JPH06501386A - Y chromosome-specific polynucleotide probe for fetal sex determination - 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] Field of invention: Y chromosome-specific polynucleotide probe for fetal sex determination The present invention relates to the genetic sex determination of mammals, and more particularly to Y-specific polynucleotide proteins. Concerning the sex determination of anti-separated animals using a tube.

Background of the invention The ability to determine the sex of an embryo immediately after fertilization has many uses in the livestock and dairy industry and in veterinary medicine. It brings many benefits. Advances in Liver Transplantation in the Dairy and Beef Cattle Industries Embryo and Development There is an increasing need for methods to rapidly determine the sex of cells obtained from early-stage embryos. I set it.

The commercial efficiency of livestock and dairy operations is increased by establishing pregnancies with embryos of the desired sex. improved. The advantage of predominant female offspring for the dairy industry is that It would be advantageous if the sex of the embryo could be routinely determined prior to implantation. What are the benefits of sexed embryos? Placement of livestock based on desired characteristics such as size, weight, and increased milk production. Consists of many options, including replacement options. In addition, specific diseases, such as human X-linked disease (X-ct+romosome-) ) and similar diseases in other mammals affect individuals of one sex more strongly. To tell If so, early determination of the sex of embryos that are individuals with such diseases would be particularly effective. provide valuable information on which decisions can be made to proceed further with the outbreak.

In addition, effective sex determination of conceptuses in vivo may be of economic importance; It also has important economic uses. Artificial or autotransplantation in dairy and animal husbandry In pregnancies that occur through natural mating, the initial determination of the sex of the embryo or fetus is determined by the embryo or fetus of the desired sex. or consider terminating the pregnancy if the fetus is not humiliated.

Determination of the sex of an embryo or fetus after fertilization is necessary for health or economic reasons. It is important to determine the sex as soon as possible. Growth arrest occurred during late pregnancy If the risk to the female's life and health increases significantly. In livestock, reproductive efficiency The required amount of upper adult (to carry the embryo) is low and poses a risk to the female's life and health. is commercially inefficient.

Advances in reproductive biology now allow for in vivo or in vitro Determine the sex of any embryo that occurs at any given time, and also determine the sex of an embryo or fetus in vivo. All related pregnancy and developmental arrest should be determined as early as possible and reliably determined. All risks and expenses can be avoided.

Mammalian sex is determined by the presence or absence of a complete Y chromosome or some functional part of this chromosome. Determined by The gene is located on the Y chromosome that controls the formation and development of the male phenotype. exist. Therefore, the sex of an individual mammal depends on whether this genome contains a specific DNA sequence. whether or not these sequences are particularly relevant to the Y chromosome, which encodes the genes responsible for sex determination. It depends on whether you include that part or not.

Therefore, the sex or presumed sex of a mammal is determined by the Y-specificity in the DNA of an individual mammal. It can be determined by genetic analysis.

Alternatively, it may be due to unrelated but genetically linked sequences, particularly those linked to the Y chromosome. and, preferably, the sex is determined by a sequence adjacent to a male-determining gene; Reduce potential errors during genetic recombination analysis.

Prior to the present invention, many researchers hybridized preferentially or exclusively to male DNA. identified a DNA sequence that Kunke I et al., rsci ence J 19L 1189-1190 (1976); hop) et al., rNatureJ 303.831 (1983); Hergner Vergnaud et al., rBrit, Med, J, J 289.73-7 6 (1984); Lau et al., The LancetJ, January 7, 1984.14-16 pages; Gosden et al., rTheL ancetJ, December 25, 1982.1416-1419 Page: Poste Bostick et al., rNatureJ272.324 (1978) See. These DNA sequences have not been functionally characterized, and these sequences are It is not known whether it can hybridize with other species.

Sperm separated by sex chromosomes containing these are isolated, and these sperm are used to grow eggs. Fertilization is one method currently used to control the sex of embryos. It takes Sperm isolation methods require preparations of spermatozoa that contain more than 99% of the sperm or spermatozoa that carry sex chromosomes of only one sex. Significantly limited by difficulty in obtaining. Currently, there is no existing method to separate sperm according to sex. Known methods involve obtaining a mixture of spermatozoa containing approximately 75% or more identical chromosomes. Not practical. Therefore, it is not economically viable to determine sex by separating sperm. limited by commercial considerations.

Fetal cells obtained several weeks after pregnancy are extracted by amniocentesis, chorionic biopsy, and Ike's method. Typing is another known method of determining sex. However, such methods are expensive. There is a risk of infection, as well as the time required to perform this type of analysis. commercially restricted.

Ike's prior art research addressing this issue has been indirect and incomplete. Ellis (El U.S. Pat. No. 4,769,319 to lis et al. Open a male-specific nucleic acid hybridization probe with a sequence complementary to the target sequence. It shows. These nucleic acid sequences can be used in hybridization for embryo and fetal sex determination. It is said to be useful as a functional probe. Australian Patent Application No. 59561/ No. 86 contains a bovine DNA probe that hybridizes preferentially to male DNA. have been disclosed and further stated to be useful for sex determination of embryos and fetuses. . These DNA sequences have been shown to be species specific. International patent application No. P CT/AU87100254 uses DNA isolated from male animals rather than from female animals. Y that can hybridize with DNA derived from horse and sheep Discloses a 307 base pair nucleic acid sequence designated BRY,1 that contains specific DNA.

PCT application no. Nucleic acid isolates capable of hybridization are disclosed.

Furthermore, prior art techniques can actually achieve 100% accuracy with a small number of cells, about two or so. , in a very short time, the morula or blastocyst stage by nucleic acid hybridization; or to sex a mammalian embryo before or during implantation of the embryo for further development Any that can be used to provide the basis for polynucleotide probes for It has not been shown that a similar DNA segment 1- exists.

Disclosure of invention The present invention has SEQ ID NO: 1; BtYl and SEQ ID NO: 3; B A segment of the Y chromosome-specific DNA sequence called tY2 and up to two Nucleic acid hybridization is performed with an amount of DNA equal to that obtained from embryonic cells. Corresponding RN that allows sex determination of bullfox embryos more quickly and with practically 100% accuracy It arises from the discovery of the A sequence. Sequences are repeated to varying degrees and are unrelated. The number of repeats varies between species and is stably inherited. Furthermore, the nucleic acid of the present invention Use probes to sex embryos at an early stage, at the time of embryo transfer or in less than a day. I can do that. The present invention enables the early, essentially specific sex determination advantage of bovine embryos to be exploited. It is possible to achieve.

The present invention includes SEo 10 No.]; BtYl and SEQ [D NO: 3; B. A rapid, reliable and economical method of embryo-derived cells, called tY2. This is achieved by enabling sex determination and providing a sensitive Y-chromosome probe. Ru. The probe of the present invention uses DNA from a small number of cells, about two, of an animal/Uno. is sensitive enough to determine the sex of fetuses and embryos using It can be used for sex determination. by nucleic acid hybridization or die cut analysis. Fetal sex determination is essentially 100% accurate, but this is only done a few weeks into pregnancy. This poses a serious risk to the fetus and mother. Thus, to determine the sex of the fetus These known methods have significant drawbacks compared to the early embryonic sex determination made possible by the present invention. Exists.

The polynucleotide probes of the present invention have been described through association with bovine DNA.

Certain of these sequences are found in male DNA compared to female Bos species (cows). Because these preferentially bind to It's more effective than that. Their prominence also means that they are present in high copy numbers and in females. Arrangements between individual elements that are present in multiple copies in males and are stronger than those that are further arranged By showing similarities.

Furthermore, the present invention applies such DNA sequences to determine the sex of ruminants/foxes. , from the male DNA of such a species, a reasonably wide range of species is found, rather than females of this species. Includes a method for isolating a nucleic acid sequence that hybridizes to a male nucleic acid.

The sequence is used in nucleic acid hybridization probes for sex determination of embryos and cells. provide the following information.

Definitions and abbreviations DNA-deoxoliphonucleic acid RNA-liponucleic acid Polynucleotide - one or two MDNA or RNA DTA ethylene Cyaminetetraacetic acid Tris) Lis(hydroxymethyl)aminomethane rl) m per minute Number of revolutions m+n mmol, solenoid concentration wm mm Optical density measured in 0, D, X nanometer DEAE diethylaminoethane TE 10mM Tris-HCl pH 8, 01mM EDTADTT Dithiothrey Thor M9 minimal plates, Maniatis iatis) etc. (19g2) rMolecular CIonlng, AL ab　ManuaI　formula　J LB Luria Bertani Broth A TP Adenosone triphosphate Refer to Anbinlin 5SPE Maniatis etc. covered with amp 150mg/region. Light.

Denhardts (too x composition such as 7 near tastes) SO 3 Sodium dodesol sulfate Detailed description of preferred embodiments of the invention The present invention includes hybridization probes for fetal sex determination in mammals. Nilobes containing specific nucleic acids can be detected using hybridization technology. may be labeled as such or used in unlabeled form; such nucleic acids and methods for isolating and identifying probes and probes; and procedures for fetal sex determination in mammals. Also disclosed is a method using the probe.

The nucleic acid sequences of the invention can be single-stranded or double-stranded DNA or RNA, or DNA or and RNA.

Sequences may be labeled or unlabeled. The sequence of the labeled nucleic acid is Nucleic acid when a labeled nucleotide is replaced by the corresponding unlabeled nucleotide is the array that has. For example, if the DNA is a deoxyuridylate residue at position 5, When labeling with a non-radioactive marker such as Chin, the sequence of the labeled DNA is All bistin-labeled deoxyuridylate was replaced by thymidylate. It is identical to that of DNA. The sequence of DNA and RNA is thymidylic acid in DNA. Except for salts, all deoxyliponucleotides or corresponding liponucleotides in RNA Otide is substituted and all thymidylate in DNA is replaced with uridylate. The case is the same.

Preferred nucleic acid probes of the invention are subjected to hybridization or similar conditions. If carried out, the all-male DNA of the bullfinch species is significantly more widespread than the all-female DNA. hybridizes to a wide range. The probe of the present invention is an all-purpose bull DN. Hybridization during which detectable hybridization occurs at A Detection of female DNA through hybridization under extremely strict conditions Do not hybridize as expected.

The nucleic acid probe of the present invention is synthesized from chromosomal DNA derived from embryonic cells of the species to be tested. It is preferable to hybridize under strict conditions. fewer probes DN found on the Y chromosome of animals such as cows, sheep, and goats Corresponds to all or part of the A sequence.

The basic characteristics of the nucleic acids of the invention are unlabeled and labeled - wood-like morphology. If all of the bovine species under these or nearly similar hybridization conditions are Preferentially all male DNA rather than female DNA with a probability greater than 0.99 hybridization.

In particular, it hybridizes only to bovine-derived nucleic acid sequences that include the Y chromosome DNA sequence. We present and describe two types of nucleic acids isolated from bulls that can be transfected.

The nucleic acid isolate is a DNA sequence that includes a portion of the Y chromosome DNA sequence of a bovine mammal. corresponding to SEQ IDN0:1; BtYI and SEQ10, respectively. NO: 3.

Denoted as BtY2.

In nucleic acid hybridization probe technology, nucleic acids with different sequences are , directly hybridizing under similar conditions to the same "target" nucleic acid. or well known. The two nucleic acids contain at least one segment of the target nucleic acid. at least about 12 nucleotides in the sequence that are complementary or nearly complementary to the sequence over a sufficiently long hybridization period to include the octide segment. detectably hybridize under severe conditions. hybridize The physical basis for these complementary or nearly complementary segments is This is base pairing between members. The time for hybridization to occur is strictly controlled. Under certain conditions, two nucleic acids containing precisely complementary base-pairing segments are tested against each other. If the value of hybridization is kept constant so that it can be Increased departure from exact complementarity may occur during base-pairing segments, but sufficient Base pairing continues as the base pairing segments of the two nucleic acids become longer (and even higher). Hybridization decreases as hybridization conditions become less stringent. occurs to the extent that it becomes detectable. Furthermore, blobbing of the probe nucleic acid ( pr.

The outer segment of the bing) segment is the hybrid between the probe and its target. Significantly altering the sequence without appreciably reducing the extent of dization, but complementary to target segments in different targets in the sample to be located. No or nearly complementary probing segments are introduced. Blobin If the outer segments of the hybridization segment significantly change in length, The speed of the application may also vary.

In the present invention, "almost the same sequence" refers to two types of single M nucleic acid segment 1 (a ) both form a base-pairing double helix with the same segment, and (b) 0 ．． The melting points of the two types of double helices differ by 10″C in the 5×5SPE solution. It means to do something. The two types of double-stranded nucleic acid segments are either of these segments. When one type of chain has "almost the same sequence" as one type of chain in another segment [have almost identical sequences]. Any labeling method known in the art may be used in this book. Suitable for carrying out the invention.

Use of nucleic acids as hybridization probes of the invention facilitates detection This is done by labeling the probe for this purpose. The nucleic acids of the present invention are similar to biotin. It is preferable to label it with a non-radioactive label so that it can be detected. Other bromodeoxy Non-radioactive labels such as uridine can also be used.

Radioactive isotopes can also be used to label the nucleic acid probes of the invention. . Preferably, the probe is labeled with 32P.

and other radioactive labels such as 3H, 14c, 35S, and 125I. Can be used movably. The label may be, for example, one or more isotopically labeled deodorants. A sample of DNA is nicked in the presence of oxynucleotide-5-triphosphate. This can be easily carried out by translation.

An alternative to the use of non-radioactive or radioactive labels is to chemically label the nucleic acids of the invention. Is Rukoto. For example, deoxyuridylic acid to replace thymidylate residues Conventional nick transduction of a nucleic acid biotinylated at position 5 of the uracil moiety in the presence of salt. The ration method is appropriate. The resulting labeled probe is located at the thymidylate residue. Contains biotinylated uridylate. The resulting labeled probe is located at the thymidylate residue. Contains biotinylated uridylate at the base of the streptavidin (str) against biotin. Any number of commercially available detection systems utilize the binding of eptavidin). It can be detected via the biotin moiety by the system. For example, Singer and Ward's rProc, Natl, Acad, Sci, U , S.A., J. 79.7331-7335 (1982). detection system For example, Bethesda Research Laboratories, located in Isselsburg, Maryland, USA. Bethesda Re5search Laboratories , Inc.) and Enzo Biochem, Inc., located in New York, New York, USA. Cal's Company (Enz.

Commercially available from Biochemicals, [nc, ).

The present invention also relates to any contiguous portion of 12 or more nucleotides or SEQ ID N O3: Includes any and all sequences shown in l-4.

Any exemplary sequence using such a nucleic acid as a hybridization probe Alternatively, similar sequences may be detected in bovine and non-cow species. It takes again Nucleic acid sequences can be synthesized using commercially available DNA synthesizers, e.g. Abright Biosystems located in Foster City (Foster C1ty) Abs obtained from Applied Biosystems Inc. Wright Biosystems (Applied Biosystems) 380A In some cases, it is synthesized using a DNA synthesizer. Nucleus less than about 12 Such nucleic acid probes containing otides have utility as hybridization probes. Usability is limited. Accordingly, preferred probes of the invention include nucleic acid isolates herein. 12 or more nucleotides of any contiguous portion of any and all sequences described This includes:

Various preferred embodiments include recombinant DNA comprising all or part of the sequences shown. Use of the molecule for cloning, amplification and/or production of the contained and further claimed sequences. Vectors that can be propagated in host prokaryotic or eukaryotic cells for the present purpose Also included. Any number of a wide range of vector molecules can be used depending on the intended use of the nucleic acid. There are cases where it is used. Examples of such vectors include pT2f8U or pTZ19u, and Bluescript (St. The vector molecule may contain molecules such as prokaryotic cells (e.g. and eukaryotic vectors, plasmids, phagemids, shuttle vectors, and bacteriophores. Includes lyophage, etc.

Is the RNA corresponding to all or part of the sequence as described in the inhydrotranscription system? including, but not limited to, bacteriophage RNA polymerases. manufactured using any method well known in the art, using be.

Any of a number of commercially available polymerases such as T3, T7 or SF3 may be used as appropriate. be. Melton et al.'s rNuc, Ac1d, Res, J12. 7035-7056 (1984), and Taylor et al. 　1oche+n.

Biophys, Acta' 442, 324-330. Therefore, various preferences In suitable embodiments, the nucleic acid isolates of the invention contain predominantly Y chromosome-specific DNA and RNA sequences. pre-hybridized and therefore useful in determining the genetic sex of mammals. Contains NA and RNA sequences.

SEQ ID NO: l; B tYl was cloned from bovine DNA 1 , an 859 kb PstI fragment. restriction ) Fragment SEQ ID NO: 1; B tY l is subcloning Genomic DNA from individual male and female mammals to be preserved and then conserved to the public By hybridizing to the Southern plot of A, it was confirmed that it is male-specific. was shown and further repeated in cattle and pond animals. SEQ [ ONO: 2 is SEQ fD NO: 1; corresponding DNA timidity in BtYl SEQ with uracil substituted [DNO:I]; Compatible with the DNA sequence of BtYl. The corresponding double-stranded RNA is shown.

SEQ rD NO: 3; Bt Y2 is a 3.71kb 5acI fragment. This is a cloned fake DNA. SEQ fD NO: l: B tYI f Used to isolate B t Y2 5E910 NO: 3; Contained within the Bt Y2 fragment.

SEQ ID NO: 3; The 5acI restriction fragment of BtY2 is a subclone. Genomes from individual male and female mammals are isolated and then commonly stored. This was demonstrated by hybridization to a Southern blot of genomic DNA.

SEQ [D NO: 4 is SEQ [D NO: 3]; Correspondence in B tY2 5EQID substituted with thymidine or uracil in DNA NO: 3: B t Indicates double-stranded RNA corresponding to the DNA sequence of Y2, which is repeated in cattle. Ta. SEQ rDNO:I; BtYI and SEQ ID NO:3:B t Y2 means SEQ ID NOS: I and 3 if indicated. means a specific sequence. These also include ring-closed and linear SEQ [DNO:l +BtYI and SEQ ID NO: 3; BtY2 and nucleotide replaced with, added to, or from the associated sequence shown. Deletion mutants are included, however, in this case, the mutant or any sequence SEQ IDNO 3: Hybridizes with all or part of 1 to 4. such mutants DNA sequences have been inserted, deleted, or inserted into a population of individuals through recombination or rearrangement. or alleles and/or cis mutants generated by point mutations. There are cases where this occurs. Alternatively, such variants may include, for example, exonucleases. Deletion of a fragment of DN A by 7, 16 site-directed mutagenesis, or D Addition of DNA sequences by linking portions of NA together or template-independent Alternatively, it can be produced artificially by adding template-dependent DNA polymerase. .

The production and use of the nucleic acids of the invention are detailed in the Examples below.

In a preferred embodiment, the nucleic acid isolate is used as a hybridization probe and Y-stained. Body-specific DNA and RNA sequences as well as results, e.g. bullfinch or pond stage animals. to detect the sex of embryonic or fetal cells. In a related use, the nucleic acid probe of the present invention Changes in the abundance and/or sequence of the corresponding sequences in individual mammals using can be detected. Such uses may be useful, for example, for paternity testing of male offspring. be. Various cell types analyzed using the nucleic acid isolates and methods described here A useful example is to apply these to fractionated sperm; In this case, using the nucleic acid isolate of the present invention, various fractions of spermatozoa having a Y chromosome are tested. You can try it out.

Sex staining of embryos, fetuses, or sperm or other cells using the nucleic acid probes of the present invention In a preferred method of determining chromoplast content, remove the cells of the sample for the assay. do. DNA and/or RNA can be extracted from these using known methods. You can do something like that. (1982) rMolecular Cl oning A Laboratory Manual J Cold Spring Ha Cold Spring Harbor Laboratory See. The isolated DNA and/or RNA is then applied to the nitrocellulose. or its derivatives can be directly immobilized on membranes. Or again, DNA and/or RNA is electrophoresed through a gel matrix and then transferred to a similar It can be fixed to a membrane.

The membrane-bound nucleic acids are then labeled with a detectable marker as described above. hybridize with any or all of the nucleic acid isolates of the invention Ru. Labeled nucleic acid isolates bind to nucleic acids on membranes using conventional methods well known in the art. , for example, by autoradiography. Labeled isolate or target sample When hybridizing to a similar sequence, the sex cannot be definitively determined to be male. Ru. When using this method, amplified target DNA may be used. Saiki (S aiki) etc. rsscience” 230, 1350~+354(+986), and Saiki et al. Nature J 324.163-166 (1986). Light.

Another method using the nucleic acid probes of the invention is to remove cells from a sample for assay. Use nucleic acids that are not extracted from obtained by heating such cells in an alkaline solution. Transfer the solution to Zeta-Probe membrane J (B io-Rad trademark) such as charge-mod if i ed) Filter on a nylon membrane. Describe the DNA fixed on the membrane using the method described above. hybridize with a nucleic acid isolate of the invention as described above.

Other methods of the invention include isolating DNA from tissue or cell samples and DNA is immobilized on a support substrate, and the immobilized DNA is hybridized with a nucleic acid isolate of the present invention. unbound nucleic acid isolates are washed from the support substrate, followed by conventional methods. Detecting the binding of a nucleic acid isolate to DNA bound by or for determining the sex chromosome composition of cell samples.

Synchronous or metaphase chromosomes, or Y chromosome in fixed cells When determining the presence or absence of such cells, a preferred system of principles is to Y chromosome-specific nucleic acid isolation of the present invention Hybridization is carried out under conditions that allow the body and the nucleic acid isolate to bind to complementary DNA sequences. including turning on the Unbound nucleic acids were washed to remove them and 5aiki etc. As described in ce J 230.324.163~+66 (+986), Nuclei were isolated using conventional in 5 itu hybridization methods. Detection of binding of acid isolates can also be applied.

Additionally, conventional methods useful for amplifying levels of target DNA can be used to synthesize the nucleic acid probes of the invention. It can be used in combination with For example, isolated from tissue or cell samples. denatured DNA to separate the coding and non-coding strands. A corresponds to a chain of 12 or more nucleotides from any nucleic acid probe sequence of the present invention. The annealed DNA is annealed with a synthetic polynucleotide chain, and the annealed DNA is This sequence is used in a greenhouse with NA polymerase to spread the polynucleotide chain through Furthermore, this sequence is repeated a desired number of times to amplify the level of the target DNA. Ru. Detection of the target DNA in the amplified sample is then performed using well-known procedures for this technique. This can be done by any number of conventional methods. For example, DNA is placed on a supporting substrate. and immobilize the immobilized DNA with the nucleic acid of the present invention to a labeled nucleic acid probe or complementary sequence. hybridize under compatible conditions, and remove unbound probe from the supporting substrate. and then or alternatively, the labeled nucleotide precursors are treated with DNA polymerase. For greenhouse applications, the sample is fractionated by electrophoresis on a gel matrix and then labeled target DNA. A sequence can be detected. Hybridization is well known through this technique. Conducted under standard conditions. Malljatj S et al. (1982), rMo Regular Cloning A Lab.

Reference Manual J Cold Spring Harbor Laboratory.

Polymerase strand growth prior to hybridization with the nucleic acid probe of the invention Amplification of target DNA using reaction (PCR) is useful for determining the sex of embryos, fetuses, etc. There are other methods that can be used. Target using polynucleotide primers from target sequence Amplify the DNA sequence that occurs between the DNA primer sequences. Amplified target sequence These are fixed on a membrane and detected using the usual hybridization method described above. It can be analyzed using

The amplified target sequence can also be immobilized using a silver reagent or a label such as ethidium bromide. Electrophoresis is performed by staining in a gel matrix using quasi-dyes and then visualized under UV light. Furthermore, a wide variety of tissue types can be visualized using the nucleic acid isolates of the present invention. or a kit for detecting the presence or absence of Y chromosome-specific sequences in a cell sample. may constitute or form part of. Nucleic acid isolates can be radioactive or non-radioactive They may be labeled with a wide variety of labels, including markers. The kit also includes reagents. Contains dilution buffers, labeled compounds, solid supports for assays, etc. It may contain a well-known number of suitable ingredients without being limited to.

Example Preparation of genomic DNA Blood samples (FML) were collected in 0.07 ml of 15% E by generally accepted veterinary methods. Sterile container containing DTA potassium salt [Vactainer Becton Deisoquinson (Va Collect in a cuttainer-Becton-Dickinson) and sterilize 5. Transferred to a zero control culture tube. Red blood cells were mixed with 35 ml of 17 mM Tris HCI, p )17.65.Add 140mM ammonium chloride solution (prewarmed to 37°C) The mixture was dissolved and incubated at 37°C for 10 minutes. Heat the sample at 4°C. - Centra-8R (rEc Centra-8R) swinging centrifuge Hackett rotor (CswInging bucket rotor) for 10 minutes Centrifugation was performed at 2.000 rpm. Beret, consisting primarily of nucleated white blood cells (pellet) was resuspended in physiological saline (0.85% NaCL in sterile H2O). The solution was stirred and spun as before. Remove the saline supernatant and add 2 ml of the pellet to the 100mM Tris HCI, pH 8,0, 40mMEl)T, Af It was resuspended in I-lium salt cloth. Equal volume of dissolved mixture (JoomM Tris H CI, pHs, o, 40mM EDTA and 0.2% 5DS) were added. The samples were cooled at 4°C overnight. 4ml of IOrnM Tris HCI, p H8,0, Proteinase K (Boehringermann Boehringer! Jannheim] at 1 m/ml Add to the lysed cell suspension at a concentration of 65°C with intermittent mixing. It was kept moist for 2 hours. Standard Phenol Chloroform Isoamyl Alcohol (Pct) Extraction was carried out as follows: redistilled phenol (BRL) was added to the phenol effluent. The solution was saturated with Tris HCI, pH 8.0, until the pH exceeded 7.5. Hido Roxyquinoline was added to 0.1%. Phenol in chloroform:isoamide alcohol (24:1). An equal volume of the mixture (Pct) was extracted. was added to the sample. Genomic DNA: PC! Rotate the mixture at low speed (on a commercially available rotator) 0 rpm) for 20 minutes. A violent vortex (vo (rtexing) with Pct. Sorvall the sample The centrifuge was spun at 10.00 Orpm in an HB40-tar. Upper part, DN Remove the aqueous phase containing A with a widebore pipette. Then, as mentioned above, there is no detection between the interphase or the aqueous DNA phase and the organic PCI phase. I re-extracted it until it was. DNA was added to 1/10 volume of 3M sodium acetate (pH 7.0). ) and 2-2.5 volumes of 95% ethanol to precipitate. Genome DN Pellet A by centrifugation at 5.000 rpm at 4°C in an 8840 (DNA from all ponds was pelleted at 10.00 rpm). Eta The nol is removed, redissolved in 1 ml of sterile H2O and reprecipitated as above.

These pellets were washed twice with 70% ethanol, vacuum dried and sterilized 1 (20 and determine the DNA concentration by measuring O,0,260 with a spectrophotometer. did.

Genomic DNA (50-10100u in 4-fold excess (i.e. 4U enzyme/ugDNA) A) Pstl or Sac I restriction enzyme [New England Biolabs ( New England Biolaf+s)) provided by the manufacturer. Digestion was carried out for 4 hours at 37° C. in buffer solution. Digested DNA was subjected to standard Pct extraction and Purified by ethanol precipitation.

DNA'A was dissolved in sterile H2O to a concentration of 0.5 mg/111.

electrophoresis Genomic DNA on agarose gel [0.8-1.2% agarose, Bio-Rad] Molecular biology grade (Bio Rad Molecular biol.

gy-grade)) Biorat horizontal DNA subcell (Bi o Rad Horizontal DNA Sub Ce1l) It migrated.

Tank buffers are 40mM Tris acetate pH 8 , o, 2mM EDTA and 0.5Mg/ml ethidium bromide. D.N. A was visualized by ultraviolet fluoroscopy. The size of the digested genomic DNA is ize-cuts) as a guide and a 46 molecular weight mer. It was isolated using a car. Introducing a slit across the gel lane, DEAE cellulose (NA45, Schleicher and Schleicher) d　5chuell):] was placed. Electrophoresis is all fluorescent or adsorbed to paper. It continued until. By setting up vapor strips here and there on the gel lane. , @heavy size fraction was isolated. Clone DN digested using the same method A fragment was isolated. T, E, vortex the vapor strip 2 ×Washed. DNA was stripped in TE with NaCl added up to IM. Elution was performed by heating at 65°C for 20 minutes. Contains interesting DNA , the buffer was PCI extracted and ethanol precipitated using standard methods. Sterilize DNA14 It was reconstituted at 20°C and stored at -20°C until use.

Preparation of vector DNA Plasmid DNAΔ was lysed in alkaline cells and subjected to cesium chloride gradient centrifugation (Manja tis) according to standard methods. blue script for fractions +KS or Bluescript +SK vector (Stratagene) Cloned. Add vector DNA (5Mg) to a 4-fold excess (20 units) of the appropriate Digested with restriction enzymes.

DNA was PCI extracted and ethanol precipitated using standard methods.

o 200uI of IOd Tris heated to 75°C, pH 8 , 3.5mM MgCl and 0.1mM ZnC1* and quickly on ice. It was cooled to Calf intestine 7/rekari 7t suf 7tase (CIAP-Boeh rnger Mannheim-molecular biology gra de) at 0.5 units/ug DNA and incubated at 42°C for 30 minutes at 55°C. Incubate at 11775°C for 1° before cooling on ice for 30 minutes. moreover Two units of phosphatase were added and greenhouseed as described above. SDS up to 0.1% The mixture was extracted repeatedly with Pct and ethanol precipitated using standard methods. Ta.

DNA was dissolved in sterile H20 at a concentration of 0.5 Mg/ul and heated to 75°C for 15 min. Cool to room temperature before storing at 120"C until use.

Ligation of insert into vector DNA Recombinant DNA molecules were prepared as follows. Vector and insert DNA: 3=1 were mixed at a molar ratio of For 10ul ligation reaction, add 25mM Tris HC I (pH 7,6), 50mM MgCIz, 5mM DTT and 25% Polyester 2 ul of a solution containing tyrene glycol 8000 was added.

1 μl (7) 10mM ATP and T4 DNA ligase (6=1 0 units), adjust the total volume to 10 ul with sterile H20, and heat the mixture at 12°C. Incubated for a minimum of 8 hours. Blunt-ended molecules (representative as subcloned fragments) (target) was ligated with 1/10 of the above concentration of ATP, twice the amount of T4 DNA ligase, and The experiments were carried out using a greenhouse at room temperature and overnight. Add TE (90ul) to the ligation reaction , and then purified by standard Pc extraction and ethanol precipitation. DNA from 10 to Dissolve in 20ul of sterile H20 and use for transformation of competent cells at 4°C. Saved to.

A single colony of the following strain was inoculated into LB broth and grown overnight at 37°C with shaking. let - The late culture was diluted 1:100 with fresh LB broth. Thin Remove the cells from 0.5 to 0.0, while stirring vigorously. D, multiplied by 600 . Cells were cooled on ice for 10 minutes and centrifuged at 4°C for 0 minutes at 4.000 x g. Ta. Aspirate the supernatant and resuspend the cells to their original volume with water and centrifuge as described above. separated. This process was repeated again using half the original volume of 820. next Re-incubate cells in 10% glycerol at 1/50% of the original volume as described above. It was suspended and rotated. Resuspend the cells in the original volume of 11,500% glycerol. Transfected with cloudy (more than 5xlO+o cells/ml) siflorized genomic DNA. Use directly or further subcloning cloned DNA fragments to frozen for. Bacterial cells using bovine genome DNA,/vector constructs Transformation of cells was performed using a Gene Pulser device [BioRat (BioRad)) was carried out by high voltage electroporation using . Transfer DNA (up to 500 ng in a volume of 2 ui or less) to a 40 ul composite on ice. Mix pre-chilled (4 °C) electroporation kit with tent cells for j min. pulses were applied according to the manufacturer's settings. Immediately after giving a pulse Add 1 ml of SOC medium pre-warmed to 37°C to 15 ml of polypropylene culture. It was added to the cells transferred to the tube and shaken at 37°C for 1 hour. Cells were incubated at room temperature for 10 min. ．． 000 x g, resuspended in 1 ml of LB amp for 1 hour ( Expand (approximately 3 cell doubling times), mix with 0.4 mi of 100% glycerol, It was frozen in a dry ice ethanol bath and stored at -70'C. Then Alico Accurately prepare 4 cells before incubating them in culture medium for colony hybridization. Filtered for CFU measurement.

colony hybridization The transformed bacteria (3,000 to 5,000 cfu) were placed on an LB amp plate ( Colonies were suspended in 2a+I LB amp plated on 185 mm). The plants were kept in a greenhouse at 37°C until they had a diameter of 0.5 to 1.5 mm. The plate was diluted with 182mM Iron Filter [Hybond N+Amersh Cooled to 4°C for 1 hour before layering with am)). needle brick Align the plate and filter (a) using e pricks). I　i　gr+a+en　t') was marked. Filter 1.51i1 7 minutes in the filter vapor soaked with NaCl and 0.5M N, IOH The bacterial DNA was denatured using 1.5M NaCl, 0.5M Tris t (CI( pH 7, 2) O, J: U Filter soaked for 3 minutes in 1 mM EDTA Bebani transfer.

Repeat the final step once and then briefly wash the filter with 2X SSPE It was dried in a oven at 80'C for 1 hour.

Southern blotting and filter hybridization of DNA Transfect the agarose gel by the method recommended by the manufacturer. The capillary method (A (ania 1982) by a nylon membrane sham'). Filter [blotted DNA or colonies] - the circle containing the colony Hfts] in 50% deionized formamine. Mido, 4X SSPE, 5X Denhards solution, 0.5% SDS and 5 Pre-housed at 42° C. for 1-4 hours in a solution containing 00 ug/mi yeast RNA.

Add the probe to more than lOng/pair of prehybrid solution. It was added at a low concentration. Greenhouse filter with filter incubated at 42°C for 12-18 hours - once with 2XSSPE, 0.1% SDS for 30 min at room temperature, then twice. Finally, 1×5sPE and 0.1% SDS were incubated at 65°C for 15 min. Washed with 0.1% SDS at 65°C for 10 minutes.

This subsequent step was a highly rigorous cleaning that was not always carried out. Fi Dry the router and apply "Lightening Plus J" Intensifying screen (Kodak) ) and exposed to Kodak XAR X-ray film at -70°C for various times.

Preparation of radiolabeled probe Radiolabeled probes were added to Hodgson and Twister (Fisk). Modified as originally described by (1987, rNARJ page 6295) Prepared without. The probe was purified by Sephadex G-75 chromatography (Fadex G-75 chromatography). Lumacia) and heated at 100°C for 1 hour before use in hybridization. Denatured for 0 minutes.

Subcloning of recombinant DNA To facilitate DNA sequence analysis, recombinant clones were completed using a variety of restriction enzymes. I mapped everything. DEA the fragment to be subcloned as described above. ε isolated on cellulose paper. Prepare vector DNA with appropriate restriction enzymes. CHAP was performed as described above. Fragment with Blue Script + SK and and inserted into the KS to facilitate sequencing from both ends. terminal or vector Fragments that are incompatible with restriction sites in the polylinker region are The Klenow fragment of limerase I was treated to convert it to a blunt-ended fragment. (see the method of Maniatis). These fragments are then placed at restriction sites Blues digested with Sma I or EcoRV in the polylinker Cloned in Crypto KS.

DNA sequencing A single colony selected from an M9 ampicillin plate was used to incubate 2 ml of LB/ampicillin. The cells were inoculated into picillin medium and grown overnight at 37°C. Next, add 10ul to 1ml of LB amp and before adding 2 ul of helper phage strain M13KO7. The mixture was shaken at 7°C for 4 hours and continued shaking for 1 hour.

The culture was grown in a medium containing 10 ml of LB amp/Kanamyson (70 ug/m’i). The cells were discarded and transferred to a 50 ml culture tube and shaken vigorously at 37°C for 12 to 18 hours. 10. After centrifugation for 15 minutes at 00 rpm, 8 ml of upper groove was diluted with 1.4 ml of PEG/Na. C1 and incubated on ice for a minimum of 1 hour. Phage 10.00 It was pelleted at 0Xg for 20 minutes and resuspended in 400ul of Tε.

DNA was isolated by standard PCI extraction and ethanol precipitation (methods described above).

The DNA was dissolved in 50 ul of sterile H,0. Nucleotide sequencing is commercially available Based on the dideoxynucleotide system (T7 DNA polymerase (sequence 2.0, United States BiochemjcaIs) Performed according to manufacturer's instructions. Point the nucleotide sequence ladder Analyzed by lyacrylamide gel electrophoresis (see Maniatis). D NA sequences were analyzed using the Microgenie sequence analysis package (Be ckman) to collect and organize them. Database search can be done using [Furstal (F) Genbank software using the J sequence analysis program Internet service (Intellino Enetics, Stafford, USA) performed by Intelligenetics Inc.).

The foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding. or that various changes and improvements may be made within the scope of the appended claims. is clear.

Sequence list SEQ ID NO:I:BtY1 indicates the double-stranded DNA sequence of BtYI , in this example C means deoxycytidine-5-phosphate; G means thioxycytidine-5-phosphate; Means siguanoone-5-phosphate.

A means thioxyadenosine-5-phosphate; and T means thioxythymine. Means gin-5-phosphate.

SEQ ID NO:2 is the DNA arrangement of SEQ 10 NO:I:B tY I. The double-stranded RNA sequences corresponding to the columns are shown. In this example, C is lanthidine-5-bos 2G means guanosine-5-phosphate: A means adenosine U means urinone-5-phosphate, and U means urinone-5-phosphate. do.

SEQ ID NO: 3; BtY2 indicates the double-stranded DNA sequence of BtY2 , in this example, C, G, A, and T are SEQ [ONO:l:B t Y 1 This is the same as shown in .

SEQ ID NO:4 corresponds to SEQ ID NO:3:BtY2 C, G, A, and U are shown as SEQ [D NO:2]. It is similar to what was done.

Continuation of front page FI

Claims (1)

[Claims] 1. A labeled or unlabeled nucleic acid isolate comprising: (a) (1) single-stranded or double-stranded; PstI fragment SEQ IDNO:1; BtY1, and (2) Single-stranded or double-stranded SacI fragment SEQ IDNO: 3; Bt Y2; having a sequence nearly identical to any chain of an isolate selected from the group consisting of or (b) a subparagraph of 12 or more nucleotides in length. any fragment of any segment of (a); provided that this fragment All the DNA of males of this breed is greater than all the DNA of females of this breed. characterized by containing fragments that hybridize over a significantly wide range with Labeled or unlabeled nucleic acid isolates. Characterized by being radioactively labeled with 2.3H, 35S, 32P, or 125I The nucleic acid according to claim 1. 3. Characterized by non-radioactive labeling with biotin or bromodeoxyuridine The nucleic acid according to claim 1. 4. All DNs of females of selected breeds from the group consisting of cows, sheep, and goats Hybridizes to a significantly wider range with all the DNA of males of this breed than A. The nucleic acid according to claim 1, characterized in that it contains Characterized by being radioactively labeled with 5.3H, 35S, 32P, or 125I The nucleic acid according to claim 4. 6. Characterized by non-radioactive labeling with biotin or bromodeoxyuridine The nucleic acid according to claim 4. 7. 5. The nucleic acid according to claim 4, wherein the nucleic acid is DNA. 8. SEQ ID NO: 3; RNA corresponding to single strand or double strand of BtY2 , or SEQ ID NO: 3; any of the 12 or more nucleotides of BtY2 5. The nucleic acid according to claim 4, comprising adjacent portions. Characterized by being radioactively labeled with 9.3H, 35S, 32P, or 125I The nucleic acid according to claim 7. 10. Characterized by non-radioactive labeling with biotin or bromodeoxyuridine The nucleic acid according to claim 7. It is characterized by being radioactively labeled with 11.3H, 35S, 32P, or 125I. The nucleic acid according to claim 8. 12. Characterized by non-radioactive labeling with biotin or bromodeoxyuridine The nucleic acid according to claim 8. 13. The DNA is closed ring SEQ ID NO: 1; BtY1, straight line SBQ ID NO: 1; BtY1, ring closure SEQ ID NO: 3; BtY2, and straight line S EQ IDNO: 3; with any strand of DNA selected from the group consisting of BtY2 8. Nucleic acid according to claim 7, characterized in that it has almost identical sequences. It is characterized by being radioactively labeled with 11.3H, 35S, 32P, or 125I. The nucleic acid according to claim 13. 15. Characterized by non-radioactive labeling with biotin or bromodeoxyuridine The nucleic acid according to claim 13. 16. The DNA was ring-closed SEQ ID NO: 1; BtY1, surface line SEQ ID NO: 1; BtY1, ring closure SEQ ID NO: 3; BtY2, and straight line S EQ IDNO: 3; A claim characterized in that it is selected from the group consisting of BtY2. Nucleic acid according to range 13. It is characterized by being radioactively labeled with 17.3H, 35S, 32P, or 125I. 17. The nucleic acid according to claim 16. 18. Characterized by non-radioactive labeling with biotin or bromodeoxyuridine The nucleic acid according to claim 16. 19. A reproducible vector comprising a nucleic acid isolate according to claim 1. 20. SEQ ID NO: 3; Nucleic acid isolate encoding BtY2 or its 1 Reproducible, characterized in that it contains any contiguous portion of two or more nucleotides vector. 21. A method for determining the sex of a mammal, the method comprising: isolating DNA from the group, immobilizing the DNA on a support substrate, Claim 13: Immobilized DNA under conditions that allow the nucleic acid isolate to bind to complementary sequences. hybridizing with a nucleic acid isolate as described; Unbound nucleic acid isolates are washed from the support substrate and DNA bound to the support substrate is washed. A method for determining the sex of a mammal comprising detecting a nucleic acid isolate that binds to the nucleic acid isolate. 22. Presence or absence of Y chromosome in fixed cells or interphase or metaphase of nuclear division A method for determining the loss of cells, comprising the steps of staining fixed cells or interphase or metaphase as described above. A nucleic acid according to claim 13 under conditions that allow the nucleic acid isolate to bind to a complementary sequence. hybridize with the isolate, wash away unbound nucleic acid isolate, and , testing the binding of nucleic acid isolates to fixed cells or chromosomes in interphase or metaphase. The presence of the Y chromosome in fixed cells or during interphase or metaphase of nuclear division, including egress. How to measure failure. 23. A method for determining the sex chromosome composition of a tissue or cell sample, the method comprising: Isolating DNA from a tissue or cell sample and denaturing the isolated DNA, amplify the levels of the nucleic acid isolate; immobilizing the amplified DNA on a support substrate; The immobilized DNA is complemented by the nucleic acid isolate using the nucleic acid isolate according to claim 13. The unbound nucleic acid isolate is hybridized under conditions that allow it to bind to the target sequence. Wash and remove, and Determination of sex chromosome composition of tissue or cell samples, including detecting binding of nucleic acid isolates method. 24. A method for determining the sex chromosome composition of a tissue or cell sample, the method comprising: Isolating DNA from a tissue or cell sample and denaturing the isolated DNA, amplify the levels of the nucleic acid isolate; incubating the labeled nucleotide precursor with DNA polymerase; Isolated DNA may be ligated to complementary sequences using the nucleic acid isolate of claim 13. hybridization under conditions that Fractionate the sample by electrophoresis on a gel matrix and detect labeled nucleic acid isolates A method for determining the sex chromosome composition of a tissue or cell sample. 25. Nucleic acid isolates can be treated with 3H, 35S, 32P, 125I, biotin, or bromo characterized by being labeled with a detectable marker selected from deoxyuridine The method according to claim 21. 26. Nucleic acid isolates can be treated with 3H, 35S, 32P, 125I, biotin, or bromo characterized by being labeled with a detectable marker selected from deoxyuridine The method according to claim 22. 27. Nucleic acid isolates can be treated with 3H, 35S, 32P, 125I, biotin, or bromo characterized by being labeled with a detectable marker selected from deoxyuridine The method according to claim 23. 28. Nucleic acid isolates can be treated with 3H, 35S, 32P, 125I, biotin, or bromo characterized by being labeled with a detectable marker selected from deoxyuridine The method according to claim 24. 29. Kits for detecting the presence or absence of Y chromosome-specific sequences in tissue or cell samples a nucleic acid isolate according to claim 1 and suitable components. Mu kit. 30. Claims characterized in that the kit includes a buffer for diluting the reagents. The kit according to Box 29.