JP2000093185A - Detection of gene deletion - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect gene deletion of a cell originating from a specimen by specifying the deletion of a heterozygosity of a gene polymorphism marker of an Na/K-ATPase β2 subunit gene at the downstream region of p53 gene of a genetic specimen. SOLUTION: The gene deletion of a cell originating from a gene specimen is detected by specifying the deletion of heterozygosity in a gene polymorphism marker in an Na/K-ATPase β2 subunit gene existing at the downstream region of p53 gene of the gene specimen, thereby differentiating one or more kinds of bases in a base sequence expressed by the formula and representing the range from about 10 kb to about 16 kb at the downstream region of the p53 gene such as adenine and guanine as the 4691st base, guanine and cytosine as the 4731st base, adenine and guanine as the 4906th base and adenine and guanine as the 2649th base.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to a method for detecting a gene deletion. More specifically,
Loss of heterozygosity using a polymorphism found near the downstream region of the p53 gene, which is known as one of the leading tumor suppressor genes, alone or using a polymorphism in the p53 gene as an index The present invention relates to a detection method capable of detecting a gene deletion in a cell derived from a genetic specimen, in particular, a gene deletion related to canceration, by specifying it in combination with loss of heterozygosity.

[0002]

2. Description of the Related Art One of the abnormalities found in many cancer cells is gene deletion. There are specific patterns of gene deletion in specific cancers, suggesting the presence of tumor suppressor genes in regions where deletions are commonly found. For example, the short arm of chromosome 17 (17p) is the chromosome with the highest frequency of deletion in various cancers, and it is one of the tumor suppressor genes in which abnormalities are most frequently observed in cancer. Are known
The p53 gene is present. Most of the abnormalities found in this p53 gene are deletions of alleles and mutations in residual alleles.

[0003] Gene deletion frequently observed in the p53 gene is easier to identify than gene mutation, and is expected to be applied to analysis using clinical specimens. As an analysis method for identifying a gene deletion in a specific chromosomal region, L
OH (Loss of heterozygosity) analysis is known. This is to examine the deletion of one of the two alleles derived from the paternal and maternal sides of the cell, and to analyze the deletion of a specific region of the chromosome of the cancer cell. Conventional LOH analysis methods include a method using a Southern blot method using a polymorphic marker and a method using a PCR-RFLP method.

[0004]

The above-mentioned tumor suppressor gene p
Some polymorphic markers that can be used for cancer diagnosis and the like have already been recognized inside 53 , and these polymorphic markers are actually being applied as cancer diagnostic means. However, samples that can identify gene deletion using these polymorphic markers in p53 , that is, samples that show heterozygosity in normal cells, are about 60% of all samples, and the remaining 40% are normal. It shows homozygosity in cells and cannot perform the desired LOH analysis.

[0005] Therefore, there is an urgent need to develop another polymorphic marker for analyzing more samples. The problem to be solved by the present invention is to find a gene polymorphism marker present in a region near this gene which is directly related to the gene deletion in the p53 gene, and to deeply relate to cancer and the like using this polymorphism marker. It is an object of the present invention to provide means for detecting a gene deletion.

[0006]

Means for Solving the Problems The present inventor has made intensive studies to solve this problem. As a result, Na / K-ATPase beta2 subunit located near the downstream of p53 gene
In the gene, a polymorphic marker corresponding to the deletion of the p53 gene was found, and the present invention was completed. That is, the present inventor provides the following invention in the present application.

[0007] In claim 1, LOH in a gene polymorphism marker in the Na / K-ATPase beta2 subunit gene present in the downstream region of the p53 gene of the gene sample is specified, and gene deletion in cells derived from the gene sample is determined. Provide a way to detect.

[0008] In claim 2, LOH in a gene polymorphism marker in the Na / K-ATPase beta2 subunit gene and a gene polymorphism marker in the p53 gene, which are present in the downstream region of the p53 gene in the gene sample, are specified,
A method for detecting a gene deletion in the cell derived from the genetic specimen is provided.

[0009] In claim 3, gene polymorphism markers in Na / K-ATPase beta2 subunit gene present in the downstream region of the p53 gene, approximately downstream region of the p53 gene 10Kb~
4 of the base sequence represented by SEQ ID NO: 1 representing a range of 16 Kb
Separation of 691th base between adenine and guanine, ibid.
Separation of 731th base between guanine and cytosine, 4
3. The method according to claim 1, wherein the one or more bases selected from the group consisting of adenine and guanine of the 906th base and another of adenine and guanine of the 2649th base. 4. A method for detecting a gene deletion in the gene sample described above.

[0010] In claim 4, gene polymorphism markers in Na / K-ATPase beta2 subunit gene present in the downstream region of the p53 gene, approximately downstream region of the p53 gene 10Kb~
4 of the base sequence represented by SEQ ID NO: 1 representing a range of 16 Kb
The presence or absence of restriction enzyme BsaJ I sensitivity in the vicinity of 691 th base, M in the vicinity of the 4731 th base
whether ae III sensitivity, selected from the group consisting of presence or absence of Hga I sensitivity in the vicinity of the presence and the 2649 th base of the Hae III sensitivity in the vicinity of the 4906 th base, for one or more restriction enzymes 3. A method for detecting a gene deletion in a gene specimen according to claim 1 or 2, which is a presence or absence of sensitivity.

5. The method according to claim 1, wherein the gene specimen is a gene specimen of an individual whose normal cells show homozygosity for a polymorphic marker in the p53 gene. A method for detecting a gene deletion in the gene sample described above.

In the sixth aspect, the means for specifying LOH is a blunt-ended single-stranded DNA higher-order structure polymorphism analysis method.
A method for detecting a gene deletion in a gene sample according to any one of claims 1 to 5 is provided.

The gene deletion according to any one of claims 1 to 6, wherein the gene deletion detected by the method for detecting a gene deletion in a gene sample is used as an indicator of the presence of cancer. Provide a way to detect loss.

[0014]

Embodiments of the present invention will be described below. In a method for detecting a gene deletion of a gene sample according to the present invention (hereinafter, also referred to as the method of the present invention),
As described above, the p53 gene for which LOH is specified exists on the short arm of chromosome 17 (17p13) and
One exon encodes a protein of 393 amino acids and a molecular weight of 53K. L in this area in various cancers
OH was observed, indicating that the p53 gene is involved as a tumor suppressor gene. This gene is 4
By binding to a specific DNA sequence in a dimeric state,
It is known that the expression of various genes is regulated. It has been speculated that cancer with a mutation in the p53 gene causes abnormal cell proliferation, apoptosis is not induced, and DNA repair is impaired, so that new cancer-related gene abnormalities may accumulate.

[0015] At least three gene polymorphisms have already been found in the p53 gene. In addition,
In the present invention, the term "gene polymorphism" means a gene polymorphism in a human chromosome, and a gene has a specific site that often has a different base for each individual in its base sequence. Means that. It is said that about one in hundreds of such base polymorphisms exist on average, and by analyzing the gene and specifying this polymorphism, the paternal chromosome (allele) and the maternal chromosome can be determined. (Allele). That is, the paternal and maternal genes have different bases at specific polymorphic sites (heterozygosity)
By identifying, the two genes can be distinguished.

The heterozygosity for a specific gene polymorphism detected in normal cells of one individual is lost in a specific sample of the individual, and in the sample, the paternally derived gene and the maternally derived gene are lost. When one of them has disappeared (when identified as homozygous)
This indicates that, in the specimen, the locus containing the specific polymorphic site is deleted in one allele (referred to as allele loss).

Therefore, by utilizing this polymorphism and measuring the loss of one allele (LOH) in a heterozygote, the deletion of the gene (in the above example, the tumor suppressor gene p53 gene was Deleted) can be identified.

A gene polymorphism can be specified by its sensitivity to a restriction enzyme that usually cleaves only a specific nucleotide sequence (gene polymorphism marker, also simply referred to as polymorphism marker). In other words, it is possible to determine whether or not the base at the specific site has been replaced with a specific base by determining whether or not the nucleotide sequence of the specific site that is a genetic polymorphism is cleaved by a specific restriction enzyme. It is.

At present, polymorphisms that are found in the p53 gene, are relatively heterozygous in Japanese, and have high utility in diagnosis include:
Restriction enzyme Ha in intron 1 region of p53 gene
the presence or absence of e III sensitivity, the presence or absence of restriction enzyme BstU I susceptibility in exon 4 region of the p53 gene, and p5
Restriction enzyme Apa I in intron 7 region of 3 genes
There are known three types: presence or absence of sensitivity.

As described above, specimens that can identify gene deletion using these polymorphic markers in the p53 gene, that is, specimens in which these polymorphic markers show heterozygosity in normal cells, are Japanese Approximately 60% of the gene samples derived from the gene are homozygous, and the remaining 40% are homozygous, so that LOH analysis cannot be performed as desired.

To make matters worse, even if a polymorphic marker inside the p53 gene other than those described above can be found, the polymorphic markers are linked, and the desired heterozygosity can be determined using the new polymorphic marker inside the p53 gene. Will be difficult to find.

[0022] The present inventors have in Na / K-ATPasebeta2 subunit gene is a specific gene downstream region near the p53 gene has been found a genetic polymorphism associated directly LOH of the p53 gene.

[0023] is in the vicinity of the p53 gene, and, p
The gene polymorphism directly related to the LOH of the 53 gene is not linked to the gene polymorphism marker in the existing p53 gene, and further, by using these gene polymorphisms as an index, the heterozygosity of a specific individual can be independently determined. Can be found.

The Na / K-ATPase beta2 subunit gene is a gene that normally encodes one of the subunits of a protein that regulates the intracellular concentrations of Na ⁺ and K ⁺ (Daniel
le Malo, Erwin Schurr, Robert Levinson and Philippe
Gros (1990) Assignment of Na, K-ATPase β ₂ -subunit Ge
ne (atpb-2) to Mouse Chromosome 11.Genomics 6: 697-6
99). It has been known for a long time that this gene is present on the short arm of human chromosome 17;
The TPase beta2 subunit gene was found to be located at a very close position of about 10 to 16 kb downstream of the p53 gene.

In the method of the present invention, a polymorphism marker serving as an index for specifying LOH is the Na / K-ATPase beta
Although corresponding to the gene polymorphism existing in the 2 subunit gene, specific embodiments of the gene polymorphism and the corresponding embodiment of the gene polymorphism marker will be disclosed in Examples described later.

In the method of the present invention, the heterozygosity of each gene polymorphism marker in the Na / K-ATPase beta2subunit gene present in the downstream region of the p53 gene is not specified in normal cells of the individual, Gene deletion in a desired sample can be detected.

In the method of the present invention, in a gene sample of an individual showing heterozygosity for a genetic polymorphism marker used as a specific means of LOH, even if gene deletion is observed due to canceration or the like, normal Usually, cells and tumor cells are mixed, and when tumor cells are not present, the gene sample shows heterozygosity and the ratio of signals derived from both alleles is the same as that of normal cells. If tumor cells are present, the degree of coexistence of the tumor cells with normal cells [tumor cell contamination (Tu
mor Cellularity): described below, the attenuation of the signal derived from the deleted allele is observed.

In the method of the present invention, the heterozygosity of the genetic polymorphism marker is specified in normal cells of an individual, ie, a subject, and the loss of heterozygosity in a gene sample in the subject is specified, that is, the subject's heterozygosity is determined. Heterozygosity of normal cells and, for example, by specifying LOH in a gene specimen to be diagnosed for cancer, to identify gene deletions often found in cancer cells, and to find genes closely related to canceration and the like. The deletion can be detected. [Normal cells of a subject used as a specific subject for heterozygosity in this case include normal cells other than germ cells (particularly, white blood cells derived from peripheral blood and normal tissues of surgical specimens). ) Is used].

Further, in the method of the present invention, it is possible to detect a gene deletion in a gene sample without specifying the heterozygosity of a normal cell as described above. That is, in the method of the present invention, when the heterozygosity in the gene specimen is maintained, it indicates that no gene deletion is observed in the cells derived from the gene specimen, and conversely, no heterozygosity is observed. If not, it indicates that the gene of the individual providing the genetic sample is originally homozygous for the genetic polymorphism marker used in the identification, otherwise, the gene sample-derived cells This indicates that some gene deletion is observed.

As described above, in the method of the present invention, it is only necessary to specify whether the heterozygosity is maintained, the heterozygosity is not observed at all, or the heterozygosity is attenuated in the gene sample. In this, without previously specifying the heterozygosity in the normal cells of the gene sample provider, it is possible to detect the gene deletion of the gene sample-derived cells, the detection method of the present invention of this embodiment,
It is excellent in that the inspection is simple.

In the present invention, the term "gene sample" means any subject that can contain a gene for which LOH is to be detected. For example, when the method of the present invention is used as a basis for detecting cancer, Tissues or cells to be examined, or the gene itself obtained from them, or urine, pancreatic juice,
DNA extracted from body fluids such as duodenal fluid and the like can be mentioned. Specific selection of a gene sample can be appropriately performed depending on the target of the gene deletion detection. Further, “gene sample-derived cell” means the cell body from which the gene sample is derived. As described above, when the gene sample refers to a cell, the gene sample and the gene sample-derived cell Becomes synonymous.

As a method for specifying LOH, generally known methods, for example, an RFLP method using Southern blotting,
CR-RFLP method, HET (hetero duplex analysis)
Method, DGGE (denaturing gradient gel electrophores
is) method, DS (direct sequence) method, CCM (chemical
cleavage mismatch) method, CDI (carbodiimide modifica)
method) and a single-stranded DNA higher-order structural polymorphism analysis method using the PCR method [PCR-SSCP (single-stranded c
onformation polymorphism) method (hereinafter referred to as the SSCP method) in the present specification [see, for example, Bio Manual Series 1, Basic Technology of Genetic Engineering, Masaru Yamamoto, Yodosha (1993), etc.] ] Is that SS can be easily and accurately identified.
It is preferable to select the CP method.

In this SSCP method, a gene near the target polymorphism site (generally, a gene region of about 100 to 200 bp including the polymorphism site) is labeled using PCR primers with a label. Amplify this PC
PCR product obtained by the R method (usually double-stranded DNA)
Is denatured into a single strand by a denaturing means such as thermal denaturation, and the single-stranded DNA is analyzed using the label as an index.

When the SSCP method is applied to the analysis of LOH, a nearby gene containing the polymorphic site to be analyzed is amplified by the PCR method. After denaturing the resulting PCR product into single-stranded DNA and performing non-denaturing polyacrylamide gel electrophoresis, the sequence difference is analyzed as a change in mobility due to the difference in higher-order structure in single-stranded DNA. can do.

This analysis is usually performed by using a label added to the PCR product as an index or by staining a nucleic acid. For example, using a pre-labeled PCR primer, the primer label in the PCR product can be used as an index, and the obtained PCR product can be labeled afterwards.

The labeling substance used for these labels can be selected from commonly known labels that can be used in gene analysis, and examples thereof include radioactive substances, fluorescent substances, chemiluminescent substances, and biotin (enzyme-labeled avidin. And the like can be appropriately selected. As the labeling method, a labeling means according to the type of each of these individual labeling substances can be used.

For example, the 5 ′ end of the oligonucleotide primer for PCR is L. F. red ^TM (C
y5 ^™ ) Amidite reagent (manufactured by Pharmacia) is used as a PCR primer, and the obtained PCR product is analyzed with an AlFred fluorescent sequencer to specify LOH at a specific gene polymorphism site. Can be. When nucleic acids are stained, a fluorescent staining method such as ethidium bromide or cyber green or a staining method such as a silver staining method can be used.

As a result of such an analysis, if the peak or band of DNA derived from a cell derived from a gene specimen and / or a normal cell indicates the number derived from both alleles, the individual is identified by the gene polymorphism. Although it is "heterozygous", when the peak or band derived from the DNA of the gene sample is out of balance between the signal intensities derived from the two alleles,
It is regarded as loss of heterozygosity, and is determined as LOH.

In the SSCP method, the specific sensitivity of LOH by the SSCP method can be further improved by blunt-ending the PCR product obtained by the PCR method.

That is, a PCR product is usually a double-stranded DNA
Although it is obtained as a fragment, an extra base tends to be added to the end of the DNA depending on the nature of the Taq DNA polymerase used, and the DNA chain length of the added DNA fragment and that of the non-added DNA fragment are not identical. Often it is uniform. Therefore, even if this PCR product is analyzed after denaturation into a single strand, the detection peak derived from the DNA fragment is split,
There is a strong tendency to be multiplexed, which lowers the LOH detection sensitivity.

In order to overcome the drawbacks of the conventional SSCP method, after blunt-ending the previously obtained PCR product,
When this is analyzed, the detection peak is unified, and the discrimination sensitivity of a DNA fragment having a gene mutation can be significantly improved.

As means for blunt-ending, for example, an enzyme having 3 ′ → 5 ′ exonuclease activity such as Klenow fragment or T4 DNA polymerase and capable of repairing a single-stranded portion, the PCR product is used. It is also possible to treat the PCR product with a restriction enzyme that blunts the ends of the double-stranded DNA to remove the end portion including the single-stranded portion.

The SSCP method including the blunt-end step is performed by blunt-end single-stranded DNA higher-order structural polymorphism analysis (Blue).
nt End-SSCP method). According to the method of the present invention, Na / K-ATP present in the downstream region of the above-described p53 gene
It is possible to identify LOH in a subject's gene sample by using a gene polymorphism marker in the asebeta2 subunit gene, and to detect gene deletion in cells derived from the gene sample, for example, various types of cancer such as colon cancer, pancreatic cancer, and bladder cancer. It can be used for detection of cancer, detection of canceration tendency, and the like.

Further, by combining the gene polymorphism marker in the Na / K-ATPase beta2 subunit gene and the gene polymorphism marker in the p53 gene, cells derived from the gene sample can be further utilized by utilizing the gene polymorphism. In the above, the rate of heterozygous subjects who can detect gene deletion deeply associated with canceration or the like can be further improved.

That is, even in the case of the gene polymorphism within the p53 gene, the gene sample of the subject is “homozygous”.
Indicates that Na / K-ATP exists downstream of the p53 gene.
If the gene polymorphism in the asebeta2 subunit gene shows “heterozygosity”, p53
LOH in extragenic polymorphism was observed, p5
By being able to indirectly identify deletions of three genes, it is possible to further improve the rate of subjects who can detect gene deletions deeply associated with canceration or the like in gene sample-derived cells.

In other words, inside the p53 gene or Na / K
Identify LOH for each of a plurality of polymorphisms in the -ATPase beta2 subunit gene, which preferably do not overlap each other, and detect the gene deletion in cells derived from the gene specimen by combining the results of the identification. be able to. That is, if any one of the gene polymorphisms shows “heterozygosity”, even if the other shows homozygosity, the gene deletion such as canceration can be determined as “positive”. It is possible to further improve the case.

Conventionally, as a method for finding a gene polymorphism marker in a gene, by comparing the presence or absence of the human gene with the restriction enzyme, it can be said that the gene is polymorphic.
A method of finding a gene polymorphism marker, a method of searching for a repetitive sequence in which a polymorphism is recognized, and the like have been adopted.

However, the former method has a problem that it is difficult to detect only the gene polymorphism in the region where the restriction site exists. In the latter method, the frequency of occurrence of a repetitive sequence in which a polymorphism is recognized is not as high as that of a single nucleotide substitution polymorphism. Not only is the problem that the PCR is difficult to perform, but also during the amplification operation of the gene, PCR products with different numbers of repeats are generated, which becomes noise when detecting LOH, and the detection sensitivity tends to be low. There is also a problem in that.

Therefore, the present inventor has found that the search for polymorphic markers by directly examining the base sequences of DNAs in a specific region derived from a plurality of gene specimens makes it extremely simple and accurate. It was found that a gene polymorphism marker can be detected.

That is, the inventor of the present invention selects a site where the same portion is defined by different bases in a mixture of DNA fragments derived from a plurality of gene specimens, in which a specific gene region is amplified. Provided is a method for detecting a gene polymorphism marker as a type marker.

As a method for amplifying a specific gene region, a generally known amplification method typified by the PCR method can be arbitrarily selected, but the Long PCR method (Barnes, WM, Proc. Natl. Aca.
d.Sci.USA, 91, pp2216-2220 (1994)), and amplify the gene region between the amplification primers,
It is possible and preferred to reduce the number of PCR operations. The base sequence of the amplification primer can be selected from known sequences for detecting gene polymorphisms.

It is preferable that the number of gene specimens to be subjected to this test is sufficient for the gene polymorphism marker to become apparent. In a gene amplified product derived from a plurality of gene specimens amplified by such a gene amplification method, a site where the same site is defined by different bases (for example, n It is possible to detect, as a genetic polymorphism marker, a site whose second base is defined by both A and G).

Here, as a method for determining the base sequence of a gene, which is used for examining the base sequence of the gene amplification product, a generally known base sequence determination method can be used. For example, Maxam, AM, and Gilbert,
USA , 74, 560 (1977)), genomic sequence method (Church, GM and Gilbert, W., Pr.
oc.Natl.Acad.Sci.USA, 81 , 1991 (1984)), multiplex method (Church, GM and Kieffer-Higgins, S., Scienc
e, 240, 185 (1988)), cycle sequence method (Murray,
V., Nucleic Acids Res., 17 , 8889 (1989)). Dideoxy method
(Sanger, F., et al., Proc. Natl. Acad. Sci. USA, 74 , 546
3 (1977)) and the like, the base sequence of the gene amplification product in the desired region is determined, and the gene polymorphism marker can be detected based on the base sequence information obtained from the base sequence.

It is also possible to determine the base sequence of a gene amplification product in a desired region by using a commercially available automatic base sequence analyzer or a base sequence determination kit applying these principles.

The present inventor has proposed a method for detecting this gene polymorphism marker by using SHIPS (Sequencing Highly Informative).
Polymorphic Site) method, which is also a detection method used in Examples described later.

In this SHIPS method, it is possible to search in detail, including the presence or absence of a gene polymorphism in a specific region and its nucleotide sequence, and it is possible to reduce the number of PCRs by combining with the LongPCR method. And that the sequence primers can be used continuously, so that efficient gene search is possible and that polymorphisms and nucleotide sequences can be directly confirmed. It is possible to estimate the frequency of the polymorphism to some extent based on the fact that information such as the site can be obtained immediately and the density of overlapping bases, and the higher the frequency of the polymorphism, the easier it is to detect. And the like.

[0057]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but these examples do not limit the technical scope of the present invention. 1. Identification of atpb2 gene and measurement of distance from p53 gene In order to obtain genomic DNA near the p53 gene, a clone containing the p53 gene, which was screened from a human genomic library using a cosmid vector, was analyzed. As a result, the p53 gene was present on one side of the human genome insert of the same clone, and the atpb2 gene with a known cDNA base sequence was present on the other side. From the information on the nucleotide sequences, it was found that the p53 gene and the atpb2 gene existed in the genome in a direction in which their 3 'ends face each other.

As described above, this atpb2 gene is one of protein subunits that regulate the intracellular concentrations of Na ⁺ and K ⁺ (Na ⁺ / K ⁺ ATPase beta2 subunit
(J. Biol. Chem. 15; 264: 4613-4618,19)
89). base sequence of genomic DNA of atpb2 gene [(Juli
o Avila et al., Gene , 208 , pp. 221-227 (1998)), it is essential to search for the target polymorphism marker. 5490 bases including the sequence shown in FIG. 1 (SEQ ID NO: 1).
Each gene region is an exon in the atpb2 gene), and its gene structure was clarified (Fig. 2:
FIG. 1 is a diagram schematically illustrating an exon region shown in FIG. 1 and other intron regions.

Although the distance between the p53 gene and the atpb2 gene is less than or equal to the maximum size (about 40 Kb) that can be inserted into the cosmid vector, LongPCR was performed for the purpose of obtaining a more accurate distance.

That is, at the 3 ′ end of the p53 gene, at
At the 3 ′ end of the pb2 gene, a primer [ p53 gene side: 5′GGAGGGGAACAAAGGCTGGGAG
ACTG (SEQ ID NO: 2), atpb2 gene side: 5'CTC
TGGCTTCTTTTTAGCAAGTTATCAAC
(SEQ ID NO: 3) was set, and the gene was amplified by LongPCR using the cosmid clone and human genomic DNA as templates. The distance between the two genes was about 10.5.
Kb (FIG. 3: In FIG. 3, the p53 region and
LongPCR region inserted between atpb2 regions,
This is an area corresponding to about 10.5 Kb. This distance is
This was clarified by the electrophoretogram shown in FIG. 3).

The nucleotide sequences of these genes were determined by Thermo Sequenase Cycle Sequenceing from Amersham.
Performed using KIT and ³³ P-Labeled Primer. Also, Lon
For gPCR, see Clone Tech's Advantage Genomic
PCR KIT was used.

[0061] 2. In the atpb2 gene by SHIPS method
Search for Polymorphic Markers As described above, in order to search for genetic polymorphism markers that frequently show polymorphism, the present inventors have established a gene analysis method named the SHIPS method. This genetic analysis method
LongPCR-direct nucleotide sequencing is performed on a region having a known base sequence by mixing DNAs of multiple people. In a region where a gene polymorphism is present, base overlap is performed at a concentration corresponding to the polymorphism frequency. Is a gene analysis method for identifying a genetic polymorphism marker by utilizing the fact that

In applying the SHIPS method to the search for a genetic polymorphism in the atpb2 gene, Japanese 13
For a sample mixed with genomic DNA derived from human peripheral blood, SHIPS was performed based on the genomic nucleotide sequence information of the atpb2 gene obtained by the above .

[0063] In this embodiment, when performing SHIPS method, a signal band, for the purpose of issuing a uniform and sharp, dideoxy nucl labeled with ³³ P
Thermo Sequenase Radiolabelled Termi using eotide
nator Cycle Sequencing Kit (Amershanm) was used. The nucleotide sequence of the primer of the PCR product in which the polymorphic marker was recognized was 5′GATGAGCGAG
ACAAGTTCGC (SEQ ID NO: 4)
But 5 'TCAACACAGGCCTCACTTCC
(SEQ ID NO: 5). These primers were also used as primers for nucleotide sequence determination.

In the former (Forward) primer for determining the base sequence, the polymorphism recognized by the restriction enzyme BsaJI [FIG. 1 and FIG.
In the cosmid DNA, only one band is observed, but in the mixed DNA, two bands are observed. The portion indicated by a white circle in the electrophoresis image is a polymorphic site. Similar in Figure 5): 5 '414 base from end to a downstream exon 7, i.e., a separate] the 4691 th base of adenine and guanine nucleotide sequence of atpb2 gene shown in SEQ ID NO: 1, the latter ( Reverse), a polymorphism recognized by the restriction enzyme Hae III (FIG. 1 and FIG. 5: the 629th base downstream from the 5 ′ end of exon 7, ie, SEQ ID NO: 1) adenine and guanine at the 4906th base in the base sequence of the atpb2 gene).

In addition, during the work of confirming the nucleotide sequence, a polymorphism recognized by the restriction enzyme Mae III showing polymorphism was found at low frequency (FIGS. 1 and 6: exon 7). The 454th base downstream from the 5 'end of
Guanine and cytosine at position 4731 of the base sequence of the atpb2 gene shown in SEQ ID NO: 1).

As described above, the SHIPS method (the DN of a plurality of persons)
A, and mix the LongP
CR-direct nucleotide sequencing) was confirmed to be effective for searching for a polymorphic marker with a high frequency of heterozygosity existing in the target region.

3. Extraction of genomic DNA Extraction of peripheral blood DNA and extraction of cancer tissue and normal tissue DNA for LOH analysis are performed by digesting these starting materials with protease K and extracting with phenol / chloroform.
Davis et al. (Basic Method in Molecular Biology, Else
vir Science Publishing Co.) and Sugano et al. (Lav.Inves
t.68, 361, 1993).

4. The genomic DNA (template) extracted by PCR was added at 0.25 μg / 25.
μL, Forward and Reverse oligonucleotide primers were added at 0.5 μM each and each nucleotide triphosphate (d
NTP) in 200 μM and Tris-HCl buffer (pH 8.3) in 10
mM, KCl 50 mM, MgCl ₂ 1.5 mM, Taq D
NA Polymerase (Perkin-Elmer) at 0.625 units /
A reaction solution was prepared so as to have a volume of 25 μL, and PCR was performed under the following conditions. Initial denaturation conditions were at 94 ° C for 5 minutes,
The subsequent reaction was carried out at 94 ° C. for 1 minute, 50 ° C. for 1 minute, 72 ° C.
Cycle at 40 ° C for 1 minute, and finally at 72 ° C
For 7 minutes.

5. Blunt-End SSCP In normal PCR, a mixture of a PCR product having one extra nucleotide adenine at the 3 ′ end and a non-added one is provided at the 3 ′ end. Therefore, when this is directly subjected to the SSCP method, as if there is only one kind of base sequence, it is as if
The species appear to be present, or the sensitivity of the band (peak when a fluorescent sequencer is used) is reduced. Therefore, extra base on the 3 'side is replaced with Klenow fragm
When SSCP is performed after smoothing the PCR product by removing with ent, a clear band (peak) is obtained and the mutation or polymorphism can be easily detected (Blunt-End SS
CP method).

Specifically, 4. P obtained in
To 5 μL of the CR product, 0.5 units of Klenow fragment was added, treated at 37 ° C. for 30 minutes, and subjected to SSCP analysis. SSCP analysis was performed on PCs with Klenow fragment processing.
The R product (1 μL) was added to 10 μL of a loading solution for SSCP, denatured by heating at 80 ° C. for 5 minutes, and then subjected to non-denaturing polyacrylamide gel electrophoresis.

The composition of the loading solution was 20 mM EDT.
A, 0.05% Bromphenol blue and 90%
Formamide. The polyacrylamide gel was prepared using Tris glycine buffer (25 mM Tris. 192 mM glycine).
Containing 10% acrylamide (bisacrylamide:
(Acrylamide = 1: 30) gel was used. The electrophoresis was performed at 18 ° C. and a constant voltage of 200 V for 6 hours. The band was detected with "2D-Silver Staining Reagent II" of Daiichi Kagaku.

The above 2. Based on the base sequence information of the genomic DNA determined in the above section, exon 4 of the atpb2 gene was replaced with a primer [Forward: 5'-CTTGGAAGTGGA
ACATCTGG (SEQ ID NO: 6), Reverse: 5'-C
TGGGGACCAAGGTCATAGA (SEQ ID NO: 7)], followed by Blunt-En
d When SSCP was performed, a new restriction enzyme Hga I
(FIGS. 1, 7, and 8: the 50th base from the 5 ′ end of exon 4, ie, the 2649th base of the nucleotide sequence of atpb2 gene shown in SEQ ID NO: 1) Adenine and guanine).

6. Measurement of the frequency of heterozygosity The frequency of heterozygosity of each genetic polymorphism marker is expressed as P
It was examined by the CR-RFLP method. The primer sequences used are shown below. BsaJ I and Mae III Markers: Forward: 5'-CCAACTTCTCCCCAACCT
CAG (SEQ ID NO: 8) Reverse: 5'-ATGCAGGCAGGAGAGTG
GAT (SEQ ID NO: 9)

Hae III Marker: Forward: 5′-TTTGCTTCCCAGAGATGA
ATG (SEQ ID NO: 10) Reverse: 5'-AATTCAAAGTTCACCAAG
ATG (SEQ ID NO: 11)

Hga I marker: 5. The same primers as the primers used in Example 1 The PCR products were cleaved with restriction enzymes corresponding to the respective markers, the cleaved allele was designated as "+ allele", and the uncleaved allele was designated as "-allele" (first table,
Table 2 -a, Table 2 -b).

[0076]

[Table 1]

[0077]

[Table 2]

[0078]

[Table 3]

[0079] The frequency of observed heterozygotes, Hae
III is 0.46 (n = 196 alleles), Mae III is 0.02 (n = 202 alleles), BsaJ I is 0.44
(N = 198 allele) and Hga I is 0.46 (n = 1
86 alleles) (Table 1).

Hae III,
Among the BsaJ I and Hga I markers, a case in which any one or more of the three polymorphic markers within the p53 gene shows heterozygosity (Table 2a) and a case in which all show homozygosity (Table 2-a) Table 2-b).

The frequency of heterozygosity observed in the Japanese population in which the polymorphic marker within the p53 gene shows heterozygosity was 0.57 for Hae III (n = 98 alleles) and 0 for BsaJ I. .54 (n = 100 alleles),
In the Japanese population where the polymorphic marker in the p53 gene is homozygous, whereas the Hga I is 0.52 (n = 96 alleles), the frequency of heterozygosity observed is Hae III Is 0.35 (n = 98 alleles), Bsa
J I is 0.35 (n = 98 alleles), Hga I 0.4
0 (n = 90 alleles), indicating that no linkage was observed with the marker inside the p53 gene.

[0082] From these facts, the polymorphic markers within atpb2 gene, in performing LOH analysis of p53 gene region, when used in combination with polymorphic markers in the p53 gene, heretofore, p53 gene inside the polymorphic markers It was found that LOH analysis could be performed even on cases where LOH analysis was not possible due to homozygosity.

7. LOH analysis For normal tissues and genomic DNA derived from colon cancer tissues,
LOH analysis was performed using the atpb2 gene polymorphism marker Blunt-End SSCP (the same primers as in the above section 5) were used. After silver staining, images were captured with a scanner and analyzed for signal intensity with Kodak 1D Analysis software.

Analysis of the Hga I polymorphism marker in exon 4 of the atpb2 gene in 5 cases of colorectal cancer tissue samples from colon cancer patients in which LOH was observed in the p53 gene region showed that all 5 cases showed this polymorphism. LO at marker
H was observed [FIG. 9: In FIG.
The number 5 is the gene sample (colorectal cancer patient) number,
Represents the normal tissue around the patient's colon cancer, and T
The colorectal cancer tissue of the patient (normal tissue and cancer tissue are mixed). The numbers on the vertical axis indicate the signal intensity ratio of the two alleles when silver-stained.
If the ratio is less than 1, it indicates that there is a cell nucleus in which the heterozygosity of the polymorphic marker has been lost in the gene sample (the heterozygosity has been lost). When the number of cells is 100%, the value is 0. However, as described above, the value is hardly completely 0 because cancer tissue includes normal tissue.)

As a result, in a gene sample in which LOH was recognized as a polymorphic marker due to the p53 gene, LOH was also recognized in a polymorphic marker in the atpb2 gene in the vicinity thereof, and the present invention was found on the atpb2 gene. It has been found that the polymorphic markers related to are extremely useful as tumor markers.

8. Deterioration of tumor cell contamination and heterozygosity
Analysis of correlation with loss Using an ALFred fluorescence automatic sequencer, the percentage of DNA containing the atpb2 gene lacking in the DNA of the gene sample was determined using the Tumor Cellularity.
We examined whether it is possible to measure quantitatively.

That is, using the Blunt-End SSCP method described above, LOH was converted to a polymorphic site of exon 4 of the atpb2 gene using a Cy-5-labeled fluorescent primer and an ALFred fluorescent automatic sequencer (Pharmacia). Detected.

The nucleotide sequence of the primer used here was as follows: on the sense side: 5'-ATCCAAGCCCAAAAGAA
TGA (SEQ ID NO: 12) Antisense side: 5'-AGCTGGGTCCGGTT
GAATTG (SEQ ID NO: 13), and the 5 'end of the primer on the sense side is labeled with Cy-5.

A2allele homozygous to DNA derived from normal cells showing heterozygosity at the same polymorphic site was ligated with atpb2 DNA derived from peripheral blood lymphocytes showing homozygosity.
Assuming the DNA of a tumor cell from which the gene had been deleted, a gene sample was prepared in a stepwise manner so that the degree of contamination of the tumor cell DNA was increased by 10% (see FIG. 10; Indicate the heterozygosity of the sample).

When the degree of tumor cell contamination is plotted on the X-axis and the ratio of A1 allele to A2 allele (A1 / A2 ratio) is plotted on the Y-axis, the A1 / A2 ratio shows the increase in the degree of tumor cell contamination. A negative correlation was observed (FIG. 11). From these results, it was found that the method of the present invention can quantitatively analyze the ratio of tumor cells in which gene deletion is observed.

[0091]

Industrial Applicability According to the present invention, a change in heterozygosity using a gene polymorphism observed near the downstream region of the p53 gene, which is known as one of the potent tumor suppressor genes, alone or as a p53 gene By using the gene in combination with a change in heterozygosity using the gene polymorphism as an indicator for detection, a detection method is provided which can particularly determine the presence or absence of cancer.

[0092]

[Sequence list] SEQ ID NO: 1 Sequence length: 5490 Sequence type: nucleic acid Number of strands: double-stranded Topology: unknown Sequence type: genomic DNA Origin: Organism name: Human Gene name: atpb2 gene Sequence GCGCGGCGCC CCCGCTTCTC CGCAACCCCC CGCCCCGCGC CCGGACTCGC CCCGCGCCAC 60 CAAGATGGTC ATCCAGAAAG AGAAGAAGAG CTGCGGGCAG GTGGTTGAGG AGTGGAAGGA 120 GTTCGTGTGG AACCCGAGGA CGCACCAGTT TATGGGCCGC ACCGGGACCA GCTGGGGTAC 180 GCAGGGCCGG CACGCAAGGG GCGGGGGAAA GCCGCGGGGC GACGCCTCGG GGGCGCAGGG 240 TCCCGCCGAC GCGGCCCCAG CTCCCCTCCC GGGTCCCCGG CGTCCAGCCT CCCTGCCGGG 300 CTCTGGGCTG GGAGGGGGCC GAATCGCCAG TCTAATCTCC CCGGCTGGCC GTGCGGAGGC 360 GGAGAAAGTA GGTCACAGCC GCCTTCCCGC CCCCCGCGGA GCCCCCTCGG GCGGGGGGTC 420 GCCAGCTCCG CCTGCGTGTC CGCGCCGCGG CGCTCACACT CCCTCTCGGG GCCTGTCCGC 480 TCCACACGGG CGTCCCCCAC CTCCAAAGAG CGCCCCTTCC CTCCCTCCGG CTCTCACTAG 540 CTCCGCAGCC CCGTCTATTT TTAGCTCGTG CCCACCCCCT GGACCCTGGG AACGTTCATG 600 AGGGGGCGGG TCTTGGGGGG GGTC TCGA TC 660 CCACCGCCTG GCCTTGGGAG CCTTCTGGGA CTGCTTTGTG GGTTGGGGGG CTGCTGATAG 720 TATGAGTTTT ACCGAGGCTG CAGGTTTTGC TCCCATGTCG GTGACGGAGG GAGGAGTGGT 780 CGCTGTGGTG ATTTGTGTGC ATCAGCCAGC CAGGTGTCTG TGACAGTCGG ATGACTTGGA 840 AGCCTCCCCA GGCTGACCAT GGCAGGACTC AGGGAGCTGT AGTGGTCGGG GGTTGGGGGG 900 GTGGAGGGGG TCTGGTGACC GGCACAGGTG CAGGTGAGGG GTGGAATTCT TCCAAGAGGG 960 ATGGTCAAGC TGGGACGTTG AGACACAGGG GACAGAGGAC ACTGTGTGAC ACGATTTACA 1020 ATCTTTCCAC ACTGGGCACC GTCCCCATCA GTCCACCCAT TCGGGGCCTA CACGAAGTGG 1080 GTCCCATGCA ATCCATTCCC TCAGGGAACT CAAACTCCAG CCCCTGGGAT GAGAAGAATC 1140 CAGCAATGCT TGGGAGAGCC AGAGGACTTC ATGGAAGAAG TGTCCTCTGA GATGGAAGGA 1200 TTGGGAGTCC AGGGTGGTGG GAACAGCCGG CCCTTGGGTC CTTACTTCAG GCGGGGGAGC 1260 CATGGAGAGA TCCCACCAAG GGAAGGCTGT GGGAGATTCT GCCTTTCCTC CCTGCCTCTG 1320 CCCAGGGTGC TGGGTGTGAA CTGAGGGTGG GGTGACTGTT GAAGGTTCTA ACAAGCCGTC 1380 TCTGAGAGAT TTGTAGCTAG GCTAGTGTTA GGTCTTTCAT TTCAGGAACT GTGTTCAAAG 1440 TTTGGCTTCT GAAGGGCACC AGGAGAGAGA TGTTGCTATT CAAATCTGAG GGTCCAGTCT 1500 CTGC GGGGTG GTATGAGGGT TTGCTTGTGA ATGGTGGCCA GTACCCGCTT TAAAAGGCAC 1560 CATGCTAGCA CAGCTTTAAG CATGAGTACG AATGCAGAGG TAACAGATGT GTGCCTTGTC 1620 AGGACTATGC ATGGTTGAGA AGTTGGAAAT GTAATTGGAG GCAAAATAAC AGACCTCCAC 1680 AAGGTCGGGC TTCACTGTGC CCTAGGACCA GGAGGGGGCT GGGAGTCATG GCTAGAAGCC 1740 AGACACAACT GCCTGTTTCC AGTTTGTCTC ATTTTGCCTC CAGAGGAAGG CTCTAAGACA 1800 TCCCTGTGGC TCTGTGATCA GTCCCAGTGC AGAACTTCAG AGTGGGTAGA GGGGTGTGTG 1860 GGGATAGTTG AGGTTATGGT GGGAACCTTG GGCCCTGCTG ACCCTGTTTC CTCCTCCCTA 1920 GCCTTTATCC TCCTCTTCTA CCTCGTTTTT TATGGGTTCC TCACCGCCAT GTTCACCCTC 1980 ACCATGTGGG TGATGCTGCA GACTGTCTCC GACCATACCC CCAAGTACCA GGACCGACTG 2040 GCCACACCGG GTGAGTGTGG AGGCTCCCCC TGCCAGCTAC TCTAACTGCT CTTGTGCCCC 2100 CAAACCTCCA GAAGGAACTC ATAGTTCCTT CCAGGAGTTT GATTTTGATG ACCCAATCCC 2160 CACGTGCTTG GAAGTTCTTG AAATCTGTCC ACCTTCCCAT TTACTGCAGT TGGGAGCTGT 2220 GTGATTTGGG CATGTGGCAG ATAGCCACAG GAGATCACCC TCCCATGAAG ACGATCTCAG 2280 ATATTCACCA CTGTCCCCAC TCTGGTGTTC CCTGTGTCCA TTTTCTTCCC TCTGTGTGCA 2340 GTCCCTCATC TTATAGATAC CCCCAACTTC TGCCTTTGTT GGCTGTAGGC TTGATGATTC 2400 GCCCCAAGAC TGAGAACCTT GATGTCATTG TCAATGTCAG TGACACTGAA AGCTGGGACC 2460 AGCATGTTCA GAAGCTCAAC AAGTTCTTGG AGCGTGAGTG TGGGCCTGGT TATGTGTCAG 2520 TTCAAGACTT CGGGCAGGGG ACTGGGGACC TTGGAAGTGG AACATCTGGC CCCTGAGTCT 2580 CTCCCTCCCA CCTCTTTAGC TTACAACGAC TCTATCCAAG CCCAAAAGAA TGATGTCTGC 2640 CGCCCTGGAC GCTATTACGA ACAGCCAGAT AATGGAGTCC TCAACTACCC CAAACGTGCC 2700 TGCCAATTCA ACCGGACCCA GCTGGGCAAC TGCTCCGGCA TTGGGGACTC CACCCACTAT 2760 GGTTACAGCA CTGGGCAGCC CTGTGTCTTC ATCAAGATGA ACCGGGTATC TATGACCTTG 2820 GTCCCCAGGG TGAATGGAGG AAGGATCTGG GGACACCACC TGCAGACAAT TGCATCCTTT 2880 CACTGGGGCT AATGGGCATG AGAAAGACTT GGATGTTTGT GTAGCTGAGA GAAAAAGAGA 2940 GGTGGGACTC TTGGAGGCTA AGCTCTGCAG TTAGAAGGCT GGATGCCTTT TGTTTTTTTT 3000 TTAGACAGGG TCTTGCTATG TTGCCCAGGC TGGCCTTGAA CTCCTGGAAT TAAGCTATCC 3060 TCCCGCCTCA ACCTCCCTAG TAGTTGGGAC TACAGTCCCC GGCTTAGCTT GGTCTGGATG 3120 CCCATCTTCG ACAACTTCTT CCTCTGACTC TCTTCACCTT CCACCCTCAC TCCAGGTCAT 3180 CAACTTCTAT GCAGG AGCAA ACCAGAGCAT GAATGTTACC TGTGCTGGGA AGGTGAGTTC 3240 GTTGGGCCTT GTCTGCCTGC TCACCTGAGT GGCTCACCTG AGTGTCCTTC ATGGTTTCTG 3300 TGTAATTCAC CTGTCTCTCC CTATCTTCTT TGCTCCTAGA GGCCCCATCA CCATAGAAAC 3360 AAGGGGGTAA GAGTGGGCTT TTGGGCTCCA CTGTAGCTTG AACTCCGAGG GCCCCGCACT 3420 CCTCTTGCTT CTCTCTGGGA TGCAGAGGCC TGCTCTCCTA GGGGCCAGAC ACACGCCCTC 3480 CTCCACCAAC GCCCTGGCCT CTGGCTTCTC TCCCTAACGC TTCCACCTTC TCCTTCATTC 3540 CCAGATTGTC CGTATCGTTC GCTCTCCCTC CCATATGGCC CCCACCCGTC AGTTCCTTCT 3600 AGGTGTCTGG TAGCTGCTGA TCTGTTAGCA CCATCTGCCA CCAGTTCGTT GTCCTTGGGA 3660 CCCTGTTCCC CGCTTCCCCC CCGGTCTGTC CTTTCTAGAA ACTGGCTGCT CCCTCCACAT 3720 CCCCTTCCTT GCTTCCTATT CAACCCTTAA TCATGTATCT CTTCTTTCTT GGCTCTGCTC 3780 CAGAAACTGA TTCCTGAGGA TGGGGTAAGA ACTTGGGGTA GGAGTGGAGT AGGAGGCTTT 3840 CGTGCCATTA GCAGCCTTCA GGAGTTCCTA GAATGATGAC AGGGACAGAC CATCCCCTCA 3900 TCTACACACG CACATGCAGA CACACACACA CAGACTCACA GCTCCAGGGA GGCAGACTTG 3960 AGACAGGAAT AATTGGGGCG AAGGAAGAAC AAAAGAACAA ATGGAAGTCT GGTGAGCTCC 4020 TGGGTGCCTG CCATCCCTAA CTGGCTCACC CCCTATCTTC CTGCACCCCC ACAGCGAGAT 4080 GAAGATGCTG AGAATCTCGG CAACTTCGTC ATGTTCCCCG CCAACGGCAA CATCGACCTC 4140 ATGTACTTCC CCTACTATGG CAAAAAGTTC CACGTAAGTC CCAGGGGAGG CCCAGGCTGA 4200 TGGCGGGTGC GGGTGGTGAG CTAGGGAAGG AGGCCGCGTG CCCCAGGCCT AGACCCTGCA 4260 CTGCTTCTCC GGCCCAGGTG AACTACACAC AGCCCCTGGT GGCTGTGAAG TTCCTGAATG 4320 TGACCCCCAA CGTGGAGGTG AATGTAGAAT GTCGCATCAA CGCCGCCAAC ATCGCCACAG 4380 ACGATGAGCG AGACAAGTTC GCCGGCCGCG TGGCCTTCAA ACTCCGCATC AACAAAACCT 4440 GAGGCCCCTT CCTCCCACCC CATCTCTCTC CTGTGGATGC TCCTGGAATG TCCCTGACCC 4500 TGCCTGATCC CTCCCTCACC CACCCCAAAG GTATTTTTGA TAACAGACCT ATGACTTGTC 4560 TGAGCCTCAC ATCCTTTTCC TTGACTTCTC AACCCAGCCT GAAGTCCATT GCGGTTCCGT 4620 CACTCACCTT TCCCACCAAC TTCTCCCAAC CTCAGATCAG TCAGACAGGG AGCTGGGCTA 4680 AGATGGCCAC GGAGGAGTTA GGAGCCTTTC TAGTTCTGGT TTAGCTGTGA GAGCTATCCA 4740 CTCTCCTGCC TGCATATCCC CTGAGAGTTA TAGGAAGTGC CCACTGACCC ACCCACCCAC 4800 CTACACCCCC CGCCACACAC ACACACAAAC GTGCACACGC GTCTCATTTG ACCCCTTTGC 4860 TTCCAGAGAT GAATGTGGCA CTCCCT CCTT CCATTCCTAA GCTCTGGCCA CCGTCCCTTG 4920 ATCTCTCATA CTTTCTCCCT GTCTACACAG TCGCCATCTT GGTGACTTTG AATTTATCTG 4980 GCTCCTGGGC AGGTCTTCTC CTCCTCTCCA TCCCTATTCC CTCCTCTGAA ATGCACCCCT 5040 TTGTAATTGA GGACAAGGTG GTTCTGTGGC CTTTTCCCTC TTTGCTGGCA CGTTCTGCTT 5100 CTCACCCTCT GGTGACTCTG TGAGCTGGGA AATGAGGGAC TGGAAGTGAG GCCTGTGTTG 5160 ACCCTTCCTG AAAATCCTCT AGCAGCCCCC GACTTCAGCA GTTTCTTTCT TTGTTTTTTT 5220 GAGATGGAGT TTCGCTCTTG TTGCCCAGGC TGGAGTGCAA TGGTGCAATC TCAGCTCACT 5280 GCAACTTCCG CATCCCAGGT TCAAGCGATT CTCCCGCCTC AGGTTCCCGA GTAGCTGGGA 5340 CTACAGGCAT GTGCCACCAT GCCCGGCTAA TTTCTTTCTT TCTTTTTTTT TTTTTTTGCA 5400 TTTTTTAGTA CACATGGGGG TTTCTCCTTG TTGGTCAGGC TGGTCTCGAA CTCCCGACCT 5460 CAGGTGATCC ACCTCACTCG GC

SEQ ID NO: 2 Sequence length: 25 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GGAGGGGAAC AAAGGCTGGA GACTG 25

SEQ ID NO: 3 Sequence length: 28 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence CTCTGGCTTC TTTTAGCAAG TTATCAAC 28

SEQ ID NO: 4 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GATGAGCGAG ACAAGTTCGC 20

SEQ ID NO: 5 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA sequence TCAACACAGG CCTCACTTCC 20

SEQ ID NO: 6 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence CTTGGAAGTG GAACATCTGG 20

SEQ ID NO: 7 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence CTGGGGACCA AGGTCATAGA 20

SEQ ID NO: 8 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence CCAACTTCTC CCAACCTCAG 20

SEQ ID NO: 9 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence ATGCAGGCAG GAGAGTGGAT 20

SEQ ID NO: 10 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence TTTGCTTCCA GAGATGAATG 20

SEQ ID NO: 11 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence AATTCAAAGT CACCAAGATG 20

SEQ ID NO: 12 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence ATCCAAGCCC AAAAGAATGA 20

SEQ ID NO: 13 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA sequence AGCTGGGTCC GGTTGAATTG 20

[Brief description of the drawings]

FIG. 1 is a drawing showing a nucleotide polymorphism site, an exon region, an initiation codon and a termination codon, together with the nucleotide sequence of the atpb2 gene.

FIG. 2 is a drawing showing the structure of the atpb2 gene and the polymorphic site of the gene.

FIG. 3 is a diagram showing an agarose gel electrophoresis diagram of a polymorphic site and a restriction enzyme site in the p53 gene and the atpb2 gene region, and a PCR product amplified by LongPCR between them, and a restriction enzyme map relating thereto. It is.

FIG. 4 is a drawing in which the polymorphism recognized by the restriction enzyme BsaJI in the atpb2 gene was analyzed by the SHIPS method.

[5] The genetic polymorphism that is recognized by a restriction enzyme Hae III in atpb2 gene is a drawing of the analysis by SHIPS method.

FIG. 6 is a drawing in which the polymorphism recognized by the restriction enzyme Mae III in the atpb2 gene was analyzed by the SHIPS method.

FIG. 7: Restriction enzyme Hga in exon 4 of atpb2 gene
For the I gene polymorphism marker, the homozygous gene sample and the heterozygous gene sample
It is a figure which shows the result of having performed End SSCP analysis.

FIG. 8: Restriction enzyme Hga in exon 4 of atpb2 gene
Nucleotide sequence around the I polymorphic marker site ( Hga I + and Hg
1 is a drawing showing I-).

FIG. 9 is a drawing showing the results of LOH analysis of a gene sample in which LOH is observed in the p53 gene region for the Hga I polymorphic marker in exon 4 of the atpb2 gene.

FIG. 10 is a view showing the result of examining heterozygosity in a gene specimen prepared in a stepwise manner for the degree of tumor cell contamination.

FIG. 11 is a drawing showing the results of examining the relationship between the degree of contamination of tumor cells and the ratio of both alleles.

Continued on the front page (72) Inventor Toshikazu Yamaguchi 1361-1 Matoba, Kawagoe-shi, Saitama F-term in BM Research Institute, Inc. (reference) 4B024 AA01 AA20 BA80 CA03 CA04 CA09 DA20 EA06 FA18 HA12 HA20 4B063 QA08 QA19 QQ02 QQ08 QQ43 QQ58 QR33 QR72 QS05 QS25

Claims (7)

[Claims] 1. A method for identifying a loss of heterozygosity in a genetic polymorphism marker in a Na / K-ATPase beta2 subunit gene present in a downstream region of a p53 gene in a gene sample, and deleting the gene in a cell derived from the gene sample. How to detect. 2. A method for identifying loss of heterozygosity in a gene polymorphism marker in the Na / K-ATPase beta2 subunit gene and a gene polymorphism marker in the p53 gene, which are present in the downstream region of the p53 gene in the gene sample. And a method for detecting a gene deletion in the cell derived from the gene specimen. 3. Na / K-ATP present downstream of the p53 gene
The gene polymorphism marker in the ase beta2 subunit gene is
In the nucleotide sequence represented by SEQ ID NO: 1 representing the range of about 10 Kb to 16 Kb in the downstream region of the p53 gene, the distinction between adenine and guanine at the 4691th base, the distinction between guanine and cytosine at the 4731th base, 4906 Selected from the group consisting of a different base of adenine and guanine and a different base of the 2649th base adenine and guanine,
The method for detecting a gene deletion in a gene sample according to claim 1 or 2, wherein the method is different from one or more bases. 4. Na / K-ATP present downstream of the p53 gene
The gene polymorphism marker in the ase beta2 subunit gene is
the presence or absence of restriction enzyme BsaJ I sensitivity in the vicinity of the base at position 4691 of the nucleotide sequence represented by SEQ ID NO: 1 representing a range of about 10 Kb to 16 Kb downstream of the p53 gene;
Mae III sensitivity near base 731, Hae III near base 4906
The presence or absence of sensitivity and the presence or absence of sensitivity to one or more restriction enzymes selected from the group consisting of the presence or absence of Hga I sensitivity in the vicinity of the 2649th base.
A method for detecting a gene deletion in the gene sample according to claim 1 or 2. 5. The method according to claim 1, wherein the gene sample is p5
The method for detecting a gene deletion in a gene sample according to any one of claims 1 to 4, wherein the polymorphic marker within the three genes is a gene sample of an individual showing homozygosity. 6. The gene deficiency in a gene sample according to claim 1, wherein the means for identifying loss of heterozygosity is a blunt-ended single-stranded DNA higher-order structural polymorphism analysis method. How to detect loss. 7. The method for detecting a gene deletion according to claim 1, wherein the gene deletion detected by the method for detecting a gene deletion in a gene sample is used as an indicator of the presence of cancer. Method.