplex containing a single-base mismatch, Myers, RM, Lumelsky, N., Lerman, LS, and
Maniatis, T. (1985), Detection of single base substitution in total genomic DNA, Nature , 313 , 495-498. Further, as a research method for digesting a heteroduplex formed between a mutant and a wild type viral DNA used for detecting a mutation in viral DNA with SI nuclease, Shenk, TE , Rhodes, C., Rigby, PNa
nd Berg, P. (1975), Proc. Natl. Acad. Sci, US 72 , 989-993
There is a description in. However, Samurai Myeres, RM ... ibid (198
As described in 5), some single-base mismatches are cleaved by S1 nuclease, but many others are not. It is an object of the present invention to provide an alternative technique in the diagnostic detection of point mutations such as single base substitutions in eukaryotic genes and the like.

It is another object of this invention to provide an improved assay for clinical diagnostic detection of point mutations in human genes, such as the detection of single base substitutions in eukaryotic genes.

Moreover, the assay of the present invention does not require the synthesis of specific oligonucleotides and the use of complex blot or gel electrorheological detection techniques, thus providing a much simpler assay than those currently available. Is the purpose of.

In addition, codon 12 or 61 of human ras oncogene
It is yet another object of this invention to provide a technique or analysis for the localization of point mutations or other mutations in eukaryotic genes such as the localization of single base substitutions in E. coli.

Like the nature of specific single base substitutions involving transversions and transitions in the human ras oncogene, there is a purposeful restriction endonuclease polymorphism for the characteristics of point mutations in eukaryotic genes etc. It is yet another object of this invention to provide synthesis of oligonucleotides of existing, limited sequence or isolation of mutated genes and sequence independent techniques or analysis.

In addition, general screening for detecting the presence of a gene containing a point mutation in human cells, tissues or individuals at adult and fetal levels, such as the screening of human tumors for the presence of the ras gene containing single base substitutions. It is yet another object of the invention to provide a method.

Two alleles that differ by small genetic alterations, such as point mutations in the same cell, that determine the relative expression of both normal and mutant ras alleles that differ in single base substitution in the same human tumor cell. It is yet another object of this invention to provide an assay or technique for quantitative analysis of the relative expression of genes.

How the object of the present invention is achieved is described in the embodiments of the specification of the present invention and FIGS. 1A, 1B and 1C, and FIG.
This will be described with reference to FIGS.

Means for Solving Problems An antisense RNA molecule, which is a single-stranded gene having complementary binding properties to a specific gene synthesized in vitro, such as by the SP6 polymerase transcription system, is used as a point mutation in a eukaryotic gene. It can be used for detection of base substitution. The assay involves or consists of solution hybridization of total cellular RNA obtained from cells expressing the mutant gene with antisense, labeled RNA. The RNA-RNA hybrid is then RNa
Digested with a mixture of se and the digested hybrids are analyzed by gel electrophoresis, such as a denaturing acrylamide gel. If the RNA-RNA hybrid contains a single mismatch, the RNase recognizes the single mismatch and cleaves the heteroduplex, producing two short hybrids. In contrast, homoduplexes remain resistant to digestion. This technique is applied to certain human genetic diseases, such as certain β-thalassemia, sickle cell anemia, and proproteinemia that result in amino acid substitutions in the encoded protein as a result of point mutations. And provides an easy and rapid analysis in searching for genes containing α-1-antitrypsin deficiency as well as genes containing single point mutations in their coding regions such as the human lath oncogene.

The method of the invention described above provides a simple assay in that it does not require the synthesis of specific oligonucleotides or the use of complex blot or gel electrophoretic detection techniques. Furthermore, the present invention also includes a method of digesting with a corresponding inexpensive RNase or a mixture thereof.

This invention promises a powerful search technique that can be applied to diagnostic screening for common genetic diseases. For example, globin disease due to a single mutation could hitherto only be analyzed using synthetic oligonucleotides corresponding to gene regions containing point mutations. These analyzes are used to search for mutant ras genes in beta thalassemia or human tumors.

As mentioned above, the analysis of the present invention is directed toward detecting any point mutations or other structural mutations to oligonucleotide technology which requires a priori knowledge of the nature of the gene and the localization of the mutation. You can The technique or analysis of this invention using RNA-RNA hybridization allows the detection of any point mutations or other structural mutations in genes not known to contain mutations. Any human tumor containing a mutated ck-ras gene can be searched, for example, before identifying the presence of this mutation by other techniques. The present invention also relates to the ck-ras oncogene containing mutations at codon 61 and another site (codon 12), other ras genes (Ha-rvey and N-ras), and sickle cell anemia. Any other human gene having a mutation such as the globin gene can be searched for.

In addition, this method searches not only for single base substitutions in the coding region of the transcribed gene, but also for single base deletions or additions, and mutations such as one or more substitutions, additions or deletions of nucleotides. Show that you can. Furthermore, this method not only allows the search for these mutations, but, in addition to the nature of the mutations, i.e. the nature of the specific nucleotides involved in the mutations, this method also derives from It also allows searching for characterizations of their position in the gene.

In the practice of this invention, it is necessary to prepare cellular RNA, and the invention is applicable to these genetic mutations in the transcribed region, such as mutations in the coding region. It is shown to occur in muteins. For example, some beta-thalassemias are the result of mutations in the promoter of the beta-globin gene, and thus they cannot be searched because the mutations may not be present in globin gene transcription. However, the applicability of the practice of this invention can be extended and also applied to the search for DNA-RNA mismatch hybrids.

For example, the invention can be practiced on all genes regardless of the localization and nature of these mutations. Therefore, the practice of this invention can be conveniently and rapidly applied, especially in assays using small amounts of cells obtained from biopsies that are important for fetal diagnosis.

From the above findings of the present invention, a single chain gene showing a complementary binding property to a specific gene is allowed to act on a gene-containing test solution which may contain a short gene mutation, and then this is
At least the formed homoduplex of the hybrid duplex is not substantially cleaved, and the heteroduplex of the mutation or mismatch is substantially cleaved after digestion with an enzyme capable of cleaving near the location of the mutation or mismatch. We have completed a method for detecting genes by separating and analyzing the digested genes from homoduplexes that are not affected by.

In describing the practice of this invention, the ras gene will be emphasized. The ras gene is thought to be involved in normal cell growth in eukaryotes, and much evidence suggests that somatic mutations in these genes cause canceration in higher vertebrates including humans. (Land, H., Parada, LFand Weinberg, RA (1983), S
cie-nce, 222, 771-778). The nature of these mutations has been demonstrated to be a single base substitution appearing in a mutant ras protein at a single amino acid at position 12 or 61 which differs from the normal protein (Varmus, H. (1984), An.
n, Rev, of Genetics 18, 553-612). The appearance of different point mutations in the ras oncogene, which was activated in spontaneous cancers and was activated as a result of in vitro carcinogenicity experiments,
Most of the amino acid substitutions at or near these two sites appear in the activation of oncodin potential in the ras gene (Fasano, O., Aldrich, T., Tama-noi, R.,
Taparowsky, B., Furth, M. and Wiler, M. (1984), Proc.Nat
l.Acad.Sci.USA 81 , 4008-4012, and Seeburg, PHColb
y, WW, Capon, DJ, Goed del, DVand Levinson, AD (1
984), Nature , 312 , 71-75).

This invention is a useful method for the diagnostic detection of these mutations in malignant tumors. In accordance with the practice of this invention, the techniques or analyzes described below are provided that are based on the ability to recognize and degrade single base mismatches in pancreatic ribonuclease (RNase A) RNA heteroduplexes. The techniques described therein are useful for determining the nature and location of mutants as well as the presence of mutations in the ras gene, and also for searching for the presence or absence of normal alleles. It is useful and also allows evaluation of the relative expression of both normal and mutant ras alleles in the same tumor cell. The methods and techniques used as examples of this invention are as follows, but in no way limited thereto.

Examples Tumor and Tumor Cell Line Identification SW480 is a cell line established from human colon fibroids (cell line), and was obtained from the American National Standards Institute for Strains (ATCC). Cslu-1, SK-LU-1, SK-CO-1, PR
310 and PR371 are human lung and colon tumors and tumor cell lines and their origins are Perucho, M., G
oldfarb, M., Shimizu, K., Lama, C., Fogh, j.and Wigler, M.
(1981), Cell 27 467-476 and Nakano, H., Yamamoto.F., Nevi.
lle, C., Evans, D., Mizuno, T.and Perucho, M. (1984), Pro
c, Natl. Acad. Sci. USA .81 , 71-75, as previously described. PR310 and PR371 tumor cells were subjected to trypsin treatment of the excised tumor mass and single-cell suspension obtained in Darbetuco's modified essential medium (DMEM) containing 10% fetal bovine serum was plated on nude mice. Cultured as grown tumor cells. The cells were subcultured for 2 passages, single colonies were separated, and large-scale culture was performed. Human fibroblasts were obtained by trypsinizing small pieces of neonatal foreskin and plating cell suspensions in the medium described above.

RNA extraction Total cellular RNA was collected from Winter, B., Palatnik, C., Wi-lliams, D., Well
s, JRB, Coles, LSand Godrdon, JS (1985), J.Cell B
Guanidine-Hcl / as previously described in iol.
Prepared from cultured cells by continuous extraction with ethanol and phenol. RNA has its tissue guanidine-HCL
It was prepared from cancerous tissue in the same manner except that it was first ground into a fine powder in liquid nitrogen before being suspended in solution.

Recombinant plasmids and in vitro transcription Four recombinant plasmids, pA, used in this study
K1 Ngly, pAK1 Mcys, pAK2 Ngln and pAK2 Mhis
Is Melton, DA, Krieg, PA, Rebagliati, MR, Maniati
s, T., Zinn, K. and Gre-en, MR (1984), Nucleic Acid Re
s . As seen in 12, 7035-7056, SP6 charge RNA
A plasmid vector containing the polymerase transcription promoter, PR310 or PR371c-k-ras containing the first or second coding exons cloned into pSP65, and also these authors of radioactive RNA probes Describes synthesis. This plasmid is Yamamoto, F., and Perucho, M. (198
4), Nucleic Acids Res. 12 , 8873-8885,
Off-the-shelf constructs (PΦN1N2N3N4H and PΦD1D
2D3D4H). Instead of the above radioactive substance labeling, a labeling substance such as an enzyme, a fluorescent substance or biotin in a conventional method may be used for labeling.

Hybridization and RNase digestion Hybridization is performed using an appropriate RNA sample 1-2 mg / m
30 μl of hybridization solution (8
0% deionized formamide, 0.4M Nacl, 1mM EDTA and 40m
M PIPES pH 6.7) and 1.0 × 10 ⁵ cpm of 32p labeled RNA probe. 85 of the solution
The mixture was heated at 0 ° C for 5 minutes, immediately transferred to a water bath maintained at 50 ° C, and incubated for 12 to 16 hours.

Following hybridization, 300mM NaCl, 5mM EDT
40 μg / ml RNase in A, 10 mM Tris-Hcl pH 7.5
A (type III A Sigma) 0.3 ml and 2 μg / ml RNase T1 (grade
IV sigma) was added. 12 minutes, 60 minutes, 120 at 34 ℃
After 20 minutes, 20 μl SDS 10 μl and 10 mg / ml proteinase K
The digestion was terminated by the addition of 5 μl (Beckmam Inc.) and further incubated for 15 minutes at 37 ° C. Carrier tRNA 10
μg was added, followed by extraction with phenol-chloroform and ethanol precipitation using 300 μl of aqueous phase. The precipitated RNA was washed twice with 70% ethanol, and gel loading buffer (97% deionized formamide, 0.1% SDS and 10 mM Tris Hcl pH 7.6) 2
It was dissolved in 0 μl, heated at 85 ° C. for 2 minutes and cooled on water. Samples (10 μl each) were loaded onto an 8% acrylamide 7M urea gel, electrophoresed at 700 V and the gel was transferred to an X-ray film at -70 ° C.

In FIG. 1A, the method of the present invention for detecting a single base substitution is schematically shown. Human cK-ras gene region around the first and second coded exons (indicated by a rectangle) and codons 12 and 61 of protooncodin (cK-ras)
The positions and sequences of PR310 and PR371 oncogenes are indicated. The distance from the mutation present in codon 12 of the PR371 oncogene to the end of the first exon expressed in base pairs (bp) and the mutation at codon 61 of the PR310 oncogene to the end of the second exon The distance displayed in base pairs to is shown. These values are
It is the same as the nucleotide present in mature cK-ras mRNA. The calculation was performed by including the nucleotides of the intervening sequence. cK-ra
s oncogenes PR310 and PR371 tumor Pst IS
The au 3A 280 bp and Hind III-Xho 1 560 bp fragments were subcloned into the PstI-BamHI and HindIII-SalI site of pSP65, respectively. The antisense cK-ras RNA molecule is either Pst I (the first code decoding exon) or Hind III.
After linearizing the plasmid with (second decoding exon),
Transcribed in vitro with SP6 RNA polymerase. Radioactive label as single-chain gene labeled with radioactive substance
RNA was hybridized with PR310 or PR371 whole cell RNA. cK-ras mRNA (wavy line) and antisense
The nucleotide sequences of the 12th and 61st codons of both cK-ras RNAs are shown. Rectangles and straight lines represent anti-RNA sequences complementary to the decoding exons and introns, respectively. The arrow indicates the polarity in the 5'to 3'mRNA. The letters refer to restriction endonuclease sites. B: BamHI; Ba: BalI; H: Hind III; P: PstI; S: Sall; S
a: Sau3A; St: StuI; X: XhoI; The restriction site in parentheses represents the original site before substitution by molecular linkage.

Figures 1B and 1C illustrate the analysis of cK-ras / antisense cK-ras ribonuclease resistant RNA hybrids corresponding to the first and second cryptic exons, PR310 or as indicated on whole cell RAN. PR371
Prepared from tumor cells and shown in Figure 1A. 32P-labeled antisense c-directed by plasmid
Hybridized with K-ras RNA. Following hybridization, the sample is incubated with a mixture of RNase A and RNase T1 for 12, 60 or 120 minutes,
The resistant fragments were then analyzed by electrophoresis on a denaturing polyacrylamide gel as described above.

Figures 2A and 2B illustrate the analysis of RNase-resistant RNA hybrids corresponding to the cK-ras first coded exon, and RNA extracted from the indicated human cells was PAK1Ngl.
Hybridized with 32P-labeled anti-RNA directed by y, for 12, 60, 120 minutes (shown from left to right)
Digested and electrophoresed on a denaturing acrylamide gel as previously described. Also, genomic DNA for Southern blot hybridization analysis was extracted from PR371 tumors grown in 2 or 12 passage nude mice and derived from 12 passage tumors and separated three times separately. Extracted from cell clones. Four different N1H3T3 cell clones transformed with DNA extracted from passage 2 tumor and control 3T3 cells were observed in the same assay. Digest about 8 μg of each DNA sample with PstI,
Electrophoresed on 0.8% agarose gel. After conversion, the nitrocellulose filter was derived from Lambda LDH-15 P
R371c-K-ras oncogene EcoR-I / SmaI fragment 2.6K
It hybridized with nickel-translated pKRSm-2.6, which is a recombinant plasmid containing ilobese.

Results RNase A does not substantially cleave at least the formed homoduplex of the hybrid duplex, and the RNA heteroduplex containing mismatches such as mutations or single bases is not generated near the mutation or mismatch localization. Cleave selectively.

Human lung adenomas PR310 and PR3 formed in nude mice
The cK-ras oncogene activated at 71 was previously isolated (Nakano et al, 1984, ibid). PR
The 371 oncogene contains a mutation in the first coding exon (cysteine instead of glycine at codon 12) as described in Nakano et al, 1984, ibid. Include a mutation in the second cryptanalysis exon (histidine instead of glutamine at codon 61) (Yamamoto and Perucho, 1984,
See the same book). The oncogene subgenomic fragments obtained from these tumors were oriented in the appropriate direction to direct in vitro synthesis of antisense RNA molecules complementary to the cK-ras first and second coding exons. It was cloned into a plasmid vector.

Thus, four types of plasmids were generated (see figure). pAK1Ngly and pAK1Mcys directed the synthesis of an RNA molecule containing 114 nucleotides of the first coding exon from the splicing acceptor site to the Sau 3A site. Therefore, except for the nucleotide corresponding to the first base of the 12th codon, the sequences of the RNA molecules directed by these plasmids were the same. pAK1Ngly
Directed RNA containing a cytosine at this position, whereas the RNA directed by pAK1Mcys contained uracil. Similarly, pAK2Ngln and pAK2Mhis directed the synthesis of an antisense cK-ras RNA molecule that was identical to the entire second coding exon except for the third group at codon 61. pAK2Ngln dictates RNA containing uracil, whereas RNA dictated by pAK2Mhis contained adenine.

Radiolabeled antisense RNA molecules synthesized in vitro, directed by these plasmids, were added to PR31.
0 and PR371 tumor cells were hybridized to total cellular RNA prepared. The hybridized RNA molecules were digested with a mixture of RNase A and RNase T1 and electrophoresed on a denaturing acrylamide gel. PR310c-K-ras
When hybridizing antisense RNA complementary to the first coding exon of the oncogene with PR310 RNA,
A protected RNA fragment consisting of 114 nucleotides (predi with a predicted radioactive band corresponding to a resistant RNA molecule)
cted 114 nucleotide protected RNA fragment). In contrast, when the same labeled RNA molecule was hybridized with PR371 RNA, the mixture of RNases produced a small subband and degraded the heteroduplex. In the short-term incubation, the subband consisted of a product of the expected size produced by the mismatch cleavage of codon # 12. For long-term incubation, these subbands were shown to be smaller in size, indicating that RNase continues to cleave double-stranded RNA molecules from the ends. Conversely, using the mutant antisense ras RNA dictated by the plasmid pAK1Mcys, the RNA heteroduplex produced by PR310 RNA and hybridization was cleaved while the PR371 RNA was mediated by PR371 RNA. The resulting hybrid was largely protected.

The same method was used to study the behavior of RNA heteroduplexes or homoduplexes corresponding to the second coding exon of the cK-ras oncogene. If normal antisense lath RNA is used as a hybridization probe, PR371R
NA produces hybrids that are resistant to cleavage, whereas PR310 RNA produced heteroduplexes that are partially cleaved. When the mutant antisense ras RNA was hybridized with either PR371 or PR310 cellular RNA, the RNase mixture produced a small size of the hybrid, which is consistent with the size expected in the fragment resulting from cleavage of the hybrid at codon 61 at mismatch. Decomposed into minor fragments.

The same results were obtained, although RNase A alone was used for digestion, yielding a slightly higher backland. These results indicate that RNase A is able to recognize and cleave single base mismatches in RNA heteroduplexes, and this property searches for mutant ras genes in human tumor cells, It has been demonstrated that it can be used for characterization. It was also shown that the same mutations already characterized by arranging the oncogene isolated from transformant N1H3T3 induced by PR310 and PR371 DNA were present in these human tumor cells. Indicated.

Of the four types of single base mismatches we analyzed, U
The -U pair was found to be the most resistant to degradation. However, the other three individual mismatches: U
-C, AG and AA could be completely digested. This is because PR310 and PR371 tumors were homozygous for the mutant allele.
Alternatively, RNA obtained from several N2H3T3 clones transformed with the mutant oncogene of PR371 tumors was used for analysis. After the treatment of RNase A after hybridizing a fixed amount of the probe with a certain amount of the gene-containing sample, the density of the band quantitatively indicates the amount of the gene in the gene-containing sample.

As described above, although RNase A is described, the present invention is not limited as long as it is an enzyme capable of satisfactorily decomposing mismatch in heteroduplex, which is an RNA selective cleavage endonuclease, More specifically, at least an enzyme that selectively hydrolyzes the 3'-5'-phosphodiester bond of the pyrimidine nucleotide represented by C or U, such as a pyrimidine selective cleavage endonuclease (Pyr-PN), may be used. Further examples include ribonucleases or ribonuclease A-like enzymes, eg enzyme number (EC) 3.
At 1.27.5, RNase A is a preferable example.

Detection of mutant cK-ras oncogenes in human tumor cells Calu-1, SK-LU-1, SK-CO-1 and SW480 are specifically Mu
rray, MJ, Shilo, BZ, Shih, C., Cowing, D., Hsu, HW an
d Weinberg, RA (1981), Cell , 25, 355 , 361, two types of lung cancer that have been shown to contain the cK-ras oncogene already activated by N1H3 T3 DNA infection assay. And cell lines derived from two types of colon carcinoma. The active mutation of the cK-ras oncogene in Calu-1 and SW480 cells was reported by Shimizu, K.,
Birnbaum, D., Ruley, MAFasano, O., Suard, Y., Edlund,
L., Taparowsky, E., Goldfarb, M. and Wigler, M. (1983), Na
ture 304 , 497-500, and Capon, DJ, Seeburg, PH, McGrat
h, JP, Haylick, JS, Edman, U., Levinson, AD and Goed
del DV (1983), Nature 304 , 507-512, it has already been characterized as a single base substitution in the first coding exon that synthesizes mutant ras proteins containing cysteine and valine at codon 12, respectively. It is attached. Thus, the CalK-1 and PR371 cK-ras oncogenes contain the same mutation, while the SW480 cK-ras oncogene.
The oncogene additionally contains a G → T transversion of the second deoxyguanosine at codon 12. SK-LU-1 and SK-C0-1
The nature of the activating mutation in the cK-ras gene of tumor cells has not been reported.

The method according to the present invention was applied to the cK-ras of these tumor cells.
It was applied to analyze mutations in oncogenes. As expected, total cellular RNA was loaded with Calu-1 and SW48.
RNA-RNA cleaved by an RNase mixture when prepared from 0 tumor cells and hybridized with antisense RNA complementary to the normal first coding exon of the cK-ras gene, like RNA of PR371 cells Generate hybrids. The same results were obtained with conditioned RNA from SK-LU-1 and SK-CO-1 tumor cells. CK-ras RNA of these tumor cells
The cleavage pattern of hybrids was very similar to that of PR371 cells, although there were changes among cells with different degrees of digestion. In contrast, RNA hybrids obtained from normal human fibroblasts and PR310 tumor cells were resistant to digestion. These results confirmed the presence of a cK-ras oncogene mutant at codon 12 of the first coding exon in Calu-1 and SW480 cells, and also SK-LU-1 and SK. -CO-1 lung carcinoma cells and SK-CO-1 colon carcinoma cells are shown to contain the cK-ras oncogene activated by the mutation at this position. A similar analysis using a mutant antisense RNA directed by the plasmid pAK1Mcys further makes it possible to characterize the nature of the mutations in the cK-ras gene of these tumor cells. Calu-1 and PR3
The hybrid corresponding to 71 RNA was considerably resistant to degradation, while the other hybrids were not. Therefore, these results show that Calu-1 and PR37
While the 1c-K-ras gene contains the same mutation, SW48
Mutations in the 0 gene confirm that they are different. CK-ras of SK-LU-1 and SK-CO-1 tumor cells
It was concluded from these results that the mutation of the gene is different from that of PR371. Furthermore, a comparison of the extent of other digestions of the observed cleavage pattern is made by comparing two consecutive sequences, such as with the heteroduplex SW480 RNA formed between mutant RNA and mutant antisense RNA in these cells. These mutations appear to reside in the second deoxyguanosine at codon 12, suggesting that it contains a mismatch of selected nucleotides.

Presence and associated expression of normal ras allele in tumor cells The results also showed that PR371 tumor cells contain both mutant and normal cK-ras alleles. P
When R371 RNA hybridizes with normal antisense ras RAN, there are hybrids that are resistant to cleavage, whereas hybridization with mutant antisense RNA yields RNA hybrids that are degraded to some extent. . Performed with RNA prepared from three different PR371 cell clones isolated in culture. RNase protective expression was similar to that obtained with RNA conditioned from the entire tumor cell population. Therefore,
These results show that normal cK- in PR371 tumor cells.
We demonstrate that the presence of the ras allele is not due to tumor cell heterogeneity. The same results were also obtained with PR310 and Calu-1 cells. The result mentioned here is PR37.
1 and Calu-1 tumor cells contain a cK-ras transcriptional mutant at codon 12 which is said to be 5-10 fold more than those containing normal sequences.

Furthermore, detection using a labeled single-stranded gene RNA of a radioactive substance that shows complementary binding to RNA of eukaryotic cells has been exemplified, but in addition to RNA of this eukaryotic cell, RNA of prokaryotic cells or viruses, Single chain gene D showing complementary binding
It may be a combination of NA or RNA, or may be a combination in which the single-stranded gene showing complementary binding property to DNA of eukaryotic cells, prokaryotic cells or viruses is RNA.
Furthermore, a homoduplex that is substantially unaffected after the enzymatic treatment by using an unlabeled single-chain gene showing complementary binding in place of the pre-labeled single-chain gene showing complementary binding May be removed, the digested gene may be electrophoresed, and then the electrophoresis may be carried out by a known Southern blot method, followed by detection using the labeled substance.

EFFECTS OF THE INVENTION Studies on methods for detecting small genetic alterations, especially single point mutations, have been applied to the diagnosis of genetic disorders that are direct or indirect consequences of the aforementioned mutations in certain cellular genes. It was completely accomplished by applying these. For recent methodologies for detecting these point mutations:
Myers, RN, Lumelsky, N., Lerman, LS and Maniatis,
T., (1985), Nature, 313, 495-498. However, the methods of this invention for detecting single base substitutions in mammalian genes, as described herein, provide benefits over the currently available tools. As described herein, the method of this description is relatively simple and rapid, and allows the diagnostic search for single point mutations as well as the characterization of the nature and location of these mutants. Four of the eight possible single base pair mismatches in the double stranded RNA molecule were analyzed and it was discovered that RNase A is capable of cleaving all of these. Therefore, Mismatch RNA
The ability of RNase A to recognize and cleave a heteroduplex is dependent on the ability to cleave a mismatched DNA heteroduplex.
It is even more general than the ability of nucleases. Shenk et
See al. (1975), ibid.

Given that RNase A is capable of recognizing all possible mismatches as single-stranded RNA, its ability to cleave the hybrid will depend on the presence of a nucleic acid site in the mismatch. Human ras gene (cK-ras, cH-ra
s and N-ras) in each case, codon 12 or 6
May show synthesis of ras protein mutated in 1 39
All single base substitutions of a species contain at least one cleavage site for RNase A. However, the mechanism by which RNase A recognizes and cleaves these single mismatches is unknown. It is not known whether the presence of any mismatched nucleotides is sufficient for the nucleic acid activity of the enzyme. Of the four single mismatch base pairs analyzed, U-U mismatch was poorly recognized despite the presence of the three attack sites, while A-A was easily cleaved.
Mismatch only contains one attack site. Other mismatches, such as GU, are considered unrecognized because these bases can pair in RNA molecules. On the other hand, the ability to recognize and cleave a single mismatch in RNA hybrids has been demonstrated with RNase A, and whether or not other RNases can also cleave the mismatch RNA molecule can be examined based on the above description. It can be easily confirmed. Whether the presence of a nucleic acid attack site in mismatch is essential for cleavage by RNase A, or whether the local instability of hybrid molecules induced by mismatch results in additional cleavage sites. Unknown. In the latter case, the nature around the nucleotide sequence will affect the nucleic acid activity of the enzyme. In all cases, as already mentioned, the method of the invention introduces a point mutation in a mammalian gene which defines that the mutation occurs in the transcribed region and that the gene is expressed in at least some tissues. It has a range of applications as a clinical diagnostic screening tool for searching, determining its location, and characterizing it.

In addition, as shown here, in the practice of this invention, DNA-RN
Single-base mismatches of A hybrids can also be recognized by RNase A. This ability is based on total genomic DNA
Will be used to detect point mutations in hybrids of eukaryotic genes and other prokaryotic and viral genes. Therefore, the present invention has very general applicability as it is not restricted to mutations in the gene coding region and / or is not constrained by the efficacy of gene transcription.

The RNase protection method is also useful for analyzing the presence and expression of different alleles in the same cell. Thus, some tumor cells contain both normal and mutant ras alleles, and in PR371 and Calu-1 lung adenomas cells, the mutated translation of the gene compared to the normal translation. It was revealed that it was more expressed.

As indicated above, the practice of this invention is useful for the diagnostic detection and characterization of small genetic alterations that include point mutations such as single base substitutions in genetic material. This invention contains a mutant gene containing small genetic alterations such as single base substitutions, and total genetic material selected from the expressing organism, e.g. total cellular RNA containing mRNA and complementary genetic material, e.g. It relates to performing hybridization with antisense RNA. The resulting hybrid genetic material, formed from the added complementary genetic material and the components of the total genetic material, is then transformed into the aforesaid point mutation or mismatch to obtain two hybrids of smaller size. In order to cleave any heteroduplex hybrids formed around the presence of the genetic alteration of, the enzymatic undegradation of the hybrids enzymatically treated and formed as homoduplexes is sufficient. Excluded. The smaller size obtained by cleavage is then isolated and / or identified, thereby serving to indicate that the organism's genetic material contains a gene with a point mutation or single base substitution. Since the size obtained in the degradation can be measured, the relative position in genes of small genetic alteration such as point mutations can also be determined in this way.

This invention is also useful for characterizing the nature of these genetic alterations in the genetic material, since complementary genetic material containing the revealed genetic alterations can be constructed. Therefore, the complementary genetic material (mutant antisense RNA / mutant mRNA) has no mismatches, and therefore it is not degraded by the enzyme, so that the complementary RNA containing the specific mutant nucleotide is , Any other gene containing this particular mutation (eg, obtained from another cell) can also be detected.

An important context and utility of this application lies in the use of antisense RNA as a hybridization probe. It has been shown previously that DNA molecules labeled with radioactive or other labels in vitro by various means can be used for hybridization with DNA molecules containing point mutations or other mutations. Thus, a DNA-DNA or DAN-RNA hybrid formed by containing a single labeled strand can be digested with an enzyme having specificity for a single-stranded DNA molecule such as SI nuclease. This method provides useful detection of mutations involving relatively large regions of a DNA molecule, such as deletions, insertions or transformations, or differences in sequence between two similar but not identical DNA molecules. provide. However, other mutations involving only small differences in nucleotide sequences are single substitutions in extreme cases, ie two DNA molecules consisting of the same nucleotide sequence except for a single nucleotide are generally recognized by SI nucleases. It will not be broken or cleaved. It was surprising to our previous knowledge in the field why RNase A was discovered to readily recognize and cleave single base mismatches in RNA-RNA hybrids, and It is against knowledge. Complementary as a hybridization probe
The use of RNA molecules and digestion with RNase A work more effectively than the S1 digestion method. There are some differences in the system that can be provided for the explanation. First, RNA-RNA hybrids are DNA-DNA
It gives rise to different molecular structures other than hybrids. Thus, double-stranded DNA is predominantly present in the B form of the DNA molecule, while both RNA-RNA and DNA-DNA hybrids are 2'-.
It is present in A type because the presence of hydroxy group hinders the establishment of B type.

Second, RNA-RNA hybrids are more stable than DNA-DNA hybrids. This means that the energy required to dissolve the double-stranded molecule is higher in RNA-RNA than in RNA-DNA, and higher than in DNA-DNA. For example, TM (solubility) of RNA-RNA molecules consisting of perfect homology (identical but complementary nucleotide sequences)
Is 10 ° C higher than a DNA-DNA hybrid molecule containing the same nucleotide sequence. Furthermore, the differences observed in the two systems also cause differences in the enzymes used. However, S1 is also capable of cleaving RNA-RNA mismatch hybrids and therefore the preferential cleavage of RNA hybrids containing single base mismatches appears to be due to differences in molecular structure.

Developments in molecular biology techniques, especially SP6 or T7 bacterial offage RNA polymerase (Melton et al, 1984,
The practice of this invention is realized by recently developed techniques for synthesizing RNA molecules in vitro using ibid ibid.). Until very recently it was not possible to synthesize RNA molecules of specified length and sequence. Therefore, RNA probes have hitherto not been available for RNA-RNA or DNA-RNA hybridization studies.
However, the gene fragment (ie DN
A fragment) and a certain bacterial off RNA polymerase (S
It is now possible to develop an in vitro transcription system that synthesizes RNA molecules with constant chain length and sequence based on the construction of recombinant plasmids containing a special promoter obtained from P6 polymerase or T7 polymerase). Combined with the potency of the purified RNA polymerase, these plasmids were shown to synthesize RNA molecules in vitro complementary to the inserted gene fragment (ie with the same nucleotide sequence) as seen in the figure. Made possible Furthermore, both transcriptional and antisense RNA molecules can be synthesized depending on the corresponding orientation of the gene fragment with respect to the RNA polymerase promoter. As mentioned above, it was stated that the method of the invention can use complementary RNA molecules which are not radioactively labeled. For example, hybridization probes, RNA or DNA may be labeled with fluorescent compounds or molecules to which other small molecules (eg biotin) have been added, or by the use of enzyme complexes or specific antibodies or reagents, eg avidin for biotin. Thus, it is possible to detect low copy number genes or mRNA in cells. This is a mutation ras in non-research institutions such as hospitals
It will be of particular importance for screening human tumors for oncogenes.

Currently, there are not many genetic diseases that are known to be the result of point mutations in genes, but there are many other metabolic diseases whose component molecules are not known at the genetic level.
However, it is possible that they are the result of point mutations in as yet unknown genes as these genes have not been isolated and characterized. It is expected that many of these genes have been isolated and their alterations characterized by traditional means such as by sequencing cloned genes. In this respect, the method of the present invention, as described above, is extremely simple in its procedure, so that the rapid identification and localization of the point mutation in the gene (once the gene is isolated) can be achieved. On the other hand, it would be extremely useful. Sequencing small genes is not very difficult, but there are very large genes, and the complete sequence of that gene is labor-intensive and time-consuming. Once the approximate localization of the mutation has been determined, it will only be necessary to sequence a small region of the gene in question. Therefore, the method of this invention is extremely useful for the initial characterization of mutations responsible for certain unknown diseases, and consequently for diagnostic screening of these mutations in individuals. is there.

The following comparisons are provided as a summary of the benefits and capabilities of practicing the invention, with particular reference to other methods for detection of single point mutations such as in eukaryotic genes. One technique that may be used to detect single point mutations in eukaryotic genes involves the technique used for restriction endonuclease polymorphisms that require the Southern blot test. This technique involves a single direct method using a Southern blot and requires only small amounts of biological material and is therefore good for fetal diagnosis, however, this technique does not allow restriction endonucleases to generate cleavage sites, or It is only applicable to screen for diseases that have specific point mutations to disrupt, and only a few single base substitutions exhibit this property.

Another alternative technique involves the use of labeled oligonucleotide probes using Southern blots. This technique is useful for screening genes with unique point mutations, but it is essential to synthesize one oligonucleotide for each point mutation. Mutations in the ras gene, eg at codons 12 and 61, would result in activation of malignancy. There are 39 point mutations in all. In addition to the difficulties mentioned above, this method is technically complex and difficult to perform.

Another other technique involves plus gel electrophoresis, called denaturing gradient gels, containing solution hybridization with single-stranded DNA probes. This technique detects about 30% of single-point mutations, but it does
Since it involves the synthesis of A probe and a complicated gel electrophoresis system, it is technically complicated and difficult to carry out. On the other hand,
Although this method can detect single base substitutions in eukaryotic genes, the technique does not characterize the nature or position of the gene.

It should be noted that another other available technique along the direction disclosed herein involves solution hybridization with single-stranded DNA probes, followed by an S1 digestion system in which gel electrophoresis is performed. The S1 enzyme digestion system has been carried out in only a few cases and is therefore not a reliable method. The general technique is simple, but the overall technique is more complicated than the practice of this invention, as it is quite difficult to prepare the appropriate single-stranded DNA probe.

In the above comparison, the technique of the present invention in which digestion with an enzyme such as RNase A digestion using solution hybridization with a single-stranded RNA probe, followed by gel electrophoresis is clearly superior in many aspects Provide a way. The technique of the present invention is not only easy or convenient,
Also quick. From the perspective of practicing this invention using RNA probes and RNA preparation, this invention is essential in situations where many cells are to be started. The techniques for preparing RNA in the practice of this invention are more verbose than the methods used to make or prepare DNA.
However, from the RNA perspective, the method of this invention is superior for use with transcribed genes. This presents a problem for tissue-specific genes such as the globin gene, which is transcribed only in restricted tissues. For example, to screen for sickle cell anemia at fetal level, the method of this invention is feasible but not practical due to the risks associated with a relatively large number of fetal cell extracts. right.

As mentioned above, the method and technique of the present invention can be applied not only to the search for single base substitutions in the coding region of transcribed genes, but also to substitutions involving more than one nucleotide,
It can be applied to single base detection or detection of insertion as well as substitution including search for insertion and detection. The practice of the present invention is not limited to the above-described mutation search, but also the search for the nature of the mutation, for example, the specific nucleotide involved in the mutation, as well as the characteristics of the mutation or the localization of the mutation position in the gene. It is possible. When the corresponding gene is absent in the test solution, for example, regarding the search for genes such as viruses, the labeled single-stranded gene of the radioactive substance showing complementary binding to be used has no virus genes. It does not form a mixed double strand, is in a free state, is easily hydrolyzed by the ribonuclease used, and becomes an extremely small digested gene, which can be searched by analyzing it by electrophoresis. is there. Accordingly, a single-chain gene labeled with a radioactive substance that shows complementary binding to a specific gene, that is, a composition containing a labeled antisense single-chain gene and RNase A, is intended for the detection of a specific gene. It can be used as.

Modifications, alterations, and substitutions are possible in the practice of the invention when it is clear from the above description that technically special techniques are required without departing from the spirit or scope of the invention.

[Brief description of drawings]

FIG. 1A systematically illustrates the method according to the invention for the detection of single base substitutions in genetic material, and FIGS. 1B and 1C show labeled hybridized RNA by denaturing original acrylamide gel. Illustrates the results of molecular electrophoresis,
2A and 2B are graphical representations of the results obtained in accordance with the practice of this invention in the analysis of mutations in the cklas oncogene of human tumor cells.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Fumiichirou Yamamoto USA, 11776 New York, Port Jefferson Station, 460 Old Town Road, Universal Gardens Apartment Ments-Apartment 3-H

Claims (14)

[Claims] 1. A RNA-RNA hybrid or RN by allowing a single-chain gene having a complementary binding property to a specific gene to act on a gene-containing sample which may contain a short gene mutation.
A-DNA hybrid duplex is formed, which is then bound to R
A method for detecting a gene, which comprises treating with a NA-selective cleavage endonuclease and then separating and analyzing the treated gene. 2. The method for detecting a gene according to claim (1), wherein the gene which may contain a short gene mutation is a gene derived from a eukaryote, a prokaryote or a virus. 3. The method for detecting a gene according to claim (2), wherein the gene derived from eukaryote, prokaryote or virus is DNA or RNA. 4. The method according to claim (2), wherein the gene derived from a eukaryote, a prokaryote or a virus is RNA, and the single-stranded gene exhibiting complementary binding is RNA having complementary binding. Gene detection method. 5. The method according to claim (2), wherein the gene derived from a eukaryote, a prokaryote or a virus is RNA, and the single-stranded gene exhibiting complementary binding property is DNA having complementary binding property. Gene detection method. 6. The method according to claim (2), wherein the gene derived from a eukaryote, a prokaryote or a virus is DNA, and the single-stranded gene exhibiting complementary binding property is RNA having complementary binding property. Gene detection method. 7. An RNA-selective cleavage endonuclease,
Pyrimidine selective cleavage endonuclease (Pyr-P-
N) The method for detecting a gene according to claim (1). 8. The method for detecting a gene according to claim (7), wherein the pyridimine selective cleavage endonuclease is ribonuclease A or a ribonuclease A-like enzyme. 9. The method for detecting a gene according to claim (8), wherein the ribonuclease A or the ribonuclease A-like enzyme is an enzyme represented by enzyme number (EC) 3.1.27.5. 10. The method for detecting a gene according to claim (9), wherein the enzyme represented by the enzyme number (EC) 3.1, 27.5 is RNaseA. 11. The single-chain gene exhibiting complementary binding property is a gene having a normal sequence or a mutant sequence, and the gene which may include a short gene mutation is a gene containing the mutant sequence. A method for detecting a described gene. 12. The single-chain gene exhibiting complementary binding property is a gene having a normal sequence or a mutation sequence, and the gene which may contain a short gene mutation is a gene containing a normal gene. A method for detecting a described gene. 13. The mutant sequence according to claim (11) or (1), which has a nucleotide deficiency, a nucleotide addition or a nucleotide conversion compared to the normal sequence.
The method for detecting a gene according to the item 2). 14. An RNA-selective cleavage endonuclease
The method for detecting a gene according to claim (1), which is RNase A and the single-chain gene having complementary binding properties is labeled with a radioactive substance.