JPH03501202A - dna probe - Google Patents

Abstract

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

Description

[Detailed description of the invention] dna probe DNA probes that detect individual differences at the DNA level, so-called “hypervariable” Hypervariable probes have recently been isolated (5), Parvariaple probes can detect variable DNA in human DNA. Ru. The hypervariable region detected by this probe is a region in which the basic DNA sequence is repeated many times. It consists of things that are Hypervariable regions have been detected at various locations on many chromosomes.

The degree of variability in these parts is due to the difference in the number of repeats of the repeat sequences in a certain part.

When individual DNA is digested with a restriction enzyme that does not cut within the excess variable region, it will contain the excess variable region. A large number of DNA fragments are obtained. DNA fragments are prepared using well-known means, and the base pairs in the fragments are Can be separated according to number. In the case of overvariable regions, the size of the resulting fragment depends on the number of times the repeat sequence is repeated in that part, so restriction enzyme fragment length polymorphism arise. These fragments are subjected to hybridization with hyperbaric pull probes. detected by These are DNA prepared from tiny samples of body tissue. can be shown as a "genetic fingerprint" by a simple test.

The variability detected by this probe is so significant that only homozygous twins can be distinguished. Shows no pattern.

Hypervariable probes that can identify variable regions of specific human chromosomes and their In addition to their usefulness in identifying individuals, derivatives are useful for mapping genetic diseases. and through families where the inheritance of the above fragment has been shown to be Mendelian. It can be used to track them. With these probes, after The likelihood of future disease states can be predicted.

However, all variable regions (5) detected by the hypervariability probe Derived from autosomes. No X-chromosome bound sequences have been detected to date.

This time, we isolated a certain DNA sequence without referring to the previously reported variable sequences. did. This novel sequence hybridizes to the variable region and flanking sequences of the X-chromosome. Soybean.

According to analysis of a human-mouse hybrid cell panel, this 3, Xeen-Xpll, 2 according to in situ hybridization 2, and the estimated position is XpH, 22.

The invention therefore provides a DNA sequence characterized by: It was determined by in situ hybridization that the Xce of the human X-chromosome was Located in the n-XpH, 22 region.

This generally refers to χeen-χpH, with the pattern of restriction sites shown in Figure 1. All or part of the DNA from three regions.

It contains a region consisting of a variable number of repeats of a repetitive sequence (IIYPERXOX); This occurs when human X-chromosomal DNA is cut with a restriction enzyme that does not cut within the repeat sequence. , producing restriction fragments that vary in length from individual to individual depending on the number of times the repeat sequence exists.

See attached drawings.

FIG. 1 is a restriction map of cosmid M27 of the M27β region.

Figure 2 shows the Sau3^/HaellI restriction fragment isolated from sequence M27. , which contains the 26 bp repeat sequence HYPERXOX 3'/2 near healer.

Figure 3 is a Southern plot of EcoRI digested female DNA probed with M27β. and establish multiallelic variation. I testify.

Figure 4 is a southern sample of EcoRI-digested DNA from relatives probed with M27β. It is a lot and shows a simple link-related Mendelian path.

Figures 5 and 6 are pedigrees and pedigrees of families in which retinitis pigmentosa is known to occur. and an EeoRI digest of DNA obtained therefrom.

Figures 7 and 8 show Mspl and HpaI of X chromosome DNA probed with M27β. This is a digest of I and shows a pattern of X inactivation.

FIG. 9 is the proposed structure for the repeating array IIYPERXOX. .

In one form, this sequence is the DNA of the M27β region that includes flYPERXOX. Construct fragments.

DNA molecules or fragments thereof prepared by conventional methods for use in detecting complementary sequences It's a piece. According to one particular embodiment, the present invention provides DNA comprising the single-stranded DNA as described above. A probe in which the heavy chain DNA molecule is labeled with a detectable marker. provide the following. Commonly detectable markers include fluorescent and radioactive markers and components of chromogenic and chemiluminescent enzyme systems. These ma markers are well known in the art.

M27β is localized in a small region in the pericentric region of the X chromosome (XpH, 3→Xcen) This is useful for linkage analysis of disease loci that are thought to exist in this region. It means using a probe to scale. The degree of recombination between sequences near centromeres is more distant. This is considered to be lower than that between the sequences located at the proximal XQ. Applicable to those of the concus and proximal Xpl. Gene loci in this region include retinitis pigmentosa. sex (XpH, 3), achromatopsia (XpH), Menkes syndrome (Xpl 1-ql 1) and Wiskott -Aldrich syndrome (Xpl1-ql2).

According to another aspect, the present invention provides the D treated under hybridization conditions with an NA probe, and then this treated Analyzing DNA to detect the presence and/or size of fragments in each DNA Assay methods (particularly for determining the heritability of hypervariable regions within a family and disease status in progeny) suitable for determining the possibility). One method suitable for carrying out this kind of method is The well-known Southards blotting method is used. According to this method, processing The desired DNA is cut with one or more restriction enzymes, and the fragments are subjected to gel electrophoresis. Separate according to size by movement. According to standard methods, the DNA in the gel is transferred to a nitrocell. Transfer to a loin filter paper and remove any DNA that is complementary to the probe or contains an NA sequence. The binding probe is then hybridized with a labeled probe that also binds to the NA fragment. The target is detected by its detectable marker. There are many variations of this type of method. In this screening method, each of these would also be suitable according to the present invention. One DNA fragment will be obtained per X-chromosome, so by female DNA Usually two fragments are obtained, and with male DNA usually one fragment is obtained. family lineage By comparing the positions on the gel of the fragments obtained from Therefore, it becomes clear whether the gene was inherited, and the genetic locus in the area of 10-B Ping reveals the possibility of disease status in offspring related to X-linked diseases. There will be.

According to another aspect of the invention, screening for disease states in which an abnormal number of X-chromosomes is present. Methods are provided for testing and diagnosis. One way to carry out this method The method is to use the Sathertz blotting method. In this method, we generate The number of different length fragments is usually the number of X-chromosomes present. Therefore it exists Diagnosis can be made simply by looking at the number of fragments present.

According to this method, in the case of Klinfelter syndrome, The above probe was used to prove the involvement of both maternal X chromosomes.

The present invention provides methods for examining X-chromosome inactivation in cells obtained from diverse sources. We also provide This is the restriction enzyme Mspl and ) (1 or 2 for palI) This is possible due to the presence of the above adjacent recognition sites. The enzyme Mspl has the sequence CCGG cleaved and does this even when internal cytosines are methylated; isoschizomer HpaII at the unmethylated cccc sequence. The probes and hypervariable regions of the present invention are cleaved only when active fragments that do not contain the target site and therefore hypervariability extends to the fragments formed by them. The sex X chromosome is methylated at the target site adjacent to the binding site of the probe of the present invention. It was found that inactive X was not methylated. According to preliminary experiments Therefore, the prediction based on the effects of other enzymes, i.e., that most females Heterozygotes for all restriction fragment alleles were confirmed.

The probe according to the invention can be used in many situations - e.g. imprinting, X series in females. Chain failure and clonal analysis - X inactivation status in progeny from one somatic cell Constant methylation resulting from consistent inactivation of one of the two X-chromosomes in can be evaluated by its persistence. This type of method is similar to other (non-overvariable sex) has been reported for the X-chromosome probe (7).

The above DNA sequence was isolated by the following method.

LNnE and 011 Human-mouse hybrid cell line MOG-T cosmid vector pTM1 (1 ) was constructed using 32p radiolabeled whole human genomic DNA. A was used to screen cosmids containing human material. hybrid MO G-T contains two fragments of the human X-chromosome translocated onto the mouse chromosome, which are the only Together, these fragments span the entire X-chromosome as one human component (2). It is thought that the entire X-chromosome is involved. There are almost no differences between human and rodent neck repeats. Since cross-hybridization does not occur, human X-chromosome DNA hybridization Only clones containing the probe hybridized to the above probe, and these were autotraced. Identified by geography.

Consistently copying DNA fragments within the cosmid insertion sequence are again fragments containing repetitive sequences. Sather restriction digest clones using radiolabeled total human DNA to detect Fragments that give no signal on autoradiography, identified by Presumed to contain unique or low copy number sequences. one of these cosmids (M27) are three types of low copy number EcoRI restriction fragments - M27α1M27β and M27γ, sizes ranging from 3 kb (M27α) to 1.5 kb (M27γ 9M27β, which was found to contain - spanning ), is 2.3 kb. These are It was subcloned into the lasmid vector pUc9.

The X localization and subregion assignment of M27β are expressed in various ways. EcoRI digestion from a hybrid cell line containing the human X-chromosome of This was carried out using a hybrid mapping panel made of NA. used in this study The hybrids used were as follows.

retained human Vibrator 1 H0RL911R8B Xpter-Xq2 (2-4) (2) PIP’ X pH, 4-Xp22.1 (2) Xq26-Xqter 8 Xqter-Xp21 (2) MCP6 Xq13-Xqter ( 4) The sequence detected by M27β is a hybrid) IORL911R8B and and WAG8. This makes these subregions XpH, 3-Xq1 It belongs to 2. This position is further derived from a cell line containing the 1soXq chromosome. 0M, refined by probing DNA from cell lines containing the X chromosome Since 27β does not detect sequences present in this hybrid, this position n-XpH, 3. The Xcen-XpH described here, 3 has a breaking point. X-staining up to XpH, 4, i.e. to the area present in the hybrid PIP It should be understood that it includes areas of the body. In situ hybridization experiments further refines the position to Xeen-XpH,22, and the estimated position becomes XpH,22. It was done. The X chromosome present in the hybrid cell line has a different origin, 0M27β. WAG8 and HORL911R8B (and control male and female DN The sizes of the EcoRI restriction fragments detected in A's are all different and therefore Indicates a hypervariable region.

A striking finding is that from the cell line MOG-T, from which clones are initially derived, The EcoRI restriction fragment detected by M27β when used to examine the DNA of The length of the fragment was ~5.1 kb, unlike the expected length of 2.3 kb. Cosmid M2 7 in the 827β region (see Figure 1). M27β (HXoxS Bgl [restriction fragment to the left of site B+] detected in the genomic digest probed in l) (the constant part) is the same size as that present in Rosmid.

The right genomic restriction fragment of B1 detected by M27β and HχoxS2 (B gl II-EeoRl, Bgl II-Bgl II, Bgl II-Pv ulI and BglII-RindIII) show significant variation among different individuals. (variable part). Restriction fragment B"-E" (HXoxS2) contains a 2.8 kb deletion. It seems to include. B detected by HXoxS2 during hybrid MOG-T The gl II-EeoRI genome fragment is 0.8 kb predicted from this map. This is because it is ~3.6 kb. The large number of repetitive sequences responsible for the observed hypervariability It is likely that the following deletions are contained within this deletion.

A plausible explanation for the sequence deletion observed here is that this locus repetitive sequences involved in hypervariability in order to propagate cosmid libraries. recA' is said to be unstable in the E. coli host (E. coli ED8767). It is something. This phenomenon has been noted by several other researchers. This iterative arrangement For the purpose of isolating columns, the method reported by Nichols et al. (6) The method was adopted. They are vector pUC9 and rec A coli HBIO Specific genomes in a small but typical plasmid library using I strain A simple and rapid method for cloning DNA fragments is reported. said excess Variable repeat sequences have been shown to be stable using this system; has been used to effectively isolate supernumerary repetitive sequences specifically associated with the α-globin locus. Ta. Using this method, 4X Cell line G? Cloning 5.3 kbBgl II-EeoRI from 41416 I tried to do that.

In other forms of the invention, the probes are present in multiple copy numbers within the hypervariable repeat region. Consists of existing DNA fragments.

IYPERXOX (hypervariable repeat sequence) is responsible for the observed multiple allelic variation. This is an array.

The sequence described as M27β is primarily a consistent copying process that detects hypervariability. However, this means a large number of repeat sequences)IYPERXO)! -Mo included tj.

5au3^/HaelII restriction fragment - 3 complete copies of a 26 base pair repeat sequence and one partial copy were isolated from M27β. The entire distribution of this restriction fragment Zero-repeat motifs whose columns are shown in FIG. 2 are represented in bold type. From Figure 9, 26 salts Base pair repeat sequence 1 (YPERXOX is a complete sequence of 10 base pairs separated by 3 base pairs) It contains an inverted repeat sequence, thus forming a cruciform structure with a symmetrical stem-loop shape. It can be seen that there is a possibility that This type of sequence is particularly useful in biology when promoting recombination. important. If these sequences exist in the human genome or in the genomes of other organisms, It may be widely distributed.

Other derivatives of the 26 bp repeat sequence FIYPERXOX can be used for oligonucleotide synthesis. It consists of the following polynucleotide.

＾CATACTATCCAtl:(:ACTC(3')This derivative (oligoHY PERXOX) cannot form a stable internally folded structure and H Unlike YPERXOX, it can easily hybridize to complementary sequences.

Radiolabeled oligo 1 (YPERXOX in M27/9 digest) HYPERXOX It can hybridize to restriction fragments that carry repetitive sequences.

Polynucleotide sequences, oligos HYPERχOx and IIYPERXOX or is a complementary sequence to RYPERXOX or its complementary sequence, and includes repetitive sequences. will hybridize to the DNA fragment containing the DNA.

Oligos containing a significant portion of the HYPERXOX sequence or complementary sequences Other probes consisting of sequences such as columns also ensure specific hybridization. It is possible to hybridize to DNA fragments containing repetitive sequences as long as sufficient portions of the sequence are used to It will be. Also, HYPERXOX sequence or complementary sequence or sufficient to cause a specific mode of hybridization to a significant portion of the Probes consisting of polynucleotide sequences with moderately high homology also contain repetitive sequences. It should be appreciated that the method can be used to detect DNA fragments containing DNA fragments.

Therefore, polynucleotides suitable for detecting variable DNA fragments containing ) IYPERXOX The nucleotide probe is specific for the HYPERXOX sequence or its complementary sequence. It can be prepared from any hybridizing polynucleotide sequence. array is short The higher or lower the homology, the less specific the hybridization will be. These low specificity probes may be used to analyze the human genome or the genome of other species. Sequences related to HYPERXOX can be isolated from the entire system. But phase The degree of homogeneity should not be reduced to the extent that it binds to unrelated sequences; As shown in Figure 2, the −1 copy sequence flanking the HYPERXOX repeats is A probe consisting of polynucleotides is used to target a restriction fragment containing the HYPERXOX sequence. It breeds. Again, specific hybridization rather than the entire sequence A probe consisting only of sufficient or complementary sequences to cause I can stay. Once the DNA fragment containing the HYPERXOX sequence is isolated, it is (also used as a single copy area to flank the IYPERXOX area) Sequences that bind to the probe region can be isolated, and these can be isolated if the restriction enzyme used between the hybridization site and the HYPERXOX region or) IYPE Hypervariability with HYPERXOX unless cleaved within the RXOX sequence It can be used as a probe to detect.

Probe M27β is complementary to the sequence of the 5au3^/HaeIII fragment shown in Figure 2. by synthesizing a polynucleotide probe containing at least a portion of the The MOG-T hybrid was then isolated under the hybridization conditions described above. To explore the EeoRI-digested genomic library constructed from the Rhinoceros cell line. The probe thus obtained is then used.

Therefore, the present invention provides a restriction fragment containing variable copy number repeat sequence 1 (YPERXOX). We also provide a method for producing hybridizing probes, which consists of the following steps: .

one marker or label component, and a restriction fragment containing the repeat sequence HYPERXOX. comprising at least a portion of the sequence of FIG. 2 sufficient to specifically hybridize. Prepare polynucleotide probes.

Genomic libraries containing X chromosome DNA prepared by restriction enzyme digestion or other methods. The library is incubated under hybridization conditions.

- Identifying plasmids containing hybridized fragments.

- Using these isolated plasmids, restriction containing HYPERχOX repeat sequences Probes that hybridize to the fragments are further prepared and optionally further modified as described above. isolate the relevant probe.

Furthermore, other regions of the human genome and other organisms closely related to HYPERXOX The possibility of the existence of contiguous sequences also serves as a basis for isolating single copies adjacent to these. Become. Excess variable repeat sequences and adjacent −1 copy sequences (within <40 kb) are both This may be of considerable utility in the genetic analysis of disease loci. For example, the present inventors demonstrated that M27β and retinitis pigmentosa (8), H The proximity of YPERXOχ sequences to disease loci suggests that their subsequent isolation will also be provided.

Furthermore, as shown in Figure 9, the scales form a multiple cross structure, making it easier to columns become more susceptible to silver content and silver-white recombination. of this probe or its derivatives. Other uses include repeat motif-bound sequences in the human genome and homologous sequences. It is the introduction into the genome of another organism.

Plasmid M27 has significant elements as (1) target cells, such as neomycosis; (2) Selectivity I flanking the “marker” gene (a copy of the YPERXOX sequence, (3) a reagent comprising a sequence that allows propagation and selection of decorated plasmids in bacteria; serves as the basis for the preparation of This reagent is known as “transfection”. It can be introduced into target cells by the method described below. This is A Practica l Guide to Mo1ecular Cloning” () (Perbal, B, > 1984, Wiley Interscience.

510-512).

After “transfection”, a certain proportion of the donor sequence is recombined into a recipe. Homologous recombination (integration into the endogenome) occurs at a low frequency, but HYPERXO It will be significantly enhanced by X.

This method is useful for introducing selectable markers at HYPERXOX sites within the genome. It is for use. Further enrichment of flanking sequences, including linked genes, indicates that chromosome-mediated genes by established methods of transduction (CMGT) and genomic library preparation. achieved (e.g. Pritchard, C. and Gutsdoff). G oo -dfellow, P, N, 1987 Developopm ent　101. (see Addenda 59-65), and also I (homologous by YPERXOX) Scales enhance sexual recombination by adding other genes to the genomes of humans and other organisms. Materials can be introduced. Especially according to this method recipes with defective genes (gene therapy) to introduce a normal copy of a gene into an experimental organism, or to introduce a new copy of a gene into an experimental organism. It becomes possible to add genes (genetic manipulation).

K! Takumi 1 By further exploring EeoRI-digested RNA from a panel of 18 unrelated women, We investigated this hyperproliferative property. Prepare filters from 2.5μ9 samples of each digest. Then, the M27β insertion was investigated. Final stringency of the filter , 1ySSC10, 1$SDS. The results are shown in Figure 3. Observation The hypervariability observed was quite significant, with 17 variants in the 18 women tested. was detected. Only two women were found to be homozygous at this locus. This suggests that the degree of heterozygosity is approximately 90%. The resolution of the gel is not good enough to accurately estimate the exact size differences between alleles. However, it was an addition/deletion of a basic repeating unit with a length of about 100 to several hundred bases. This seems to be for the purpose of

dog 113- The stability of this multiple allelic variation through female meiosis indicates that it is possible to have a three-generation pedigree. was analyzed by probing the DNA obtained from These mutants have a simple sex-linked mechanism. The results are shown in FIG. 4, indicating a genetic defect.

All experimental methods used were described by Maniatis et al., Molecular C1oniB, % test manual, published by Cold Spring Harbor Laboratory. It was published in Publishers, New York (1982).

The hypervariability detected by the above probe and the position to Xeen-Xpll, 3. Location determination is important for diseases in which the locus is located in this region (retinitis pigmentosa and ataxia pigmentosa). mapping and prenatal diagnosis of We have demonstrated the importance of this marker in the analysis of other diseases (including Menga syndrome). suggest.

Fire Tribe Master 1 Study of the linkage between M27β and the mmembrane pigmentosa locus.

Retinitis pigmentosa (RP) occurs in 1 to 2 in 5000 people in the general population. R 36-38% of P patients are isolated cases, the rest are autosomal dominant, autosomal The X-linked type (XLRP), which shows recessive or X-linked inheritance, is a rice It is found in 14-22% of RP families in the country.

Linkage experiments using RFLP probe L1.28 showed that RP was close to XpH,3 Although it was shown that there is a genetic locus, not all carriers provide information. This probe provides limited information because it does not. Ll, at 28 The heterozygosity of the two Tagl alleles detected was estimated to be ~40%. Ru.

M27β detects a high degree of heterozygosity, which is estimated to be over 90%. In this analysis style, information is available for almost all individuals in each family. Once linkage to a family is confirmed, detection of suspected carriers and morbidity can occur. Prenatal diagnosis of affected individuals should be possible.

T! 4 German and European groups in which 4 membranous pigmentosas are known to be isolated. Since we were about to obtain blood samples from a large family in Goslavia, we decided to DNA isolated from samples was analyzed with H27β and alleles detected by this probe were analyzed using H27β. We investigated whether the gene was linked to the locus for this disease.

Blood samples were collected from which DNA samples were prepared using standard methods.

Figures 5 and 6 show the pedigrees of the individuals analyzed in this study and from these individuals. The autotra obtained by probing the EcoRI digest of DNA with M27β. Show geograph.

Seeking five NΩU-wan From this pedigree, females 57 and 58 are obl (obl) as they are daughters of the affected male. igate) Both are M, W, 3.4kb and 3.4kb. and a 3.0 kb allele. In each case, the sons of these individuals ( 63 and 59) have inherited the 3.4 kb allele, which is unique in this family. 0 females 56 suggesting that this disorder segregates with the 3.4 kb allele was clinically diagnosed as a carrier and also received the 3.4 kb allele. I inherited it. Therefore, for the purpose of linkage analysis, information on three cases in which no crossover is detected is required. There is meiosis, which provides Furthermore, the woman, 53, is also believed to be a carrier.

Seeking picture 1μ” Want Woman 49 will have an affected son and a daughter who will have an affected son in subsequent generations. , is an absolute carrier. She is M, W.

Affected sons 45 with 4.8 kb and 2.8 kb alleles, both children Daughters 43 and 46 carry the 2.8kb allele, which is unique in this family. This suggests that this disorder segregates with the 2.8 kb allele. affected Male 44 also inherited this allele. No crossover detected in this family4 Meiosis providing example information was recorded. Furthermore, the woman, 42, is a carrier. It appears that unilateral 47 and 48 are not carriers.

All these pedigrees provide information, and the results are simple representations of the different alleles detected. Consistent with X-linked Mendelian inheritance.

Master of Kyoshiba U's DNA was extracted from blood (B), lymphoblastoid cell lines (L>) or somatic cell hybrids (H ) was prepared by standard methods. 5μ this sample Mspl or) by 1paII , digested with buffer restriction solution (BRL) supplied by the manufacturer, and electrophoresed. It was transferred to a Hybond, N-nylon membrane (Emersham). probe DNA Labeled by two-dimensional translation as lO”dp-/μg, high After hybridization and washing, the final stringency was 0.5XS. C (at 63°C).

Details of the sample examined Number of samples: 7 types, number of alleles/X-inactivation state: 46, XY: 2 (B) + 5 (L ) 1 activity x 46, XX 3 (B) + 1 (L) 2x/-y random inactivation 48 , XXXX 1 (L) 1 active + 3 inactive - the chromosome is active) shows a single band for the Mspl digest, which is M This is because spl cleaves internal cytosines independently of methylation. Hpall In the digest, this single band is replaced by a high molecular weight substance. HpaI [ is not cleaved if the internal cytosine is methylated.

Normal women show two bands for Mspl digest.

After digestion with Hpa[, both bands are present, but their intensity has decreased and the This suggests that most women are heterozygous for X inactivation. Check.

Figure 8 - Women with a pattern of X inactivation have This is not present in the Hpal digest, and is present in the Hpal digest where high molecular weight substances are present. The other band present in the digest is Hpa [which serves as a good control for the digestion.

Somatic cell hybrids containing the M27β locus on the active X chromosome have the same pattern as males. Indicates the tone.

These results indicate that M27β was isolated after digestion of genomic DNA with Mspl/HpaI. The hybridization patterns elucidated in may be directly applicable to the evaluation of X-linked disorders in cancer and clonal analysis during tumor progression. Suggests that there is something wrong with it. High degree of heterozygosity and methylation at this locus Direct detection of pharyngeal enzymes, M spl and Hpall, is shown here. We show that the simple system we describe is a useful complement to our previously published (7).

11 sentences 1 1. Grosveld (Grosveld, F, G, > et al., Nucleic) Ac1dsRes, (1982) Vol, 10.6175-6733°2, Boyd, Y., Ann, Hum, Genet, (1987) Vol, 51゜13-26゜ 3. Bishop (B1shop, C, E, ) et al., Nature (198 3) Vol.

303.831-832° 4. Goodfellow, P, N, et al., Proc. , Natl.

Acad, Sci, USA (1982) 79.1190-1194°5, Ji JefTreys A, J, et al., Nature (1985 ) Vol, 314.67-73゜ 6. Nichols (N1chol Is R', D,) et al., Nucleic Ac1dsRes (1985) Vol, 13.7569-7578°7, Fo. - Vogelstein, B., Fearon , E, R, ) Hamilton, S, R, et al. 1987, Ca ncer Res, 47.4806-4813°8, Meitinger inger) et al., 1988 Hu+man Genetics (in press).

Engraving (old’!+:=, no ie) Shinsho (Sad to Hei G: None) turtle Ft(,,3 ・ Engraving (no change to [t;) Engraving (no changes to RjiS) −・・・・・ Engraving (no changes to the content) MspI Hpa[l kbゝb Ficr, y 46, Xt (X; 8) 46. χdicX 4s, xxxx /, 1” yF “F/C7,’S (Replacement of claims 12 to 16) 12. A claim in which the first and second enzymes are Mspl and pHpaII, respectively. The method according to item 11.

13. Any by at least -1 copy of the sequence according to claim 4 A DNA sequence consisting of a sequence of DNA whose sides are flanked sequences.

14. A DNA sequence containing multiple copies of the sequence described in claim 4 is used as a genome In the method of introducing into Is the DNA sequence intended to be inserted flanked on either side? A reagent is prepared, and the prepared reagent is labeled using a transfection method. A method consisting of introducing a target cell into a target cell.

15. Contains a label or marker component and hybridizes to the DNA restriction fragment In the polynucleotide probe, the following sequence where the fragment is read from 5° → 3' TCCTG with ATAGATAACTATCCAC (:ACTC or complementary sequence A probe containing at least -1 copy.

16. In the method for manufacturing the probe according to claim 15, the following A method consisting of steps - a marker or label component and a restriction fragment containing the repeat sequence HYPERXOx A sufficient number of sequences according to claim 3 to hybridize specifically. A polynucleotide probe consisting of at least a portion of a spider is prepared.

A genomic library containing X chromosome DNA prepared by restriction enzyme digestion or other means. The library is incubated under hybridization conditions.

Identification of plasmids, cosmids, or hepatocytes containing single-hybridized fragments Ru.

- Using these isolated plasmids, Probes that hybridize to the restriction fragments are further prepared and, if desired, further Isolate the appropriate probe.

Procedural amendment book phantom

Claims (1)

[Claims] 1. A DNA sequence characterized by: -It was determined by in situ hybridization that the Xce of the human X chromosome was It is located in the n-Xp11.22 region. - This generally refers to Xcen-Xp11 with the restriction site pattern shown in Figure 1. ．． All or part of the DNA from three regions. - It contains a region consisting of a variable number of repeats of repetitive sequences, which is the human X chromosome D When the NA sequence is cut with a restriction enzyme that does not cut within the repeat sequence, the number of occurrences of the repeat sequence is reduced. Restriction fragments with different lengths are produced depending on the number of individuals. 2. Contains a marker or label and consists of the DNA sequence according to claim 1 DNA probe. 3. Isolates or clones having the following formula read in the 5'→3' sense Polynucleotides in converted or synthesized form [sequences available] or a polynucleotide of complementary sequence to the above. 4. Isolates or clones having the following formula read in the 5'→3' sense: polynucleotide in the form of a sequence [there is a sequence] or in the polynucleotide 5.5'→3' sense of the complementary sequence to the above. The polynucleotide in isolated or cloned form has the following formula as read by [There is an array] or a polynucleotide of complementary sequence to the above. 6. The polynucleotide sequence or polynucleotide sequence according to any one of claims 3 to 5 and a label or marker component. probe. 7. The following sequence from 5' to 3' including a label or marker component [Sequences available] or a polynucleotide probe that hybridizes to a complementary sequence. 8. The following sequence containing a label or marker component and read from 5' to 3' [There is an array] or in a single copy region of a DNA restriction fragment containing two or more repeats of complementary sequences. Polynucleotide probe that hybridizes. 9. Loci linked to M27β or other genes containing HYPERXOX sequences In a method for determining the disease state of an individual with a locus, restriction enzyme-digested chromosomal DNA is Restriction fragments containing a variable number of repeats of the sequence or complementary sequence of claim 4 probed under hybridizing conditions by a probe that hybridizes to the piece; and The processed DNA is analyzed for the presence of restriction fragments from chromosomal DNA and/or consists of detecting the size by comparing it to DNA from a similarly treated standard. Method. 10. In a method for measuring the number of X chromosomes in an individual's genome, restriction digested DNA of the individual A or DNA from a tissue sample to the sequence or complementary sequence according to claim 4. Hybridization is achieved by probes that hybridize to restriction fragments containing a variable number of repeats of the sequence. Alleles detected by probing under hybridizing conditions and analyzing processed DNA A method consisting of measuring the number of genes. 11. In the method for determining the X inactivation state, the methyl of the nucleotide within the recognition sequence at least once by cleaving the DNA regardless of the Request a DNA sample digested with a first restriction enzyme that produces a restriction fragment containing a copy of hybridize to a restriction fragment containing at least a single copy of the sequence described in section 4. If the recognition sequence contains methylated nucleotides, If so, use another DNA sample digested with a second restriction enzyme that does not cleave DNA with the above probe. Each X-stain is probed with A method consisting of determining the inactivation state of color bodies. 12. Claimed wherein the first and second enzymes are Msp1 and HpaII, respectively. The method according to scope item 11. 13. by at least a single copy of the sequence according to claim 4. A DNA sequence consisting of a sequence of DNA that is flanked on its sides. 14. A DNA sequence containing multiple copies of the sequence according to claim 4 is inserted into the genome. by at least a single copy of the sequence described in paragraph 4 of the same. From a DNA sequence intended for insertion flanked on either side and target the prepared reagent by transfection method. A method consisting of introducing into cells. 15. A polymer that contains a label or marker moiety and hybridizes to a DNA restriction fragment. In the renucleotide probe, the following sequence is read from 5' to 3' of the fragment. [There is an array] or a probe containing at least a single copy of a complementary sequence. 16. The method for manufacturing a probe according to claim 15, comprising the following steps. How to: - a marker or label component and a restriction fragment containing the repeat sequence HYPERXOX; A sufficient number of sequences according to claim 3 to hybridize specifically. A polynucleotide probe consisting of at least a portion of a spider is prepared. - Genomic library containing X chromosome DNA prepared by restriction enzyme digestion or other means. The library is incubated under hybridization conditions. - Identification of plasmids, cosmids or phages containing hybridized fragments Ru. - Using these single plasmids, restriction containing HYPERXOX repeat sequences Probes that hybridize to the fragments are further prepared and optionally further modified as described above. isolate the relevant probe.