CN-120866534-B - Design method and application of pig chromosome end centromere probe

Abstract

The invention discloses a design method and application of a pig chromosome terminal centromere probe, and belongs to the technical field of molecular biology. The invention utilizes bioinformatics means to screen out a probe specifically combined with the terminal centromere of the pig chromosome from preliminary searching, comparison and screening out a sequence with higher repetition times and stronger specificity, and the nucleotide sequence of the probe is shown as SEQ ID NO. 1. The invention develops an in-situ fluorescence hybridization prefabrication reagent by utilizing a pig primary cell line and an immortalized cell line, and the result shows that the probe can be well combined with the current DNA region. The design method of the pig chromosome centromere probe provided by the invention can be used for designing most of probes, and the developed pig chromosome end centromere probe has good specificity, good hybridization effect and high detection accuracy, and provides a new visual angle and method for further researching pig chromosome rearrangement and translocation.

Inventors

• LIU JIANFENG
• MA YIJIA
• FENG YU
• ZHANG JING
• Ni Ruinan

Assignees

• 中国农业大学

Dates

Publication Date

20260508

Application Date

20250725

Claims (9)

1. 1. A probe for detecting a pig chromosome terminal centromere is characterized in that the nucleotide sequence of the probe is shown as SEQ ID NO. 1.
2. 2. A fluorescent in situ hybridization kit for detecting a pig chromosome end centromere, comprising the probe of claim 1.
3. 3. The fluorescent in situ hybridization kit according to claim 2, wherein the fluorescent in situ hybridization kit further comprises an RNase working solution, 0.1% NP-40, 0.5% Triton X-100, a denaturing solution, and a hybridization solution; The denaturing solution is 70% deionized formamide, 20% ddH 2 O and 10% 20 XSSC; The hybridization solution is prepared by mixing 10% dextran sulfate, deionized formyl, tween-20 and ddH 2 O according to the mass ratio of 1:5:0.01:3.99.
4. 4. Use of the probe according to claim 1 for the preparation of a kit for detecting chromosome end-of-pig centromeres.
5. 5. The use of the probe according to claim 1 for the preparation of a kit for detecting chromosome end-of-line centromeres of porcine primary small intestine epithelial cells and porcine kidney cell lines.
6. 6. Use of a probe according to claim 1 for the preparation of a kit for detecting rearrangement and ectopic of porcine chromosomes 13-18 for non-disease diagnostic purposes.
7. 7. The use according to any one of claims 4 to 6, wherein the probe binds to the target region of the pig chromosome end centromere based on fluorescence in situ hybridization, and the position and structural changes of different chromosome end centromeres are accurately identified by fluorescence.
8. 8. A method for performing in vitro fluorescence in situ hybridization of a chromosome on porcine cells for non-diagnostic purposes, comprising the steps of inducing a mid-stage of the chromosome in cultured porcine cells by using colchicine, mixing the probe according to claim 1 with hybridization solution, performing hybridization reaction, and detecting the terminal centromere region in the porcine cells.
9. 9. The method of claim 8, wherein the probe and the hybridization solution are mixed according to the volume ratio of 1 (30-40), and placed in a dark place at 80 ℃ for thermal denaturation for 20min, and then placed on transfer ice for standing for 5-10 min; the hybridization solution is prepared by mixing 10% dextran sulfate, deionized formyl, tween-20 and ddH 2 O according to the mass ratio of 1:5:0.01:3.99; The hybridization reaction conditions were 30℃for 20h.

Description

Design method and application of pig chromosome end centromere probe Technical Field The invention relates to the technical field of molecular biology, in particular to a design method and application of a pig chromosome terminal centromere probe. Background Pig is one of important domestic animals, and the promotion of genetic breeding and reproductive performance thereof has been the focus of research in the field of zootechnical science. Chromosome is a carrier of genetic material, and the structural and numerical stability of the chromosome is critical to maintaining genetic stability of a species. However, structural variations such as rearrangements and translocations may occur in porcine chromosomes under the influence of natural or human factors. The mechanisms by which chromosomal rearrangement and translocation occur are complex and are mainly related to DNA double strand breaks and repair processes. When DNA is damaged and double strand breaks occur, the cell initiates DNA repair mechanisms. If an error occurs in the repair process, it may cause abnormal reconnection of the chromosome fragment, thereby causing chromosome rearrangement and translocation. Furthermore, the lack of separation, abnormal exchange, etc. of chromosomes during meiosis may also lead to the creation of these structural variations. Chromosome rearrangement during long-term evolution of different pig breeds can reflect the history of speciation and make it possible to determine phylogenetic relationships to reconstruct ancestral karyotypes and to discern mechanisms of genomic evolution and function. Chromosome rearrangement and translocation may affect germ cell formation and embryo development in pigs. For example, translocation may result in the occurrence of pairing abnormalities in the chromosomes during meiosis, thereby producing unbalanced gametes. After fertilization of the unbalanced gametes, the problems of embryonic death, abortion or fetal malformation and the like can be caused, and the reproductive efficiency of pigs is reduced. In addition, certain chromosomal rearrangements and translocations may affect gene expression and regulation, thereby affecting the growth and phenotypic characteristics of pigs. The traditional detection method for the rearrangement and translocation of the pig chromosome mainly comprises chromosome karyotype analysis, G banding technology and the like. Chromosome karyotyping primarily judges whether chromosome abnormality exists or not by observing the morphology, number and structure of chromosomes, and cannot identify which chromosome is abnormal. The G banding technology can further display the banding characteristics of the chromosome, and improve the accuracy of detecting the structural variation of the chromosome. However, the resolution of these methods is relatively low, and may be difficult to detect for some minor chromosomal rearrangements and translocations. Centromeres are specific regions on the chromosome, consisting mainly of repetitive DNA sequences and related proteins. It plays a key role in the separation of chromosomes, can bind to spindle microtubules, and ensures accurate distribution of chromosomes into daughter cells during mitosis and meiosis. Research shows that the centromere sequence has certain conservation and variability in the species evolution process. In translocated chromosomes, the position of the centromere may change, and by comparing differences and similarities in centromere sequences between different pig species or closely related species, the evolutionary history and genetic relationship of pig chromosomes can be understood in depth. With the development of molecular biology technology, fluorescence In Situ Hybridization (FISH) and other technologies are gradually applied to the detection of the structural variation of the pig chromosome, so that the sensitivity and the accuracy of the detection are greatly improved. However, there is currently no established method for designing chromosome centromere probes in pigs, and probes for detecting chromosome centromere at the ends of pigs based on FISH. Disclosure of Invention The invention aims to provide a design method and application of a pig chromosome end centromere probe, which are used for solving the problems existing in the prior art, the design method of the pig chromosome centromere probe provided by the invention can be applied to the design of most probes, and has the advantages of good specificity, good hybridization effect and high detection accuracy. In order to achieve the above object, the present invention provides the following solutions: The invention provides a pig chromosome terminal centromere probe, and the nucleotide sequence of the pig chromosome terminal centromere probe is shown as SEQ ID NO. 1. The invention also provides a fluorescence in situ hybridization kit for detecting the pig chromosome terminal centromere, which comprises the pig chromosome terminal centromere