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FISH method of one piece and multiple target of cotton

A multi-target, cotton technology, applied in the field of molecular cytogenetics, can solve the problem that the probe and the target DNA have no homologous sequence and are difficult to tell

Inactive Publication Date: 2010-09-01
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this way, when this fixed probe hybridizes on the chromosome of a certain species and its close relatives, once no signal is generated after FISH detection, it is difficult to define whether it is due to human manipulation or there is indeed no signal between the probe and the target DNA. Homologous sequences are hard to tell

Method used

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  • FISH method of one piece and multiple target of cotton
  • FISH method of one piece and multiple target of cotton
  • FISH method of one piece and multiple target of cotton

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 FISH experiment of a piece of cotton with multiple targets

[0020] Experimental materials: diploid cotton species Raymond cotton, Thurber cotton, Davidson cotton and tetraploid cotton species sea island cotton, yellow brown cotton and Darwin cotton were all from the Cotton Research Institute of the Chinese Academy of Agricultural Sciences.

[0021] Experimental reagents: cellulase Onazuka R-10 and pectinase Pectolyase Y-23 were purchased from Solarbio; probe labeling kits Bio-Nick Translation Mix and DIG-Nick Translation Mix were purchased from Roche.

[0022] 1. Cotton genomic DNA extraction

[0023]Preheat nuclear lysis buffer at 60°C (add 2% β-mercaptoethanol v / v before the experiment), take about 2g of young leaves, put them into a frozen mortar immediately, add liquid nitrogen to the mortar and quickly grind them into powder . Quickly put it into a 7mL centrifuge tube, add about 3mL of extraction buffer preheated in a 60°C water bath, and shake well. ...

Embodiment 2

[0038] The preparation used in Example 1 was hybridized with the 428k20 sequence and the 150D24 sequence as a double-purpose probe, and the hybridization result obtained was as follows figure 2 Shown: The red signal of the 150D24 sequence appears in all chromosomes of Raymond cotton ( figure 2 -B) and the centromere region of the smaller chromosome (subgroup D) of sea-island cotton, because it overlaps with the green signal produced by the 428k20 sequence in the centromere region of the smaller chromosome (subgroup D) of sea-island cotton, so sea-island cotton is relatively Centromeric regions of small chromosomes (subgroup D) are seen as synthetic signals in yellow ( figure 2 -A), it can also be confirmed that the 150D24 sequence is from its donor species Raymond cotton, but it is inconsistent with the 428k20 sequence.

Embodiment 3

[0040] The yellow-brown cotton and Darwin cotton are both tetraploid cotton species, both of which have 52 chromosomes, and the recognition probe 45S is added. There are 3 pairs of 45S rDNA loci in the yellow-brown cotton, one of which is very large and almost covers the entire cotton. The short arm of the chromosome, while the other two pairs are very small; the three pairs of 45S rDNA loci in Darwin cotton are basically identical. The target probes are diploid D group Thurber's genome and Davidson's cotton genome respectively, and salmon sperm DNA is used as blocking, and the intensity of hybridization signal is used to compare the Thurber's cotton and Darwin's cotton respectively. What is the ratio of cotton gDNA to Davidson cotton gDNA.

[0041] The mitotic metaphase chromosomes of the yellow-brown cotton and Darwin cotton produced in the same slice were probed with Thurber's cotton gDNA, and the hybridization results were as follows: image 3 Shown: From the recognition ...

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Abstract

The invention belongs to the field of cytogenetics, specifically relating to a FISH method of the one piece and multiple target of the cotton, which comprises the following steps of: 1) putting chromosome samples of different cotton types on one carry sheet glass to produce a piece in a mixing way; and 2) simultaneously performing fluorescence in situ hybridization with the same probe, selecting and photographing the division phases of the chromosomes of the different cotton types under the same visual field when acquiring a fluorescence in situ hybridization result image. The method guarantees the consistency of external environment (the hybridizing or eluting temperature, humidity and time, and the content of each ion in solution, etc.), a manual operation step and the like, in the hybridization step of the target chromosomes of the different cotton types, and avoids the experimental error caused by the manual operation or the external condition when respectively performing the fluorescence in situ hybridization, therefore, the FISH experimental result obtained by the method is relatively true.

Description

technical field [0001] The invention belongs to the field of molecular cytogenetics, in particular, the invention relates to a multi-target FISH method for a piece of cotton. Background technique [0002] Chromosomal in situ hybridization technology is based on the principle of complementary base pairing of nucleic acid molecules. The radioactive or non-radioactively labeled exogenous nucleic acid probes are paired with the denatured single-stranded DNA on the chromosome, and then undergo a series of detection methods. A technology that displays the position of the nucleic acid sequence to be tested on the chromosome. In the initial stage of chromosome in situ hybridization technology, radioactive isotope-labeled probes were used, but with the emergence of fluorescein substances, fluorescein was used to detect hybridization signals, and the hybridization results were observed under a fluorescent microscope, that is, fluorescence in situ hybridization (FISH). in situ hybridi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 杨晓芳王坤波刘方王春英黎绍惠张香娣王玉红
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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