Fluorescence in situ hybridization method for positioning 45S rDNA on plant chromosome

A fluorescence in situ hybridization and chromosome technology, applied in the fields of cytogenetics and molecular biology, can solve the problems of cumbersome steps, laborious experimental results, time-consuming, etc., and achieve the effects of simplified pretreatment steps, simple components, and simple methods.

Active Publication Date: 2013-11-27
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the development of fluorescence in situ hybridization, multicolor fluorescent labeling has become possible. Using multicolor fluorescent in situ hybridization technology, a variety of differently labeled probes can be positioned on chromosomes at the same time, and s

Method used

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  • Fluorescence in situ hybridization method for positioning 45S rDNA on plant chromosome
  • Fluorescence in situ hybridization method for positioning 45S rDNA on plant chromosome
  • Fluorescence in situ hybridization method for positioning 45S rDNA on plant chromosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] FISH Localization of 45S rDNA on Metaphase Chromosomes of Rye

[0054] 1. Chromosomal Specimen Preparation

[0055] (1) After the rye seeds germinate, the root tip grows to 0.5cm.

[0056] (2) Cut off the root tip and pretreat it with saturated p-dichlorobenzene aqueous solution for 5 hours at room temperature.

[0057] (3) The root tip was hypotonic with 0.075mol / L potassium chloride hypotonic solution for 30 minutes, and then fixed with 3:1 (methanol: glacial acetic acid) fixative for 20 minutes.

[0058] (4) Wash three times with distilled water, 5min each time, to fully wash away the fixative.

[0059] (5) Using 2.5% (w / w) pectinase and cellulase mixed in a weight ratio of 1:1, the root tip was dissociated at 37°C for 1 hour.

[0060] (6) Wash off the enzyme solution with distilled water, hypotonic with 0.075mol / L potassium chloride hypotonic solution for 30min, and fix with 3:1 methanol: glacial acetic acid fixative for 20min.

[0061](7) Prepare chromosome spe...

Embodiment 2

[0085] FISH Location of 45S rDNA on Metaphase Chromosome of Poplar

[0086] 1. Chromosomal Specimen Preparation

[0087] (1) Cut poplar branches and soak them in tap water, change the water every day until the root tip grows to 1cm.

[0088] (2) Cut off the root tip and pretreat it with saturated p-dichlorobenzene aqueous solution for 3 hours at room temperature.

[0089] (3) The root tip was hypotonic with 0.075mol / L potassium chloride hypotonic solution for 30 minutes, and then fixed with 3:1 (methanol: glacial acetic acid) for 40 minutes.

[0090] (4) The root tip was washed three times with distilled water, each time for 5 minutes, and the fixative was fully washed away.

[0091] (5) Using 2.5% (w / w) pectinase and cellulase mixed in a weight ratio of 1:1, the root tip was dissociated at 37°C for 1 hour.

[0092] (6) Wash off the enzyme solution with distilled water, hypotonic with 0.075mol / L potassium chloride hypotonic solution for 30min, and fix with 3:1 methanol: gla...

Embodiment 3

[0116] Embodiment 3: comparative embodiment

[0117] classic in situ hybridization

[0118] FISH localization steps of 45S rDNA on rye metaphase chromosome:

[0119] 1. Chromosomal Specimen Preparation

[0120] (1) After the rye seeds germinate, the root tip grows to 0.5cm.

[0121] (2) Cut off the root tip and pretreat it with saturated p-dichlorobenzene aqueous solution for 5 hours at room temperature.

[0122] (3) The root tip was hypotonic with 0.075mol / L potassium chloride hypotonic solution for 30 minutes, and then fixed with 3:1 (methanol: glacial acetic acid) for 20 minutes.

[0123] (4) The root tip was washed three times with distilled water, each time for 5 minutes, and the fixative was fully washed away.

[0124] (5) Using 2.5% (w / w) pectinase and cellulase mixed in a weight ratio of 1:1, the root tip was dissociated at 37°C for 1 hour.

[0125] (6) Wash off the enzyme solution with distilled water, hypotonic with 0.075mol / L potassium chloride hypotonic solu...

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Abstract

The invention discloses a fluorescence in situ hybridization method for positioning the 45S rDNA on a plant chromosome. The fluorescence in situ hybridization method mainly comprises the following steps of preparation of a chromosome specimen, preparation of 45S rDNA probes, fluorescence in situ hybridization of the chromosome and hybridization signal detection. The provided method for probe marking and hybridization is simple, convenient, quick and accurate, and mainly solves the problem about complex and cumbersome marking of the existing probes for fluorescence in situ hybridization. The probes provided by the invention are oligonucleotide probes which are not marked any more, and a fluorophore for modification can be added during synthesis of the probes, and compared with the prior art, the method is simple and the cost is low. By utilizing the technology, FISH positioning of the 45S rDNA on the chromosome can be completed within 3 hours.

Description

technical field [0001] The invention belongs to the technical field of cytogenetics and molecular biology, and relates to a simple and fast fluorescence in situ hybridization method for locating 45S rDNA on plant chromosomes. Background technique [0002] Fluorescence in situ hybridization is a well-developed biological research method at the molecular and cellular levels. The labeled probes can locate and characterize the target sequence by binding to targets such as chromosomes, interphase nuclei, or DNA fibers. and relative quantitative analysis. Ribosomal RNA gene (rDNA) is a tandem repeated multi-copy sequence, mainly including two parts of 18S-5.8S-25S rDNA (45S) and 5S rDNA, which are different transcription units, each forming a tandem repeat sequence and located at A specific location on a chromosome. 18S-5.8S-25SrDNA (45S) and 5S rDNA are widely distributed in plants, have highly repetitive and highly conserved characteristics, and can be used as an accurate mole...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 陈成彬王春国宋文芹
Owner NANKAI UNIV
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