KR100444432B1 - A diagnosing probe for Freemartin - Google Patents

Abstract

PURPOSE: A probe for diagnosis of freemartin is provided, which is specific to Y-chromosome of cow and has a small nucleotide sequence, so that the diagnosis duration can be reduced, and the accuracy of diagnosis can be improved with a small amount of blood without additional treatment of blood sample. CONSTITUTION: A primer for isolating the nucleotide sequence of SEQ ID NO:3 or its complementary sequence from the bovine genomic DNA comprises one nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2 and their complementary sequences. The probe for diagnosis of bovine Y-chromosome comprises the nucleotide sequence of SEQ ID NO:3 or its complementary sequence and is used in FISH(fluorescence in situ hybridization) method. A kit for diagnosis of bovine Y-chromosome and freemartin comprises the probe of SEQ ID NO:3 in at least one vessel.

Description

A diagnosing probe for Freemartin}

The present invention relates to a nucleic acid sequence capable of specifically hybridizing to a bovine Y-chromosome and a probe capable of confirming the presence of a bovine Y-chromosome including the same. In addition, the present invention relates to a method for diagnosing sex discrimination or primate of a bovine using a nucleic acid sequence that specifically hybridizes to a Y-chromosome, and the present invention also relates to a probe comprising a bovine Y-chromosome specific nucleic acid sequence. The present invention relates to a kit for confirming the presence of Y-chromosome in cells and tissues or diagnosing primate in an individual.

Primate is a form of intersex in ruminants, especially cattle, in which the majority of born females become infertile due to lack of normal development and development of female genitalia (David et al., 1976). . This cause is due to placental fusion and vascular system confluence at the time of benign fetal implantation. The fetal and male fetal placental vascular convergence occurs around 20 gestation and the female sex differentiation occurs about 40 to 50 gestation. Morphological changes in the ovaries are caused by specific secretory substances of differentiated males, resulting in hormonal abnormalities. In addition, cellular exchange occurs from vascular system confluence resulting in all fetuses becoming XX / XY chimeric individuals (Dominguez et al., 1990; Shanker and Bhatia, 1983; Biggers and McFeely, 1966).

These pritins show morphological abnormalities such as uterus, ovary, fallopian tubes and vagina in females, and various abnormalities occur such as individuals having testicular-like structures, male seminal vesicles, and byproducts such as the prostate gland. Breeding becomes impossible (Sattar, 1977; Laster et al., 1971; Rajakoski and Hafez, 1964).

On the other hand, new breeding techniques are being developed as a strategy to accelerate the rate of improvement early, as well as to establish an efficient selection and breeding system of excellent genetic resources in the domestic livestock industry, and some of them are becoming practical. to be. Embryo transfer and twin induction are the most widely used and commercialized propagation cutting-edge technologies. Unfortunately, many of the cattle produced using these techniques are heterozygous, and most of the females are produced as primates, which are almost impossible to breed, causing economic losses.

The incidence of primates produced in heterosexual pairs has been reported to be as low as 82.5% and as high as 95% (Zhang et al., 1994; David et al., 1976). 5-18% of heterosexual pairs are normal females. This suggests that it is possible that the abnormality of the reproductive organs and the degree of maturation of primate are very different from person to person.The timing of vascular conjugation of heterosexual pairs, the exchange of unknown specific inducers or inhibitors It is thought to be mainly influenced by the appearance, timing and location of implantation in benign uterus (Khan and Foley, 1994; Sattar, 1977; Greene et al., 1977).

As such, culling all females born as heterosexuals as primates will also cause economic losses. Therefore, in the case of individuals produced with heterosexual pairs, early diagnosis of female prematin needs to be preceded. This early diagnosis can contribute to the substantial income increase of livestock farmers by minimizing economic losses and by devising economic uses of the produced pritin individuals.

Such methods for detecting primate include clinical diagnostic methods for measuring the length and quality of the vulva, cytogenetic diagnostic methods for analyzing XX / XY cell chimerism in blood, and polymerase chain reaction (hereinafter referred to as polymerase chain reaction). Molecular genetic diagnostic methods such as PCR).

Specifically, as a clinical diagnostic method, it is possible to check whether the chorion is mixed by irradiating the placental membrane, and another method is to insert a blunt object such as a thermometer into the vagina to examine the length of the conduit. If the probe is inserted into the vagina, the distance the probe should be about 3 to 5 cm, and in the case of primartin can be diagnosed from about 1/3 of it. Primate's external genitalia are female, but internal genitalia usually show male sexuality. According to Wilkes et al. (1981), in reproductive anatomy, some primates may appear similar to normal females, although the mutations are very large and distinctly male-made. There are some limitations in the diagnosis of primartin by such clinical methods because in some cases, it is slightly sung and has a quality close to normal.

Meanwhile, in the case of primartin, cytogenetic diagnostic methods for individuals have been widely used based on a mixture of sex chromosomes due to vascular anastomosis at fetus. This involves taking blood from individuals and performing karyotyping as white blood cell cultures. In other words, karyotyping results in diagnosing primatin as a mixture of cells with XX and XY chromosomes. Cytogenetic diagnostic methods have the advantages of high accuracy and reliable diagnosis, but the cumbersome culturing process and separation of chromosomes takes a long time and technical training for karyotyping.

Recently, the diagnosis of primate using a PCR method has been introduced as a molecular biological technique, the principle of which is to enzymatically synthesize and amplify a specific nucleic acid in vitro (in vitro). That is, in the case of primate, the Y chromosome is accompanied to amplify the Y-chromosome specific DNA and confirm the amplification. So far, Y-chromosome-specific DNA in cattle has been very diverse and many reports have been made. International application PCT WO86 / 07095 describes three probes capable of discriminating males of the Bos genus, in particular the males of Hereford and Holstein species. A first probe corresponding to a 6 kbp RsaI fragment, a second probe corresponding to a 4 kbp RsaI-EcoRI fragment of male bovine DNA, and a third probe corresponding to a 2.2 kbp EcoRI fragment of male bovine DNA are identified. Meanwhile, US Pat. No. 5,328,827 discloses a probe of Y-chromosome specific nucleic acid sequence having a size of about 49 bp. In addition, US Pat. No. 4,769,319, Australian Patent Application AU59561 / 86, International Application PCT / AU87 / 002543, and International Application PCT / AU89 / 0029 disclose nucleic acid sequences that can hybridize to Y-chromosome specific DNA sequences of bovine male or ruminants. Presenting. The nucleic acid sequences of the Y-chromosome specific probes of the prior art are all used as diagnostic probes for discrimination of sex, for most of them, such as PCR.

The PCR technique is an advanced technique that is very convenient and useful for sex discrimination of a small number of cells or embryos. This technique can amplify male-specific DNA sequences in a short time and provides precise and robust information about sex. However, PCR has some drawbacks such as misjudging from amplification of nonspecific sequences or binding by contamination, and can lead to serious errors, especially in the diagnosis of primate. In other words, there may be confusion with male individuals as amplification of Y-chromosome specific DNA only, and in extreme cases, amplification is rarely performed depending on the mixing ratio of XX cells and XY cells.

Thus, the present inventors attempted to construct non-radiolabeled probes using nucleic acid sequences of small size and specific to the male Y-chromosome of the prior art and used for the diagnosis of primate. As a FISH method using this probe, not only the presence or absence of Y-chromosome, but also a small amount of blood was able to diagnose primate in a short time, and a diagnosis rate of 100% was confirmed to complete the present invention.

In order to overcome the problems of the prior art and to improve the convenience of use and the accuracy of the diagnostic results, the present invention provides a nucleic acid capable of specifically binding to the Y-chromosome (SEQ ID NO: 3) and a diagnostic method using the same. I would like to.

In another aspect, the present invention provides a primer pair of SEQ ID NO: 1 and SEQ ID NO: 2 for separating bovine Y-chromosome specific nucleic acid sequences from bovine genomic DNA.

In another aspect, the present invention is to provide a probe that specifically hybridizes to a bovine Y-chromosome comprising the nucleic acid sequence of SEQ ID NO: 3.

In yet a further aspect the present invention seeks to provide a method for producing bovine Y-chromosome specific non-radioactive label probes.

In a still further aspect, the present invention provides a method for diagnosing primate using the probe as a FISH technique.

In another aspect, the present invention is to provide a diagnostic kit for diagnosing the presence of bovine Y-chromosome or primate using a nucleic acid of SEQ ID NO: 3 or a probe comprising the same.

1 shows the results of FISH on the metaphase of G-banded bovine males. (a) shows the G-banding medium-phase chromosome, and the arrows indicate the X chromosome and the Y chromosome, respectively. (b) shows the result of the FISH analysis of (a) using the probe of the present invention, where Y-chromosome emits yellow fluorescence. The left figure of (c) is the standard karyotype of the G-banded bovine Y-chromosome, the middle figure is actually the G-banded Y-chromosome, and the right figure is the probe on the Y-chromosome by FISH using the probe of the present invention. The fluorescent junction site of (Yp12-13) is shown.

(A) and (c) of FIG. 2 are chromosomal samples isolated from cultured whole blood, and (b) and (d) are photographs showing the results of FISH performed using uncultured white blood cells as samples. to be. (a) and (b) are for samples derived from males, and (c) and (d) are for samples derived from females.

Figure 3 shows the expression patterns of FISH fluorescence conjugated in individuals identified with pritin.

Definition of Terms

As used herein, the term “Y-chromosome” refers to males, not females of mammals, and for this reason it is believed that some genes exhibiting male characteristics are present. About 95% of the Y chromosome sequences belong to a repeat sequence, in which there is a male-specific region (MSY, Male-Specific Y-chromosome). The diagnostic probe of the present invention can be used to determine the sex of an individual by specifically binding to the bovine Y-chromosome.

As used herein, the term "probe" refers to a diagnostic probe and includes a nucleotide capable of specifically binding to a Y-chromosome. It can also be labeled for ease of detection.

As used herein, "PCR (Polymerase Chain Reaction)" refers to a method of amplifying a desired DNA molecule in vitro by specifically repeating synthesis of a specific DNA site. This method can selectively amplify only a part of DNA from large DNA and visualize it as a clearly visible band on commonly used agarose gel or polyacrylamide gel. In addition, the 20-30 base sequence which complementarily binds to the well-known DNA sequence used as a template in PCR execution and becomes the primer of PCR amplification is called "primer." Typically, the primer is composed of approximately 50-60% GC%, evenly distributed four bases, it is preferable to avoid the continuous arrangement of purine or pyrimidine. Avoid three or more consecutive G or C primers because they can bind to unwanted sites, minimizing the formation of the 'primer-dimer' by the complementary binding of the 3 'end in a pair of primers. For efficient cloning of the PCR product, the base of the restriction enzyme may be included at the 5 'end of the primer.

As used herein, "genomic DNA" refers to a collection of genetic information of a particular individual, and "bovine genomic DNA" refers to all gene collections constituting bovine DNA.

As used herein, the term "Fluorescence In Situ Hybridization" refers to a method of identifying a probe of a target sample by labeling a specific DNA probe with a fluorescent material and conjugating it with a target DNA. to be. Specifically, in situ hybridization is a method of recognizing where a particular nucleic acid is located on a chromosome or on a cell or tissue. In situ hybridization may be combined with immunochemical techniques to confirm the presence or absence thereof. Its principle is that nucleic acids have complementary binding properties, so if a mixture of DNA or RNA probes with a specific sequence and single-stranded DNA is slowly melted after heat fusion and cooled slowly, base pairs between the probe and the target molecule in the region of homology are present. Will form. Previously, the bonding technique has been mainly labeled with radioactive materials and searched through autoradiography. Recently, however, a method called fluorescence conjugated bonding method (FISH) has been developed by applying a fluorescent material using a fluorescent material as a non-radioactive material labeling method.

The present invention starts from identifying a repeat nucleic acid sequence specific for bovine Y-chromosome. Specifically, the present invention includes nucleic acid sequences that specifically hybridize to a bovine Y-chromosome, more specifically, a sequence of SEQ ID NO: 3, a complementary sequence thereof, or a fragment thereof.

The present invention includes PCR primers for separating nucleic acid sequences that specifically hybridize to bovine Y-chromosomes from bovine genomic DNA. More specifically includes primers comprising the sequence of SEQ ID NO: 1 and SEQ ID NO: 2.

The present invention includes a probe capable of diagnosing the presence or absence of a Y-chromosome of bovine Y-chromosome comprising a nucleic acid sequence of SEQ ID NO: 3, a complementary sequence thereof or a fragment thereof.

The present invention includes the presence or absence of the Y-chromosome of a bovine, including a process for labeling the nucleic acid sequence of SEQ ID NO: 3, its complementary sequence or fragments thereof, and a method for preparing a primate diagnostic probe.

The present invention performs PCR using a primer comprising a nucleic acid sequence of SEQ ID NOS: 1 and 2 or a complementary sequence thereof to isolate a nucleic acid sequence that specifically hybridizes to a bovine Y-chromosome from bovine genomic DNA, the isolated nucleic acid sequence, Preparing a probe by labeling its complementary sequence or fragment thereof; Preparing a sample from the cells of the individual to be diagnosed; And a method for diagnosing the presence of Y-chromosome or primate, including performing the hybridization reaction of the probe with the sample and analyzing the probe.

In addition, the present invention includes kits for diagnosing sex discrimination or primate comprising a probe capable of ascertaining the presence of a Y-chromosome of a bovine comprising a nucleic acid sequence of SEQ ID NO: 3, a complementary sequence thereof or a fragment thereof.

Hereinafter, the configuration and implementation of the present invention will be described in detail.

Isolation of Y-Chromosome Specific Nucleic Acids

Nucleic acid sequences capable of specific hybridization to the bovine Y-chromosome of the present invention include single- or double-stranded DNA or RNA, hybrids of DNA and RNA. Specifically, the nucleic acid sequence that can specifically hybridize to the Y-chromosome of the present invention includes the nucleic acid sequence of SEQ ID NO: 3, its complementary sequence, or a fragment thereof.

Methods for producing nucleic acid sequences that specifically bind to bovine Y-chromosomes of the present invention include chemical synthesis. DNA sequences can be synthesized by, for example, chemical methods using oligonucleotide synthesizers such as the Applied Biosystems 380A DNA synthesizer (Applied Biosystems, Inc., Foster City, Calif., U.S.A.). It can also be obtained by cloning, amplification and / or expression in host eukaryotic or prokaryotic cells using recombinant DNA techniques.

In addition, another method of constructing the Y-chromosome specific DNA sequence of the present invention includes reverse transcriptase polymerase chain reaction (RT-PCR). That is, after extracting total RNA from bovine genomic DNA, the RNA is used as a template, and oligonucleotides having specific primers, more preferably, nucleotide sequences of SEQ ID NOs: 1 and 2, are used as primers. It can be obtained by performing. Or from a bovine genomic DNA, by performing PCR using a specific primer, preferably oligonucleotides having the nucleotide sequences of SEQ ID NOs: 1 and 2, as primers.

Specifically, one form of the method for preparing the Y-chromosome specific hybridizing nucleic acid sequences of the present invention includes preparing a bovine genomic DNA; Using the prepared bovine genomic DNA as a template, using oligonucleotides having the nucleotide sequences of SEQ ID NOs: 1 and 2 as primers, and performing a PCR, and separating and identifying the PCR product.

Sense: 5'-AAGCAGCCGATAAACACTCCTT-3 '(SEQ ID NO: 1)

Antisense: 5'-ATCAGTGCAGGGACCGAGATG-3 '(SEQ ID NO: 2)

The PCR method may be performed by a conventional method, and the resulting PCR product may be separated by a buffer-gradient or an electrolyte-gradient method. Sequencing of isolated nucleic acid sequences can be performed and confirmed by dideoxy chain termination methods (Sanger et al., 1997). The present invention comprises a 190 bp base sequence (SEQ ID NO: 3) isolated from bovine genomic DNA identified by the above method.

aagcagccga taaacactcc ttggacacat ctcggtccct gcactgatcc aggctggatc ctgcccttgt tcatcccgac agacactccc gaggccacag cacagtccct gccatgaccc

aggctgggtc tgcctttgtt caagcccccg acaaacactc ttgagcccac atctcggtcc

ctgcactgat (SEQ ID NO: 3)

Construction of Y-chromosome diagnostic probe

The nucleic acid sequence (SEQ ID NO: 3) may be used as a hybridization probe capable of detecting Y-chromosome specific DNA or RNA sequences. The Y-chromosome specific hybridization probe of the present invention comprises the nucleic acid sequence of SEQ ID NO: 3, its complementary sequence or fragment thereof.

Nucleic acid sequences included in the Y-chromosome specific hybridization probes of the present invention can be used with or without labeled. Preferred nucleic acid sequences of the present invention hybridize only to bovine total male DNA when hybridization is performed under the same conditions. The probe comprising the nucleic acid sequence of the present invention does not hybridize with whole female DNA when hybridization is performed under stringent conditions, while hybridization occurs with whole male DNA.

Probes comprising nucleic acids of the invention can be hybridized with cDNA of cells derived from embryos, fetuses or adults of cows to be diagnosed, DNA of blood or tissues, chromosomes of sperm. Probes of the invention hybridize only with a portion of the DNA present specifically on the Y-chromosome in cattle.

Probes comprising nucleic acids of the invention hybridize at least 99% of the frequency of bovine male DNA under the same hybridization conditions, both labeled and unlabeled. The probe of the present invention can change the nucleic acid sequence of SEQ ID NO: 3 or add nucleic acid fragments at both ends thereof without changing the degree of hybridization specifically to bovine Y-chromosome.

Nucleic acid sequences used as hybridization probes of the invention can be labeled for ease of detection. Preferably, the nucleic acid of the present invention may be labeled with a non-radioactive material such as Biotin, Digoxigenin (hereinafter referred to as Dig). In addition, other non-radioactive labels such as bromodeoxyuridine may be used. Radioisotopes can also be used to label nucleic acid probes of the invention. Preferably, the probe may be labeled with ³² P, and other radioactive elements such as ³ H, ¹⁴ C, or ¹²⁵ L may be readily used. Labels can be labeled using, for example, Nick-Translation of DNA samples in the presence of one or more deoxy-nucleoside-5-triphosphatase labeled with isotopes.

Another labeling method is chemical labeling of nucleic acids. Random Primed Labeling uses Klenow fragments of E. coli polymerase with 5 'to 3' polymerase activity and hexanucleotides provided as polymerase primers of DNA. Since all DNA sequence combinations are present in the hexanucleotide primer mixture, the primers bind about 80-100 bases each in the DNA framework, and the new DNA strands contain DNA in a reaction mixture containing labeled and unlabeled nucleic acids. Synthesized by the cleno fragment of polymerase. This method can be used for small amounts of DNA (e.g. 10-200 ng) because the DNA is not denatured during the reaction, and one or both strands of DNA can be used as a template for the reaction, It can be applied to the labeling of small DNA fragments (100-500 bp).

Nick translation method uses DNAaseI and E. coli DNA polymerase which can bind labeled nucleotides to both strands of DNA. The most important variable of this reaction is the activity of DNAaseI, which nicks the two strands of DNA. If too small nicks are formed, the labeled probes are large and difficult to bind. If too large nicks are formed, the labeled probes are small and cannot be tightly coupled. Commercially available enzyme mixes containing DNAase I and E. coli DNA polymerase can be used to bind more than 50% of the label in 60-90 minutes and to form probe lengths of 200-400 bp. It is preferable to use

The labeling method of the probe used in the present invention is a PCR-Dig labeling method. The PCR-Dig labeling method is a method of labeling non-radioactive materials in combination with the PCR method, and can obtain a probe labeled with a large amount of Dig from a limited amount of DNA. In order to obtain a successful labeling probe using this method, the condition of PCR is most important, which may vary depending on the target DNA and the primer. In other words, the concentrations of the target DNA, primers, Mg ions and polymerases, the number of repeats and the temperature of PCR are decisive factors.

Specifically, the preparation of the diagnostic probe used in the present invention uses a PCR-Dig labeling method, wherein the sequence number 1 and the sequence are present in the presence of a PCR reaction mixture containing the Y-chromosome specific nucleic acid sequence of the present invention as a template and containing a fluorescent substance. Performing PCR using the primer of No. 2; And identification of PCR products.

Fabrication of Diagnostic Specimens

Cell samples are prepared to confirm the presence of Y-chromosome or primate using the diagnostic probe of the present invention.

Diagnostic specimen preparation obtains blood from an individual in a cow to be diagnosed and uses it as a sample. That is, the blood may be cultured by heparin treatment to induce a metaphase, or may be directly stored and fixed without being cultured. The fixed cells can then be dropped onto a slide to prepare a sample. When the blood is cultured using a microscope on the prepared sample, chromosomes and interphases are observed, and when not cultured, interstitial cells of leukocytes are observed.

Specifically, the method for producing a preferred chromosome sample of the present invention comprises the steps of collecting blood from the individual to be diagnosed; Culturing the collected blood and inducing a medium phase; Separating the cells by centrifugation after culturing, storing them, and centrifuging again to harvest the cells and immobilizing the cells; And dropping the immobilized cell suspension onto a slide to produce a chromosome sample.

In addition, another method for producing a sample of the present invention is the process of collecting blood from the individual to be diagnosed; Centrifuging the collected blood to separate leukocyte cells, storing and immobilizing them; And dropping the immobilized cell suspension onto the slide to produce an interstitial sample.

Hybridization and Diagnosis of Specimens Using Probes

The presence of a Y-chromosome in a sample can be confirmed by hybridizing the Y-chromosome specific nucleic acid of the present invention or a diagnostic probe labeled therewith with DNA present in a tissue or cell sample to be diagnosed. This is based on the base-pairing of the DNA present in the probe and the sample. Hybridization of the DNA in the probe and the sample occurs because the specific DNA sequence is present in the sample.

Diagnosis of the present invention can be targeted to embryos, fetuses or adult tissues or cells, blood, sperm and the like. In particular, the presence of the Y-chromosome in the sample can be confirmed, and it can be usefully used to confirm the presence of the primate in the heterozygous by confirming the presence of the Y-chromosome from the blood or tissue sample of the female.

Specifically, the method for diagnosing primate of the present invention comprises the steps of: separating DNA from a tissue or cell, fixing the separated DNA onto a support matrix; Hybridizing nucleic acid probes with immobilized DNA; Washing and removing the isolated nucleic acid from the support; And detecting a nucleic acid bound to the immobilized DNA.

The present invention includes a FISH method using a nucleic acid that specifically hybridizes to a Y-chromosome to confirm whether a female of a heterozygous female is primate. Specifically, the primate diagnostic method of the present invention comprises the steps of separating nucleic acid sequences from bovine genomic DNA using primers comprising the nucleotide sequences of SEQ ID NOs: 1 and 2; Preparing a probe by labeling the separated nucleic acid sequence with a fluorescent material; Collecting and culturing blood from an individual to be diagnosed, inducing a medium phase, preparing a chromosome sample through storage and fixation, or storing and fixing leukocyte cells without culture to prepare a sample; And hybridizing the diagnostic probe to a sample and detecting the result.

Fluorescent materials in the probe fabrication process include biotin, Dig, and the like. In particular, since Dig is a steroid that exists only in plants called digitalis purpurea and digitalis lanata , anti-Dig antibodies have the advantage of not being able to bind with other biomaterials at all. Hybridized Dig-labeled probes can be detected by anti-Dig antibodies bound with alkaline phosphatase, peroxidase, fluorescein or rhodamine. Specific Dig-labeling processes include Dig-PCR probe synthesis methods, which are primers capable of amplifying and amplifying the Y-chromosome specific nucleic acid sequences of the present invention, Dig-11-dUTP or Dig-11- PCR is performed using UTP as a substrate.

The process of preparing the chromosome sample may be blood from an individual to be diagnosed. Blood is collected in a heparinized container, and the collected blood is cultured in a medium to which antibiotics and the like are added, and includes a process of inducing the medium to the end of the culture. After culturing, the cells are separated by centrifugation and stored therein, and after the storage process, the cells are fixed by centrifugation, and the fixed-treated cells are dropped onto the slide. On the other hand, when the collected blood is not cultured, a sample of the interstitial cells can be prepared only through the storage treatment and the fixed treatment.

Hybridizing the sample with the diagnostic probe includes removing the RNA from the sample and then dehydrating the sample. Hybridization mixtures comprising the probes are prepared and dropped on chromosomal specimens to allow denaturation and hybridization processes. Detection of probes can be detected using anti-Dig antibodies after removal of isolated probes that do not bind to chromosomal specimens. Anti-Dig antibodies that do not bind to the probe-Dig present in the sample may be removed and examined by fluorescence microscopy.

Diagnostic kit

Isolated nucleic acids of the present invention or diagnostic probes comprising the same may constitute a kit or form part of a kit for detecting the presence or absence of a Y-chromosome in various tissues or cell samples. Diagnostic probes included in the kits of the invention comprise the nucleic acid sequence of SEQ ID NO: 3, its complementary sequence or fragment thereof. In addition, the nucleic acid may be labeled including a non-radioactive marker.

Kits of the present invention include essential components well known in the art, including, but not limited to, buffers for dilution, reagents, labeled compounds, solid support for analysis, and the like. have.

Hereinafter, the examples are only for illustrating the present invention more specifically, and the scope of the present invention is not limited to these examples according to the gist of the present invention.

Example

<Example 1>

Isolation of Y-chromosome specific nucleic acid sequences

The Y-chromosome specific nucleic acid sequences of the present invention were constructed from bovine genomic DNA by PCR using the following primers.

Sense: 5'-AAGCAGCCGATAAACACTCCTT-3 '(SEQ ID NO: 1)

Antisense: 5'-ATCAGTGCAGGGACCGAGATG-3 '(SEQ ID NO: 2)

(The sense sequence has a GC content of 57.1%, the molecular weight is 6520.329 g / mole; the antisense sequence has a GC content of 34.8%, and a molecular weight of 7077.712 g / mole)

That is, amplification of the nucleic acid sequence of the present invention was carried out using a PCR reaction mixture containing a total volume of 25 µl containing the primers and the following components: 2.5 µl of 10X PCR buffer, 2.5 mM dNTP 2 Μl, 2 μl of Taq polymerase (0.5 unit / μl) (Takara, Japan), 2 μl of each of said primers (10 pmol / μl), 2 μl of bovine genomic DNA (25 ng / μl), 12.5 μl of ddH ₂ O.

The PCR reaction was performed at 97 ° C. for 2 minutes with minimum denaturation, 40 cycles consisting of 1 minute at 94 ° C., 1 minute 30 seconds at 56 ° C., and 1 minute 30 seconds at 72 ° C., and the final stretching process at 72 ° C. 5 minutes was performed. PCR products were identified by electrophoresis on 2% agarose gel and bands were separated. The nucleotide sequence method of the isolated DNA was purified by QIAquick PCR purification Kit (QIAGEN Inc., CA, USA) and confirmed by 1.5% agarose gel, and only a pure band of about 200 bp was cut out to obtain a gene clean kit (Bio 101 Inc., CA, USA). This was inserted into TA-cloning vector pCR21 (Invitrogen Life Technologies, San Diego, Calif., USA) and the nucleotide sequence of the inserted DNA was confirmed by dideoxy chain termination (Sanger et al., 1997). Sequence analysis was performed using an automatic base sequencer (ABI PRISM 377, PE Biosystems, Foster City, CA, USA) using USS kit and ³⁵ S-dATP (800 Ci / mmol, Amersham) according to the instructions of the sequencing kit. Analyze as.

The nucleic acid sequence of the present invention identified through the above process was 190 bp nucleic acid (SEQ ID NO: 3), which was submitted to the National Center of Biotechnology Information (NCBI) maintained by the US government on May 21, 2003. Registered on GenBank as of 16 June 2003.

Fabrication of Probes

The nucleic acid sequence of the present invention can be labeled and used as a diagnostic probe. PCR-Dig probe synthesis was used for the label for fluorescence expression of the probe. That is, using a nucleic acid sequence of the present invention as a template using a primer capable of amplifying it, a new label-probe labeled with fluorescent material was prepared. 50 μl of the total volume of the PCR reaction mixture was placed in a tube, mixed lightly, and PCR was performed under the same conditions as above to label the probe. PCR reaction mixture: 5 μl PCR buffer, 5 μl PCR DIG mixture, 0.75 μl enzyme mixture (2.6 unit) (above Boehringer-Mahnheim, Indianapolis, IN, USA) and primers of SEQ ID NO: 1 and SEQ ID NO: 2 (10 pmol / μL) ) 3 μl each, ₂ μl (100 ng) of purified 190 bp genomic DNA, 31.25 μl ddH ₂ O.

Sample fabrication

For preparation of chromosomal specimens from blood cultures, 5 ml of blood was drawn into the jugular vein of each individual using a heparinized vacuum vessel. For the verification of the present invention, a pair of heterosexual couples are being raised at Jinju Industrial University (150, Chilam-dong, Jinju-si, Gyeongsangnam-do, Korea), and 17 heterosexual pairs in Boseong, Gyeongnam Chilbuk and Chilseo, and Sichuan, Gyeongnam Province.

RPMI 1640 (> Invitrogen Life Technologies, San Diego, CA, USA) with 0.5% blood per subject added 10% FBS, 1% pokweed fission promoter, 0.1% penicillin-streptomycin The culture was incubated for 72 hours in a 37.5 ° C., 5% CO ₂ incubator, and treated with 0.1 μg / ml of Colcemid (Invitrogen Life Technologies, San Diego, CA, USA) 50 minutes before the end of the culture to induce the medium phase.

After incubation, cells were separated from the culture medium by centrifugation (1000 rpm, 200 g), and the separated cells were stored in 0.06 M KCl for 15 minutes in a 37 ° C constant temperature water bath. After storage, the cells were harvested by centrifugation again, followed by fixation by dropping Carnoy's liquid (Acetic acid and Methanol mixed at 1: 3 (v / v)) dropwise. . The fixed cell suspension was dropped in 5-6 drops on a cold-stored slide to prepare a chromosome specimen.

On the other hand, the sample preparation from the non-cultured blood cells were sampled only with the interphase cells through the storage and fixation treatment, without undergoing the culture process after collecting the blood.

<Example 2>

Sample analysis using the FISH method

Cultured or uncultured cell samples to be published on FISH were added with 5 μg of RNase A (Sigma Chemical Co., Saint Louis, MO, USA) in 50 ml of 2 × SSC solution and 30 in a 37.5 ° C. water bath. RNA was removed by treatment for a minute. The cell samples were then washed, dehydrated with 80%, 90% and 100% ethanol and dried.

A hybridization mixture was prepared in which 5 µl formamide and 4 µl of 4x hybridization solution were mixed with 1 µl (100 ng or more) of the labeled probe, and used for denaturation and hybridization of the probe.

The hybridization mixture was dropped onto the specimen and covered with an 18 × 18 mm cover slide and sealed with rubber cement. The denaturation was performed for 10 minutes on a 72 ° C. aluminum block, followed by hybridization at 38.5 ° C. for 1 hour. The treated specimens were washed with 72 × 2 × 2 SSC solution for 5 minutes, washed with PN buffer (0.1% Nonidet P-40 / 0.1% sodium phosphate) at room temperature for 2 minutes, and dried.

Detection of the probe was performed by adding 2.5 μl of anti-Dig-fluoroescein to 1 ml of PNM buffer (5% non-fat milk / PN) to prepare an anti-Dig-fluorosane solution, which was 20 μl. After dropping on a sample and covered with a cover slide, it was treated for 30 minutes on a 38.5 ° C aluminum heating plate. Thereafter, washing with PN buffer three times to remove unbound anti-Dig-products and dried in the dark.

Background staining was performed using propidium iodide (PI) solution (0.3 μg PI / ml anti fade solution), and 20 μl of each slide was added to each slide, followed by a cover slide and a fluorescence microscope. (Olympus Ax-70 with WIB filter). Speculum was photographed with a digital camera and analyzed using an image analysis program (MetaMorph, Universal Imaging Co., PA, U.S.A.).

<Example 3>

Identification of FISH Probes

To confirm whether the probe of the present invention is specifically conjugated to the Y-chromosome, FISH was applied using the probe and the male cell sample by the method of <Example 1> and <Example 2>. That is, chromosomal specimens were prepared from bovine male cells, subjected to G-banding, and the same images were published in FISH.

Figure 1 shows the results of FISH for the same phase as the mid-phase of the G-banding male, showing that the probe of the present invention specifically conjugated on the Y chromosome. In addition, the G-band idiogram of the small Y chromosome, the G-banded Y-chromosome, and the FISH chromosome were compared and analyzed to determine the chromosomal junction locus of the probe more specifically. Was found to be mainly distributed in cattle Yp12-13 (Fig. 1, c). Figure 1 (a) shows the G-banding medium-phase chromosome, the arrow represents the X chromosome and Y chromosome, respectively. (b) shows the FISH analysis of (a) using the probe of the present invention. Yellow fluorescence is the Y-chromosome. In addition, the left figure in (c) shows the standard karyotype of the G-banded bovine Y-chromosome, the middle figure is actually the G-banded Y-chromosome, and the right figure shows the Y-chromosome image FISH using the probe of the present invention. It can be seen that the chromosomal junction of the probe is at Yp12-13.

Analysis of Y-chromosome detection using FISH

In order to confirm the reliability of the probe manufactured according to the present invention, FISH was performed on chromosome samples in which bovine whole blood was cultured and white blood cell samples which were not cultured.

The results are shown in FIG. 2 (a) and 2 (c) are samples of cultured whole blood, and (b) and (d) are samples of uncultured white blood cells. (a) and (b) are for samples derived from males, and (c) and (d) are for samples derived from females.

As shown in FIG. 2, in the male sample, not only the medium phase but also the uncultured interphase phase, one or more FITC fluorescence expression may be observed for each cell, whereas the female sample shows FITC fluorescence in any sample. There was no conjugation.

Through the above results, it was confirmed that the bovine Y-chromosome specific DNA probe of the present invention was specific for bovine Y-chromosome, and the presence or absence of Y-chromosome could be clearly identified using FISH technology.

<Example 4>

Diagnosis of Pritin Using Probes of the Invention

In order to confirm the usefulness of pritin diagnosis as a diagnostic probe of the present invention, FISH was performed on individuals identified as pritin by cytogenetic verification. As a result, in the case of the individual identified as primartin as shown in FIG. 3, it was possible to find a clear fluorescence conjugation pattern of the probes in some cells, unlike normal females. As expected, the XX and XY chromosomes are present. I could confirm that.

In addition, we compared the karyotype and FISH assay results for the same subject to verify the reliability of primate diagnosis using FISH. Bovine karyotyping was carried out using the whole blood culture as in the sample preparation method of Example 1 to induce a medium phase and its chromosomes were analyzed, FISH analysis was performed by the method of Examples 1 and 2.

The results are shown in Table 1 below.

Comparison of XY Cell Distribution Between Karyotype and FISH Assays for Primate and Heterozygous Males Analysis object Karyotyping (%) FISH analysis (%) Contrast axis (normal hydrogen) 138/138 ^a (100) 169/169 ^b (100) Contrast axis (normal cow) 0/143 (0) 0/151 (0) Primartin A 20/126 (15.9) 31/228 (13.6) Primartin B 63/143 (44.1) 80/188 (42.6) Heterosexual male A 37/155 (23.9) 45/187 (24.1) Heterosexual male B 14/136 (10.3) 33/212 (15.6)

Note: ^a number of medium phases with total XY chromosome / number of total analyzed medium phases, ^b number of cells showing probe fluorescence conjugation / total number of cells analyzed

As such, the results of both analyzes showed a similar pattern in almost all the analyzed subjects, i.e., the results of karyotype analysis and the results of FISH using the probe of the present invention were found to be almost in agreement. This proved the reliability of primate diagnosis as a FISH method using the probe of the present invention.

The present invention relates to sex determination and the diagnosis of primate using the bovine Y-chromosome specific DNA probe as a FISH method. This provides an advantage that the diagnosis can be easily performed in a short time.

<110> Republic of KOREA (Jinju National University) <120> A diagnosing probe for Freemartin <160> 3 <170> Kopatent In 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223 > sense primer <400> 1 aagcagccga taaacactcc tt 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> antisense primer <400> 2 atcagtgcag ggaccgagat g 21 <210> 3 <211 > 190 <212> DNA <213> Bos taurus <220> <221> repeat_region <222> (1) .. (190) <223> Bos taurus Y chromsome-specific repetitive DNA sequence <400> 3 aagcagccga taaacactcc ttggacacat ctcggtccct gcactgatcc aggctggatc 60 ctgcccttgt tcatcccgac agacactccc gaggccacag cacagtccct gccatgaccc 120 aggctgggtc tgcctttgtt caagcccccg acaaacactc ttgagcccac atctcggtcc 180 ctgcactgat 190

Claims (14)

delete A primer for separating SEQ ID NO: 3 or its complementary sequence from bovine genomic DNA comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and its complementary sequences. A probe for diagnosing the presence of a Y-chromosome of a cow, characterized by having SEQ ID No. 3 or its complementary sequence and used in the FISH method. delete delete delete delete delete delete delete delete delete delete A kit for diagnosing bovine Y-chromosomes and primates comprising the probe according to claim 3 in at least one container.