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Sex identification method for mammalian embryo

An identification method, mammalian technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of embryo damage, detection method can only detect the end point, and the identification effect is not ideal, etc. Achieve the effects of simple operation, high identification efficiency and accuracy, and increased cost

Pending Publication Date: 2021-11-02
TARIM UNIV
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Problems solved by technology

[0005] However, because some embryonic cells need to be cut out as templates during embryo sex identification, it will cause certain damage to the embryo
In order to ensure the survival rate of embryos, the number of embryonic cells should be cut as little as possible, and the identification effect is not ideal.
Moreover, traditional detection methods can only detect endpoints

Method used

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  • Sex identification method for mammalian embryo
  • Sex identification method for mammalian embryo

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preparation example Construction

[0031] The FISH method mainly includes three parts: preparation of fluorescein probe, denaturation and hybridization of probe and target DNA, and observation and identification. The probe is first combined with the reporter molecule, and after hybridization, the fluorescent dye is connected to the immunocytochemical process to generate fluorescence. This method can be hybridized and identified in the interphase and metaphase of cell division. Although this method is specific, efficient and has a low error rate, it does not It takes a long time, has strict requirements on reagents, and high identification costs, so it is difficult to apply in production practice.

[0032] According to the characteristics of DNA, the PCR method is to add appropriate amount of buffer, template DNA, 4 kinds of deoxygenated mononucleotide (d NTP) solutions, heat-resistant DNA polymerase, Mg2+ and 1 pair of synthetic primers into microcentrifuge tubes. Use DNA in vitro at 90-95°C to dehelicate, dena...

Embodiment 1

[0051] The technical scheme of the present embodiment is:

[0052] (1) Acquisition of embryos: Sex identification can be carried out for embryos produced by live embryo collection or in vitro fertilization, as well as frozen embryos.

[0053] (2) Obtaining the genomic DNA of embryonic cells.

[0054] (3) Design and synthesis of primers and probes.

[0055] Wherein, the male-specific gene primer sequence is:

[0056] ① Upstream 5'-CTATACACCGAGACAAATACCCG-3';

[0057] ② Downstream 5'-AATCGTCCCTGTATGTGAAGG-3'.

[0058] The male-specific gene Probe is:

[0059] FAM-AAGAGGCCACAGAAATCCCTTGCT-MGB.

[0060] Internal reference gene primer sequence: ①upstream 5'-GGTCCACATGGCCTCCAAG-3'; ②downstream 5'-TCCCTTTCCTCAGGGCCTT-3'.

[0061] (4) QPCR reaction: during PCR amplification, a specific fluorescent probe is added while adding a pair of primers;

[0062] PCR reaction system 10 μL: 5 μL of 2×S6 Probe qPCR Mix, 0.2 μL of upstream and downstream primers, 0.1 μL of Probe, 1 μL of DNA...

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Abstract

The invention relates to a sex identification method for a mammalian embryo. The sex identification method for the mammal embryo comprises the following steps: (1) obtaining the embryo; (2) obtaining genome DNA of embryonic cells; (3) designing and synthesizing primers and probes; (4) performing QPCR reaction: during PCR amplification, adding a pair of primers and a specific fluorescent probe at the same time; and (5) amplifying the curve and analyzing, and identifying the sex of the embryo. The sex identification method for the mammal embryo, disclosed by the invention, is a real-time fluorescent quantitative PCR (Quantitative Real-time PCR) probe method, is simple in design, only needs a pair of primers and is added with a specific fluorescent probe at the same time, so that the limitation that only end-point detection can be carried out in a traditional method is effectively solved; the method has the advantages of less sampling, high sensitivity, stronger specificity, rapidness, simplicity, convenience, effective solving of a PCR pollution problem, good universality, high automation degree and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a sex identification method for mammalian embryos. Background technique [0002] The technical methods of sex control in mammals have developed rapidly along with the in-depth study of sex determination theory, from the individual level, the cellular level to the molecular level. Due to the development of in vitro fertilization, nuclear transfer and cell fusion technology, the source of embryos has been greatly increased. Therefore, as one of the sex control technologies, the role of embryo sex identification is becoming more and more significant. The optimization and maturity of this technology will be widely used in future production practice. is of great significance. [0003] At present, among embryo sex identification methods, cytological methods mainly determine the sex of embryos by analyzing the chromosome composition of some embryo cells (XX for females and XY f...

Claims

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Application Information

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IPC IPC(8): C12Q1/6879C12Q1/686C12N15/11
CPCC12Q1/6879C12Q1/686C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 高庆华方翟陶维昆
Owner TARIM UNIV
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