Oligonucleotide probe kit for detecting rye chromosomes and use method thereof
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An oligonucleotide probe and chromosome technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of complex procedures, lack of large sample research, and low resolution, Achieve the effect of reducing costs and promoting chromosome engineering
Active Publication Date: 2019-10-22
NANJING AGRICULTURAL UNIVERSITY
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However, due to the complexity and low resolution of the previous technical procedures, there is still a lack of systematic large-sample research on the analysis of rye chromosome diversity to quantitatively describe the heterozygosity and heterogeneity of rye chromosomes
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[0028] The rye kit includes TAM-oligo-6, FAM-(AAC)10, FAM-pSc119.2-1, 1 tube of dry probe powder, 1 bottle of 1000ml 2×SSC buffer and 1 stainingjar.
[0029] The method of use includes the following steps:
[0030] (1) Dye solution configuration: Dissolve 3 tubes of dry powder with 30 μl ddH2O respectively, add 30ml 2×SSC to the stainingjar first, then add 1 μl of probes in each tube in turn, and mix well.
[0031](2) Place the chromosome preparations in the dye solution, stain at 37°C for 12 hours (overnight), take out the preparations, rinse with 2×SSC buffer for 5 minutes, then rinse with pure water for 3 minutes, add 6.5 μl to each piece after air-drying Cover with DAPI glue, cover with a cover glass, observe and take pictures under a fluorescent microscope to obtain chromosome pictures. According to the established rye variety reference karyotype ( figure 1 ), after staining with the dye solution, all chromosomes can be ...
[0033] Probe Stain Kit ONPM#6 for simultaneous detection of wheat and rye chromosomes, including TAM-labeled pAs1-1, pAs1-3, pAs1-4, pAs1-6, AFA-3, AFA-4, oligo-6 and FAM Labeled (AAC)10, pSc119.2-1, 1 tube of dry probe powder, 1 bottle of 1000ml 2×SSC buffer and 1 staining jar.
[0034] The method of use includes the following steps:
[0035] (1) Dye solution configuration: Dissolve 13 tubes of dry powder with 30 μl ddH2O respectively, add 30ml 2×SSC to the staining jar first, then add 1 μl of each probe into the staining jar, and mix well.
[0036] (2) The method of use is the same as in Example 1. After ONPM#6 staining, according to the established Huixianhong-Jingzhou rye diploid karyotype ( figure 2 ), which can identify all wheat and rye chromosomes in the wheat background, Figure 5 It is the first dyeing effect, Figure 6 It is the fifth dyeing effect.
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Abstract
The invention belongs to the technical field of biology, and provides an oligonucleotide probe kit for detecting rye chromosomes and a use method thereof. The oligonucleotide probe kit can quickly andaccurately identify the diversity of the rye chromosomes, and is conductive to rye variety identification and variety right protection. The kit combines with six wheat oligonucleotide probes developed in the earlier stage to simultaneously identify wheat and rye chromosomes in a wheat background, which provides a simple, economical and efficient tool for wheat-rye chromosomeengineering. An oligonucleotide dyeing solution prepared by the disclosed kit can be circularly used for batch dyeing, which greatly simplifies a fluorescence in-situ hybridization procedure and reduces the cost, also provides a powerful tool for automatic and high-throughput analysis of the chromosomes, and has important research and application potential.
Description
technical field [0001] The patent discloses an oligonucleotide probe kit for detecting rye chromosomes and its application method, belonging to the field of biotechnology. Background technique [0002] Fluorescencein situ hybridization (FISH) is a new technology developed in the late 1980s. It is widely used in the field of plants for chromosome identification, karyotype analysis, phylogenetic analysis, phylogenetic relationship analysis, and chromosome identification. A powerful tool for genomics research (Jiang JM and Gill BS. Current status and the future ofluorescence in situ hybridization (FISH) in plantgenome research. Genome, 2006, 49(9): 1057-1068). In recent years, the development and utilization of oligonucleotide probes has greatly simplified the FISH procedure and promoted the popularization and application of this technology. Oligonucleotide probes can be designed based on the genome sequence, and the sources of probes are abundant and the development cost is...
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