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KR-20260065034-A - PCR technique for improving the accuracy of sex determination of Korean cattle fetuses

KR20260065034AKR 20260065034 AKR20260065034 AKR 20260065034AKR-20260065034-A

Abstract

The present invention relates to a PCR technique for improving the accuracy of sex determination of Hanwoo fetuses. Specifically, the present invention allows for the pre-planning of farm operations by determining sex using the blood of a pregnant cow, and enables verification of how the feeding performance of the newborn calf changes by providing nutritional supplementation to the cow two months prior to calving.

Inventors

  • 박병기
  • 신종서
  • 권응기
  • 차현지
  • 손기활
  • 이소희
  • 김영래

Assignees

  • 강원대학교산학협력단

Dates

Publication Date
20260508
Application Date
20241031

Claims (12)

  1. A composition for sex determination of a Korean beef fetus comprising, as an active ingredient, a primer having a nucleotide sequence represented by SEQ ID NOs 1 to 4.
  2. In paragraph 1, Primers having the nucleotide sequences represented by SEQ ID NOs 1 and 2 are the first primers for sex determination of Hanwoo fetuses, and Primers having the nucleotide sequences indicated by SEQ ID NOs 3 and 4 are second primers for sex determination of Hanwoo fetuses, and Composition for sex determination of a Korean beef fetus.
  3. In paragraph 2, Characterized by the first primer and the second primer being used in sequence, Composition for sex determination of a Korean beef fetus.
  4. In Paragraph 3, The composition used together with the first primer mentioned above is, Characterized by comprising 3uL of DNA, 2uL of primer, 2uL of PCR buffer (excluding MgCl₂ ), 1uL of 10mM dNTP, 1uL of 20mM MgCl₂ , 0.2uL of DNA Taq, and 10.8uL of distilled water. Composition for sex determination of a Korean beef fetus.
  5. In Paragraph 3, The composition used together with the second primer mentioned above is Characterized by comprising 2 µL of DNA of the product amplified using the first primer, 2 µL of primer, 2 µL of PCR buffer (excluding MgCl₂ ), 1 µL of 10 mM dNTP, 1 µL of 20 mM MgCl₂ , 0.2 µL of DNA Taq, and 11.8 µL of distilled water. Composition for sex determination of a Korean beef fetus.
  6. In any one of paragraphs 1 through 5, The above composition is based on plasma separated from the blood of Korean cattle, Composition for sex determination of a Korean beef fetus.
  7. How to determine the sex of a Hanwoo fetus through the following steps: Step 1: Collecting blood from a pregnant Hanwoo; Step 2: Separating plasma from collected blood; Step 3 for extracting DNA from separated plasma; Step 4, amplifying a target sequence using primers having nucleotide sequences represented by SEQ ID NOs 1 and 2; Step 5 for obtaining the amplified product; Step 6, amplifying the target sequence using primers having the nucleotide sequences represented by SEQ ID NOs 3 and 4 on the product obtained in Step 5; 7 steps to determine the sex of a Hanwoo fetus by verifying the amplification product.
  8. In paragraph 1, The above 4 steps are, Characterized by proceeding by a Polymerase Chain Reaction (PCR) method under conditions of 95℃ for 5 minutes, 94℃ for 30 seconds, 58℃ for 30 seconds, 72℃ for 30 seconds, 72℃ for 10 minutes, and standing at 4℃. Method for determining the sex of a Hanwoo fetus.
  9. In paragraph 1, The above 6 steps are, Characterized by proceeding by a Polymerase Chain Reaction (PCR) method under conditions of 95℃ for 5 minutes, 94℃ for 30 seconds, 56℃ for 30 seconds, 72℃ for 30 seconds, 72℃ for 10 minutes, and standing at 4℃. Method for determining the sex of a Hanwoo fetus.
  10. A primer used for PCR amplification to determine the sex of a Hanwoo fetus, having a nucleotide sequence represented by SEQ ID NOs 1 to 4.
  11. In Paragraph 10, Primers having the nucleotide sequences represented by SEQ ID NOs 1 and 2 are the first primers for sex determination of Hanwoo fetuses, and Primers having the nucleotide sequences indicated by SEQ ID NOs 3 and 4 are second primers for sex determination of Hanwoo fetuses, and Characterized by the first primer and the second primer being used in sequence, Primer.
  12. A kit for sex determination of a Hanwoo fetus comprising primers having nucleotide sequences represented by SEQ ID NOs 1 to 4 as active ingredients.

Description

PCR technique for improving the accuracy of sex determination of Korean cattle fetuses The present invention relates to a PCR technique for improving the accuracy of sex determination of Korean beef cattle fetuses. Cattle are monotetes, giving birth to only one calf per year. If the newborn calf is female, the farmer can sell the cow with a high parity and raise the new calf to utilize it as a breeding cow; if the newborn calf is male, it can be raised for six months and then sold to a fattening farm. As such, the sex of the newborn calf influences the farm's operational strategy. To date, ultrasound is the most frequently used method for early determination of fetal sex in cattle. However, the ability to determine fetal sex decreases as pregnancy progresses and is only possible between 56 and 98 days after ovulation (Heyman et al., 2002). Furthermore, the use of ultrasound to determine fetal sex in cattle is limited due to technical constraints, increased costs, and animal safety and management issues (Hylan et al., 2009). Invasive methods for sex determination include chorionic villus sampling (CVS) and amniocentesis, which carry risks of premature birth and death. Lo et al. (1997) discovered that fetal DNA is present in the plasma and serum of healthy mothers. According to their findings, fetal DNA in maternal plasma accounts for an average of 3.4% of total DNA in early pregnancy and 6.2% in late pregnancy (Lo et al., 1998), and is cleared at a very rapid rate after birth (Lo et al., 1999). It is a well-known phenomenon that fetal nuclei reach the maternal peripheral circulation through the placental barrier. Therefore, pregnant females possess fetal DNA circulating during pregnancy, depending on the temporal relationship between pregnancy and birth and the increase in fetal DNA concentration in maternal plasma (Lo et al., 1998). Thus, it is believed that the presence of fetal DNA in the plasma of pregnant cows can predict the fetal sex in cattle. Currently, research is underway on sex determination methods using fertilized eggs and artificial sex control through in vitro fertilization. However, these methods are currently applicable only to breeding cows conceived via in vitro fertilization and cannot be used on breeding cows conceived through natural or artificial insemination. Furthermore, there are many cases where fertilized eggs fail to implant properly due to sex determination or artificial sex control, resulting in miscarriage or failure to conceive. However, if blood is utilized, this approach can be applied not only to in vitro fertilization but also to breeding cows conceived through artificial insemination and natural pregnancy. PCR is a technique capable of amplifying specific genes, and it has the advantage of being able to amplify fetal DNA within the plasma of a mother cow. Since sex determination via PCR is performed using male-specific target sequences, specific band amplification cannot be observed in the case of females. Therefore, it is determined that if the fetus in the womb is male, specific band amplification will appear, and if it is female, specific band amplification will not appear. We intend to determine sex through the amplification of these specific bands. Therefore, in this invention, by using the blood of a pregnant cow to determine the sex, the farm's operational plan can be planned in advance, and by providing nutritional supplementation to the cow two months before calving, it is possible to check how the feeding performance of the newborn calf changes. Figure 1 is a figure showing the experimental results of a comparative analysis of whole blood and plasma in one embodiment of the present invention. FIG. 2 is a figure showing the result of accurately expressing a DNA band to be confirmed in plasma using the primer of the present invention in one embodiment of the present invention. Figure 3 is a figure comparing the results of the third PCR step, in which the temperature is adjusted to 57°C or 58°C using the first primer of the present invention in one embodiment of the present invention. Figure 4 is a figure comparing the results of adjusting the temperature of the third PCR step to 56°C, 57°C, or 58°C using the second primer of the present invention in one embodiment of the present invention. Figure 5 is a figure comparing the results of performing the 2nd PCR of the present invention in one embodiment of the present invention, where the concentration of MgCl₂ in the buffer composition is 1 mM or 1.5 mM. Figure 6 is a figure showing the results according to the type of primer when performing the 2nd PCR of the present invention in one embodiment of the present invention. Figure 7 is a figure showing the results of electrophoresis performed after PCR to confirm β-actin expression, in which DNA was extracted from plasma and whole blood of non-pregnant Korean cattle in one embodiment of the present invention. Hereinafter, the present invention will be described in det