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Probe group for detecting avena plant chromosome, and kit and application

A chromosome and probe group technology, applied in the application field of fluorescent in situ hybridization probes, can solve the problems of indistinguishable genetic relationship between chromosomes, difficult recycling of slices, and affecting the strength of hybridization signals, etc. Easy to slice, easy to prepare, and easy to operate

Inactive Publication Date: 2018-01-12
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

GISH, the genome in situ hybridization technique, uses the total genome sequence as a probe to mark chromosomes. Probe preparation requires a large amount of high-concentration genomic DNA. The limitation is that genome chromosomes cannot be distinguished when they are closely related. In addition, the blocking ratio will affect the hybridization signal. Strong or weak, it is difficult to recycle the film, the repeatability of the experiment is not strong, the picture is in color

Method used

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  • Probe group for detecting avena plant chromosome, and kit and application
  • Probe group for detecting avena plant chromosome, and kit and application
  • Probe group for detecting avena plant chromosome, and kit and application

Examples

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Effect test

preparation example Construction

[0060] 1) Preparation of chromosome slide specimens of plants of the genus Avena: Soak the seeds of plants of the genus Avena in double-distilled water, place them in an environment at 4°C for 24 hours, absorb excess water, and culture them at a constant temperature of 22°C until the root tip grows to 1.5 to 2.0 cm, cut 1 to 1.5 cm of root tip tissue, followed by N 2 O treatment for 5 hours, glacial acetic acid treatment for 5 minutes, and then stored in 75% alcohol at -20°C;

[0061] Wash the root tip tissue preserved at low temperature with double distilled water, cut out the root tip meristem 1-2mm, put it in the enzymatic hydrolysis solution at 37°C for 45 minutes, remove the enzymatic solution, wash with double distilled water, 75% alcohol in turn , 95% alcohol, and 100% alcohol to clean the root tip meristem and then dry it at room temperature; add glacial acetic acid to the root tip tissue to make a suspension; drop the suspension on a glass slide and dry it at room tem...

Embodiment 1

[0068] This embodiment adopts (ACT) in the kit 6 The probes were used to mark six species of Avena diploid plants (AA genome): Avena wiestii, Avena longiglumis, Avena brevis, Avena nuda, Avena strigosa, Avenacanariensis. (ACT) 6 The base sequence of the probe is: 5'-ACTACTACTACTACTACT-3', the 5' end of which is labeled with FAM, showing a green signal, and the amount of the probe is 0.5 μL. The hybridization signals of the above-mentioned 6 diploid plants of the genus Avena can be clearly observed through a fluorescence microscope. In the distribution images of their signal sites, except for the four signals of Avena brevis, the rest are two signals, as shown in figure 1 As shown, the scale bar in the figure is 5 μm, and the arrow points to (ACT) 6 hybridization signal.

Embodiment 2

[0070] This embodiment adopts (ACT) in the kit 6 The probes were used to label three tetraploid plants of the genus Avena (AABB genome): Avena barbata, Avena abyssinica, Avena vaviloviana. (ACT) 6 The base sequence of the probe is: 5'-ACTACTACTACTACTACT-3', the 5' end of which is labeled with FAM, showing a green signal, and the amount of the probe is 0.5 μL. The hybridization signals of the above three tetraploid plants of the genus Avena can be clearly observed by fluorescence microscopy, and each species contains four signals, such as figure 2 As shown, the scale bar in the figure is 5 μm, and the arrow points to (ACT) 6 hybridization signal.

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Abstract

The invention provides a probe group for detecting an avena plant chromosome and a kit and an application. The probe group comprises four probes of Aml, (ACT)6, (GAA)6 and (TTG)6, wherein the base sequence of Aml is 5'-GATCCATGTGTGGTTTGTGGAAAGAACAC ACATGCAATGACT CTAGTGGTT-3'; the base sequence of (ACT)6 is 5'-ACTACTACTACTACTACT-3'; the base sequence of (GAA)6 is 5'-GAAGAAGAAGAAGAAGAA-3'; and the base sequence of (TTG)6 is 5'-TTGTTGTTGTTGTTGTTG-3'. The probe group consists of 18-51bp oligo-sequence probes for recognizing avena plant chromosomes, and is beneficial to establishment of stable andconvenient fluorescent in-situ hybridization reaction systems.

Description

technical field [0001] The invention belongs to the application field of fluorescent in situ hybridization probes, and in particular relates to a probe group, a kit and an application for detecting chromosomes of plants of the genus Avena. Background technique [0002] Existing methods for detecting chromosomes of Avena species include C-banding, GISH, FISH, and the like. C-band can color heterochromatin, which is usually located around the centromere and often contains DNA with highly repetitive sequences. The picture observed by this method is black and white, and the signal position is limited to around the centromere. GISH, the genome in situ hybridization technique, uses the total genome sequence as a probe to mark chromosomes. Probe preparation requires a large amount of high-concentration genomic DNA. The limitation is that genome chromosomes cannot be distinguished when they are closely related. In addition, the blocking ratio will affect the hybridization signal. S...

Claims

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Application Information

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IPC IPC(8): C12Q1/6841C12N15/11
Inventor 罗小梅周永红陈亮万文林刘俊成
Owner SICHUAN AGRI UNIV
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