KR20140088363A - Marker composition for sex determination of Bovine Embryo - Google Patents
Marker composition for sex determination of Bovine Embryo Download PDFInfo
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- KR20140088363A KR20140088363A KR1020130000174A KR20130000174A KR20140088363A KR 20140088363 A KR20140088363 A KR 20140088363A KR 1020130000174 A KR1020130000174 A KR 1020130000174A KR 20130000174 A KR20130000174 A KR 20130000174A KR 20140088363 A KR20140088363 A KR 20140088363A
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Abstract
Description
The present invention relates to a marker composition for discriminating sex of a bovine embryo, a sex-differentiating kit for identifying a bovine embryo containing the marker, and a method for distinguishing the same.
Not only the demand for beef is rapidly increasing due to the increase in national income and the improvement of eating habits but also the tendency to prefer high quality beef with good quality along with the well-being trend. Among the factors affecting the quality of food, the gender of livestock is also identified as one of the major factors in quality determination. In other words, it has been found that there is a difference in meat quality due to physiological differences such as sex hormone secretion according to the sex of the food animals. Generally, males are known to have poor meat quality compared to females. therefore. If the sex of the calves born can be artificially controlled to produce only 100% female calves, the Hanwoo industry and the dairy industry will have tremendous internal and external benefits. Therefore, the best technique among modern reproductive technology is to determine the gender in advance and increase the productivity and improvement of livestock by planned mass production of the desired female (female) expectant and expectant.
Until recently, various genetic engineering techniques have been disclosed to determine the sex of mammalian embryos, but the technology has not yet been secured enough to commercialize them in Korea.
The present inventors have made efforts to develop a novel marker composition capable of discriminating the sex of a bovine embryo. As a result, the present inventors have found that a marker capable of discriminating the sex of a bovine embryo with a small amount of DNA using a Y-chromosome- By confirming, the present invention has been completed.
It is an object of the present invention to provide a marker composition for discriminating sex of a bovine embryo.
Yet another object of the present invention is to provide a sex discrimination kit and a discriminating method of a small embryo containing the marker.
In order to solve the above problems, the present invention provides a primer set comprising a base sequence of SEQ ID NOS: 1 and 2; And a probe comprising the nucleotide sequence of SEQ ID NO: 3. The present invention also provides a marker composition for distinguishing sex of a bovine embryo.
In addition, the present invention provides a sex discrimination kit and a discriminating method of a bovine embryo including the marker.
The marker composition according to the present invention can discriminate the sex of a bovine embryo only with a trace amount of DNA of a few pg level and can be usefully used in the Hanwoo industry and the dairy industry.
Figure 1 shows the melting curve of a sry-specific fragment identified using an LC-PCR (Roche LightCycle real-time PCR) assay.
(M: size marker, 1: 1000 ng, 2: 100 ng, 3: 10 ng, 4: 1 ng, and 5: 0.1, respectively) using DNA isolated from bovine blood. ng, 6: 0.01 ng, 7: 5 pg, 8: 2.5 pg).
FIG. 3 is a diagram showing real-time PCR results according to concentration using DNA isolated from blood of a hydrogen source.
Hereinafter, the present invention will be described in more detail.
The present invention provides a primer set comprising a base sequence of SEQ ID NOs: 1 and 2; And a probe comprising the nucleotide sequence of SEQ ID NO: 3. The present invention also provides a marker composition for distinguishing sex of a bovine embryo.
In the present invention, the term "marker" refers to a substance that can discriminate the sex of a bovine embryo. Examples of the marker include a polypeptide, a nucleic acid (e.g., mRNA), a lipid, a glycolipid, a glycoprotein, a sugar (monosaccharide, Oligosaccharides, etc.) and the like.
As used herein, the term "primer" refers to a primer that is hybridized under appropriate conditions in a suitable buffer solution (for example, four different nucleoside triphosphates and a polymer such as DNA, RNA polymerase or reverse transcriptase) Refers to single stranded oligonucleotides that can serve as a starting point for DNA synthesis. The appropriate length of the primer may vary depending on the intended use, but is usually 15 to 30 nucleotides. Short primer molecules generally require a lower temperature to form a stable hybrid with the template. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template.
In the present invention, "forward primer" and "reverse primer" bind to 3'-terminal and 5'-terminal of a specific site of a gene amplified by gene amplification reaction, respectively, Quot; primer "
As used herein, the term "probe" means a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides that can specifically bind to a complementary nucleotide sequence. . The probe may be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. In order to perform real-time PCR, a fluorescent marker probe can be used. More specifically, when a TaqMan probe is used, an oligonucleotide modified at the 5 'end with a fluorescent substance and at the 3' end with a quencher substance is added to the PCR reaction solution.
The primers or probes of the present invention can be chemically synthesized using methods known in the art such as, for example, the phosphoramidite solid support method. Further, it can be modified by methylation, capping or the like by a known method.
The primer set and the probe according to the present invention specifically bind to the Y-chromosome specific sry gene in bovine embryos, and the sex of the bovine embryo can be quickly and accurately discriminated from a trace amount of DNA of several pg.
The present invention also provides a primer set comprising a nucleotide sequence of SEQ ID NOS: 1 and 2; And a probe comprising the nucleotide sequence of SEQ ID NO: 3.
The term "kit" of the present invention includes a genomic DNA derived from a sample to be analyzed, a primer and a probe set of the present invention, an appropriate amount of DNA polymerase (for example, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermusfiliformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), dNTP mixture, PCR buffer solution and water. The PCR buffer may contain KCl, Tris-HCl and MgCl 2. In addition, components necessary for conducting electrophoresis to confirm whether the PCR product is amplified can be further included in the kit of the present invention. At this time, the MgCl 2 concentration greatly affects the specificity and yield of the amplification, and it can be preferably used in the range of 1.5-2.5 mM. Generally, when Mg 2 + is excessive, nonspecific PCR amplification product is increased, and when Mg 2 + is insufficient, the yield of PCR product is decreased. The PCR buffer solution may further contain an appropriate amount of Triton X-100.
In addition,
(a) separating DNA from the bovine embryo;
(b) a primer set consisting of the nucleotide sequences of SEQ ID NOS: 1 and 2, using the DNA isolated in step (a) as a template; And a probe comprising the nucleotide sequence of SEQ ID NO: 3; And
(c) identifying the product amplified in step (b) and discriminating the sex of the bovine embryo.
In the step (a), the DNA includes both gDNA and cDNA.
In step (a), the DNA may be separated by a phenol / chloroform extraction method, an SDS extraction method, a CTAB separation method (Murray et al., Nuc. Res. 4321-4325, 1980) Or may be performed using a commercially available DNA extraction kit, but the present invention is not limited thereto. If the target sample is mRNA, total RNA is isolated according to a method commonly used in the art, and cDNA is synthesized and used.
The amplification of the target sequence in the step (b) can be performed using polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), ligase chain reaction (LCR), Gap-LCR, repair chain reaction, transcription-mediated amplification (TMA), self sustained sequence replication, and hybridization of the target polynucleotide sequence The primers used in this study include selective amplification of target polynucleotide sequences, consensus sequence primer polymerase chain reaction (CP-PCR), arbitrarily primed polymerase chain reaction (AP-PCR) Nucleic acid sequence based amplification (NASBA), strand displacement amplification and loop-mediat amplification ed isothermal amplification (LAMP), and the like. General PCR methods are well known in the art and commercially available kits can be used. In addition, the amplified target sequence may be labeled with a detectable labeling substance. The labeling substance may be a substance that emits fluorescence, phosphorescence, or radioactivity, but is not limited thereto.
In the step (c), the amplified products may be identified by various methods commonly used in the art. For example, the amplified products may be identified by electrophoresis, but the present invention is not limited thereto. In the present invention, the gel used for electrophoresis can be a gel which can be used in electrophoresis. Agarose gel or polyacrylamide gel can be used as a representative example. After electrophoresis, electrophoresis results can be analyzed by silver staining.
Hereinafter, the present invention will be described more specifically based on examples. It will be apparent to those skilled in the art that the embodiments are only for describing the present invention in more detail and that the scope of the invention is not limited by these embodiments in accordance with the gist of the present invention.
Example One. DNA Preparation of samples
For biopsy of Bovine Embryos, when the cells 16 to 32 after fertilization, zona pellucida (zonae pellucidae) the degradation of 0.5% trypsin, and pipette the blastomeres Ca 2 +, from the (free) DPBS solution that does not contain Mg 2 + . Whether or not nuclei were present in the isolated cells was confirmed by fluorescence staining using a Hoechst 33342 die.
In addition, the remaining cells were continuously cultured until development into blastocysts, and then DNA was isolated by boiling in the blastocysts and the blastocysts. More specifically, the biopsied embryo was placed in a PCR tube and 12 μl of lysis buffer (20 mM Tris.Cl (pH 7.4), 0.9% Tween 20, 0.9% Nonidet) was added (option: And then spun down. The cells were then heated for 15 minutes in a heating block preheated to 95 ° C and stored at -20 ° C until used.
In addition, DNA was isolated from blood samples of cancer and hydrogen using GeneAll (Seoul, Korea) Cell SV kit.
Example 2. Real time PCR reaction
2-1. primer And Probe design
Real-time PCR primers and probes for sex determination of bovine embryos are available under GenBank accession number AB039748 Bos Designed based on the tetsis determining factor SRY sequence of taurus , manufactured by SigmaProligo. The specific sequences are shown in Table 1 below. The fluorescent reporter material 6FAM was labeled at 5 'of the probe, and BHQ1, which is a quencher material, was labeled at 3'.
2-2. Preliminary experiment
In order to confirm the reactivity of the primer prepared in Example 2-1, the specific binding force to the sry gene was verified by comparing Tm values using SYBR green. The Tm value is a peculiar value determined by the GC content of the amplified DNA fragments. Samples showing Tm values in the range of ± 0.5 ° C can be judged from the same DNA. The results are shown in Fig.
As shown in FIG. 1, Tm values of 86.78 ± 0.2 ° C for hydrogen DNA and 79.70 ± 0.18 ° C for blood DNA of cow were confirmed. Thus, the primers prepared in Example 2-1 It was confirmed that the sry gene was specifically bound and reacted normally.
2-3. PCR reaction
The PCR reaction was carried out using the DNA isolated from the blood sample of bovine obtained in Example 2-1. A more specifically, the dilute each concentration of DNA, DN AccuPower TM PCR PreMix (Bioneer, Daejeon, Korea), of 10 pmole primers (SEQ ID NOS: 1 and 2) mixing the (total 20 ul), then 5 minutes at 94 o C After preincubation, the reaction was repeated 30 times at 94 ° C for 30 seconds, 58 ° C for 30 seconds and 72 ° C for 45 seconds, and then reacted at 72 ° C for 7 minutes. PCR products were confirmed by electrophoresis on 1% agarose gel. The results are shown in Fig.
As shown in FIG. 2, a band of 300 bp corresponding to the sry gene was confirmed up to a DNA concentration of 100 pg, but it was not confirmed when the DNA concentration was less than 100 pg.
2-4. real time PCR reaction
The PCR reaction was carried out using the DNA isolated from the blood sample of bovine obtained in Example 2-1. More specifically, 4 μl of LightCycler Taqman Master (Roche Diagnostic), 0.5 μM of primer (SEQ ID NOs: 1 and 2), 0.2 μM of Taqman probe (SEQ ID NO: 3) and DNase free H 2 O After incubation at 95 ° C for 10 minutes, the reaction was repeated 45 times at 95 ° C for 10 seconds, 58 ° C for 40 seconds, and 72 ° C for 1 second and then cooled at 40 ° C. The reaction was terminated for 30 seconds. Ct values were analyzed by quantification mode (LightCycler Ver 4.1). strandard curve was obtained and the result of Ct value 37 or more was not taken. The results are shown in Fig.
As shown in Fig. 3, the sry gene could be confirmed until the DNA concentration was 5 pg.
In addition, the DNA isolated from the bovine embryo obtained in Example 2-1 (the embryo half-seeded cells isolated from the cells in the 16-32 cell anomaly and the DNAs obtained by developing the same embryo cells to the blastocysts and then separated) And real-time PCR was performed in the same manner. The results are shown in Table 2.
(300 bp DNA fragment)
(Hydrogen, 100 ng)
(Cancer, 100 ng)
# One
# 2
# 3
As shown in Table 2, two of the three samples were female and one was male.
From the above experimental results, it was confirmed that when real-time PCR was performed using the primer and the probe set of the present invention together, the microorganism could be rapidly and accurately determined from a trace amount of DNA of several pg.
<110> Sahmyook University Industry-Academic Cooperation Foundation <120> Marker composition for sex determination of Bovine Embryo <130> s <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> sense primer <400> 1 tagccataca ccgagacaaa tacc 24 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> antisense primer <400> 2 gtccgccgaa atccgtgtag 20 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 3 atgttacctt attgtggccc aggcttgtcc 30
Claims (6)
(b) a primer set consisting of the nucleotide sequences of SEQ ID NOS: 1 and 2, using the DNA isolated in step (a) as a template; And a probe comprising the nucleotide sequence of SEQ ID NO: 3; And
(c) identifying the product amplified in step (b) and discriminating the sex of the bovine embryo.
The amplification of the target sequence in step (b) may be performed using a polymerase chain reaction (PCR), a reverse-transcription polymerase chain reaction (RT-PCR), a ligase chain reaction (LCR) repair chain reaction, transcription-mediated amplification (TMA), self-sustained sequence replication, selective amplification of target polynucleotide sequences, consensus sequence priming (PCR), arbitrary primed polymerase chain reaction (PCR), nucleic acid sequence based amplification (NASBA), and primer-based amplification Strand displacement amplification and loop-mediated isothermal amplification (LAMP). ≪ RTI ID = 0.0 > Wherein the method comprises the steps of:
Wherein the confirmation of the amplification product of step (c) is performed by electrophoresis.
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