Fluorescence in situ hybridization method of prunus mume chromosome

A fluorescence in situ hybridization and chromosome technology, which is applied in the field of fluorescence in situ hybridization of plum blossom chromosomes, can solve problems such as the application of FISH technology that has not been reported, and achieve the effect of improving the success rate of hybridization and high hybridization specificity

Active Publication Date: 2012-11-21
BEIJING FORESTRY UNIVERSITY
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  • Description
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Problems solved by technology

FISH technology has been successfully used in humans, animals and a few plants. So far, there has been no report on the application of FISH technology in plum blossoms.

Method used

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  • Fluorescence in situ hybridization method of prunus mume chromosome
  • Fluorescence in situ hybridization method of prunus mume chromosome
  • Fluorescence in situ hybridization method of prunus mume chromosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] 1. Preparation of chromosome slide specimens

[0058] The stem tip of plum blossom 'Longyou' (Prunus mume 'Longyou') germinated in spring is used as the material, and its length is preferably 0.5-1cm. Chromosomal slide specimens were prepared by the wall-removing hypotonic flame-drying method, followed by Carnot’s fixative at -20°C for more than 24 hours, 0.075mol / L KCl solution before hypotonic treatment for 30 minutes, and 2.5% cellulase and pectinase enzymes Solution 110min, ddH 2 After O, hypotonic treatment was performed for 30 minutes. Perform microscopic examination under an optical microscope, and select metaphase cleavage specimens with clear images and a high degree of dispersion and store them in a -20°C refrigerator for later use.

[0059] 2. Pretreatment of chromosome slide specimens

[0060] 1) Operate in groups of 4 slides, place the chromosome slide specimen in an oven at 65°C for 1 hour, add 100 μl of 0.1 mg / ml RNaseA solution on the chromosome slide...

Embodiment 2

[0080] The difference from Example 1 is that the plum blossom variety is 'Xiaogongfen'. The steps and signal observation are the same as in Example 1.

[0081] Analysis of results: from Figure 4 and Figure 8 It can be seen that there are 6 hybridization signals when the Dig-labeled 45SrDNA probe hybridizes with the Meihua chromosome preparation, and the signal points are located on the 1st, 3rd, and 7th pairs of homologous chromosomes. Among them, the signal of homologous chromosome 1 was the strongest, the signal of homologous chromosome 7 was the weakest, and the signal intensity of homologous chromosome 3 was in the middle, with slight differences among different varieties. The karyotype formula is 2n=2x=16=10m(2SAT)+6sm. The signal points are all located at the end of the short arm of the chromosome, which can well distinguish non-homologous chromosomes with similar shapes, assist karyotype analysis to a large extent, and improve the accuracy of karyotype.

[0082] P...

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Abstract

The invention relates to a fluorescence in situ hybridization method of a prunus mume chromosome. The method consists of: taking a prunus mume metaphase chromosome as the target DNA, adopting maize 45SrDNA as a probe, using a nick translation method to label the probe, and positioning the maize 45SrDNA probe on the prunus mume chromosome clearly. Based on the prior art, the invention improves the co-denaturation method, and employs the two steps of probe denaturation, and chromosome and probe co-denaturation to complete the denaturation process, thus greatly improving the hybridization success rate. The invention determines an appropriate denaturation temperature and denaturation time, a hybridization time and an elution method, and has high hybridization specificity, so that the FISH technology can be well applied in chromosome detection of prunus mume and other small chromosome plants, thus providing a new approach and method for in-depth study of the prunus mume chromosome.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a fluorescence in situ hybridization method for plum blossom chromosomes. Background technique [0002] Fluorescence in situ hybridization (Fluorescence in situ hybridization) is an important in situ hybridization method developed in the late 1980s. It is a new molecular cytogenetics technique formed by replacing radioactive isotope labels with non-radioactive fluorescent labels. At present, it is one of the most direct methods for constructing a cytogenetic map, the most direct method for studying the specific position of a DNA sequence on a chromosome, and has become an effective means for studying the association between a specific DNA sequence and a chromosomal region. This technology has been widely used in many scientific research fields, such as DNA physical mapping, chromosome mapping, construction of cytogenetic mapping, cytological identification of transgenes, detec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 张启翔陈晶鑫杨冰洁罗乐杨炜茹
Owner BEIJING FORESTRY UNIVERSITY
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