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In-situ hybridization probe and method for identifying barley chromosome set by adopting same

A fluorescence in situ hybridization and chromosome technology, applied in the field of molecular cytogenetics, can solve the problems affecting the test process and effect, complex target sequence process, unsatisfactory labeling effect, etc., to achieve good detection effect, good sensitivity, and easy to distinguish Effect

Active Publication Date: 2017-05-10
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the process of amplifying target sequences by labeling is complex, time-consuming and laborious, and the effect of labeling is often unsatisfactory due to the influence of various factors
Especially when carrying out a large number of test operations, this method will greatly affect the test process and effect
At the same time, the method of carrying out chromosome in situ hybridization experiment by marking and amplifying the target sequence needs to denature the chromosome of the target material, which not only increases the test process, but also easily shifts the chromosome position on the glass slide during the processing

Method used

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  • In-situ hybridization probe and method for identifying barley chromosome set by adopting same
  • In-situ hybridization probe and method for identifying barley chromosome set by adopting same
  • In-situ hybridization probe and method for identifying barley chromosome set by adopting same

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1 Analysis of Barley Subtelomere Repeat Sequence and Determination of Oligonucleotide Probe Sequence

[0037] 1. Extraction of barley genomic DNA

[0038] Select the young leaves of Baudin and CN4027 to be tested, and use the column method (TaKaRa MiniBEST PlantDNA Extraction Kit) to extract DNA. The extraction steps are as follows:

[0039] a) Take 150 mg of fresh young leaves, grind them into fine powder with liquid nitrogen, quickly add 500 μl of Buffer HS I and 10 μl of 50×DTT Buffer and mix well, then add 10 μl of RNase A (10 mg / ml), shake and mix well, and then Incubate in a 56°C water bath for 10 minutes.

[0040] b) Add 62.5 μl of Buffer KAC and mix thoroughly. Place on ice for 5 minutes and centrifuge at 12,000 rpm for 5 minutes. Take the supernatant, add the same volume of BufferGB as the supernatant, and mix well.

[0041] c) Place the Spin Column in the Collection Tube, and move the solution to the Spin Column. Add 500 μl of Buffer WA to the Spi...

Embodiment 2

[0061] Example 2 Method of oligonucleotide probe Oligo-442A01 marking barley chromosome in ND-FISH method

[0062] 1. Chromosome preparation of barley root tip cells

[0063] Choose root length 1-2cm root tip after germination in N 2 Treated in O for 4h, inactivated by acetic acid, ddH 2 O wash, cut out the meristematic area, and enzymolyze it in the enzyme mixture (cellulase: pectinase = 1:1) at 37°C for 37 minutes, absorb the enzyme solution and use ddH 2 O and absolute ethanol were washed twice. Add 20 μl of acetic acid to each root tip and stir well until the cells appear in suspension, and drop 10 μl of the suspension on each slide. Examine the chromosomes under an optical microscope, select slides with clear and well-dispersed chromosomes, and mark them and store them in a refrigerator at 4°C for later use.

[0064] 2. Chromosome in situ hybridization

[0065] Take out the slides to dry naturally, mix the hybridization solution at 0.35μl 1OD·ml for each slide -1 Th...

Embodiment 3

[0067] Example 3 Oligonucleotide probe Oligo-442A01 and (AGG) 5 Establishment and application of barley genome identification method in ND-FISH method

[0068] Select the prepared glass slides in Example 2 to dry naturally, and mix the hybridization solution at 0.35 μl 1OD·ml for each slide -1Oligo-442A01, 0.35μl 1OD·ml -1 (AGG) 5 The ratio of hybridization solution to 9.30 μl was prepared, and the mixed hybridization solution was mixed repeatedly with a pipette gun, and 10 μl of the mixed hybridization solution was pipetted and dropped on a dry glass slide, and the coverslip was incubated in a humidified dark box at 37°C for 2 hours. After taking out, wash 2 times with 2×SSC under dark condition, ddH 2 O wash 1 time, 5min each time. After natural drying, 10 μl DAPI was added dropwise to each slide, and the coverslips were observed under the OLYMPUS BX63 fluorescence microscope for chromosomes and photographs were taken with the OLYMPUS DP80CCD camera. The above hybridiza...

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Abstract

The invention provides an in-situ hybridization probe and a method for identifying a barley chromosome set by adopting the in-situ hybridization probe. The in-situ hybridization probe Oligo-442A01 can be used for marking the barley chromosome, wherein the nucleotide sequence of the in-situ hybridization probe Oligo-442A01 is shown as SEQ ID NO.3, and the nucleotide is subjected to fluorescence labeling, so that the probe is constructed. A probe assembly composed of the probe and (AGG)5 can be used for effectively identifying the barley chromosome set under the condition that chromosome denaturation is not needed, and the method belongs to a good probe assembly method for identifying and distinguishing the barley chromosome set. The probe and the method provided by the invention can be used for accurately marking and identifying the barley chromosome set, have good effects for analyzing the morphology and the behavior of the barley chromosome, and have a good application prospect in the fields of barley molecular cytology and chromosome engineering breeding.

Description

technical field [0001] The present invention relates to the field of molecular cytogenetics, in particular to the determination and application of oligonucleotides in the process of barley chromosome in situ hybridization (FISH). Establishment of denatured fluorescence in situ hybridization (ND-FISH) reaction system and its application to identification of barley genome. Background technique [0002] Chromosomes in higher organisms have a complex structure, and there are a large number of repetitive sequences in different chromosome regions. Based on this characteristic, in the field of molecular cytogenetics, repetitive sequences are usually used to identify chromosomes. It prepares probes by fluorescently labeling repetitive sequences, and uses chromosome in situ hybridization to analyze the distribution of probe signals on chromosomes, thereby distinguishing the genome from the chromosome group. In addition, it is a common practice to analyze the behavior of chromosomes ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6841C12Q1/6895C12Q2600/13C12Q2563/107C12Q2565/601
Inventor 陈光登胡德益李廷轩张锡洲王永东郑子成余海英康亮珠
Owner SICHUAN AGRI UNIV
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