A peanut chromosome probe staining kit and its application method

A dyeing reagent and chromosome technology, which is applied in the field of peanut cytology research, can solve the problems of multi-manual operation steps, unfavorable automatic identification, etc., and achieve the effect of simple technical method and high cost

Active Publication Date: 2022-04-12
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, oligonucleotide FISH technology still contains more manual steps, which is not conducive to automatic identification

Method used

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  • A peanut chromosome probe staining kit and its application method
  • A peanut chromosome probe staining kit and its application method
  • A peanut chromosome probe staining kit and its application method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1, peanut chromosome probe staining kit

[0027] The kit includes Probe Mix dry powder, DAPI stock solution with a concentration of 100 μg / ml, 2×SSC buffer solution, DAPI buffer solution, staining jar and variety control karyotype.

[0028] The probes in Probe Mix dry powder include TAM fluorescently labeled probes DP-1, DP-7 and FAM fluorescently labeled probes DP-5, DP-8, each probe content is 0.2OD, as follows:

[0029]

[0030] 2 x SSC buffer consisting of 0.3 M trisodium citrate C 6 h 5 Na 3 o 7 2H 2 O and 3M NaCl composition.

[0031] DAPI buffer consists of 0.1M citric acid and 0.05M Na 2 HPO 4 composition.

[0032] The staining jar is a black light-proof glass staining jar.

Embodiment 2

[0033] Embodiment 2, the use method of peanut chromosome probe staining kit, comprises the following steps:

[0034] (1) Preparation of dyeing solution: Dissolve Probe Mix dry powder in 1ml 2×SSC buffer, pour it into the staining jar, and add 39ml 2×SSC buffer to make the final concentration of DP-1, DP-5, DP-7 and DP-8 Both are 0.005OD / ml;

[0035] (2) Put the slides (76×26×1mm) containing the peanut chromosomes in the staining solution of the staining jar, and stain at 35-37°C for 6 hours; take out the slides, wash them with 2×SSC buffer for 3 times, and then Add 500 μl of the mixture of DAPI mother solution (100 μg / ml) and DAPI buffer solution with a volume ratio of 2.5:500 dropwise on each slide, stain for 3-4 minutes, then wash with DAPI buffer solution for 4-5 times, and drop 5 μl of antibacterial solution. Fluorescence-quenched mounting medium, covered with a coverslip, and photographed under a fluorescent microscope.

Embodiment 3

[0036] Embodiment 3, the staining of the four red chromosomes of peanut cultivars

[0037] Adopt the method for embodiment 2, use the same dyeing vat dyeing solution to dye the peanut cultivar four-grain red metaphase chromosome, and dye to obtain the picture ( figure 1 a), and cut out each chromosome, and arrange the karyotype ( figure 1 b). from figure 1 It can be seen from the figure that after staining with the Probe Mix dye solution of the kit, there are obvious red and green characteristic signals on the chromosomes, the A genome is dominated by red signals, and the B genome is dominated by green signals, and A and B of peanuts can be clearly distinguished according to the color Genome. In addition, the banding patterns of peanut chromosomes are different, and the characteristics are as follows: In genome A, there is only a weak signal at the end of chromosome 1, weaker red and green signals at the centromere of chromosome 2, and weaker red and green signals at the ce...

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Abstract

The invention discloses a peanut chromosome probe staining kit and a method for using the same. The kit includes Probe Mix dry powder, DAPI mother solution with a concentration of 100 μg / ml, 2×SSC buffer, DAPI buffer, a dyeing cylinder and a variety control nucleus The probes in the Probe Mix dry powder include TAM fluorescently labeled DP-1, DP-7 and FAM fluorescently labeled DP-5, DP-8. The invention establishes a peanut probe staining technology and develops a probe staining kit, which is a new type of peanut banding analysis method. The method is simple, efficient and low-cost, and can be effectively used for peanut species identification and genome classification, etc., and lays the foundation for the next automated analysis and identification of peanut chromosomes.

Description

technical field [0001] The invention relates to the field of peanut cytology research, in particular to a peanut chromosome probe staining kit and a use method thereof. Background technique [0002] Plant chromosome engineering technology is the use of cytogenetic methods to artificially, purposefully and plannedly increase, decrease, replace, translocate homogeneous or heterogeneous chromosomes, or chromosome fragments, so as to create different chromosomes that meet people's needs. Structure of new plants. Chromosome engineering breeding plays an important role in the creation of plant germplasm resources, breeding improvement, and creation of genetic research materials. The construction of plant chromosome karyotype depends on the identification technology of a single chromosome. However, the backwardness of plant chromosome identification technology limits the in-depth study of plant chromosomes and the development of chromosome engineering technology. Therefore, it is...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6841
CPCC12Q1/6841C12Q2563/107
Inventor 杜培张新友亓增军崔彩红付留洋董文召汤丰收刘华
Owner HENAN ACAD OF AGRI SCI
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