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Detection method of vegetal single copy genes

A single-copy gene and detection method technology, which is applied in the field of detection of plant single-copy genes, can solve the problems of short DNA fragments, single-copy detection, difficulty of quantum dots entering the nucleus, etc., to improve sensitivity and resolution, and effectively detect , the effect of high fluorescence intensity

Inactive Publication Date: 2013-01-23
WUHAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

By searching the existing technology, there are two main methods for applying quantum dots to fluorescence in situ hybridization: one method is to use biotin-labeled probes for hybridization, and then use streptavidin-labeled quantum dots to detect (Nucleic Acids Res. 2004, 32, e28), the particle size of quantum dots marked by this method is too large, due to the existence of plant cell walls, chromosomes are highly concentrated, it is difficult for quantum dots to enter the nucleus, and cannot be applied to plant fluorescence in situ hybridization (J Nanobiotechnology, 2006, 4, 5); another method is to use quantum dot-labeled oligonucleotides, Chinese Patent Application No. 201010503053.7 "Detection method of symbiotic bacteria in sponge cells based on quantum dot fluorescence in situ hybridization ", using this method to achieve the detection of internal commensal bacteria
However, the DNA fragments coupled by this method are short (1-100bp), and can only detect repetitive sequences, not single-copy detection.

Method used

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  • Detection method of vegetal single copy genes
  • Detection method of vegetal single copy genes
  • Detection method of vegetal single copy genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1) Prepare maize interphase nuclei and metaphase chromosomes on glass slides;

[0025] 2) Synthesis of Octylamine-modified polyacrylic acid coated CdSe / ZnS quantum dots (Octylamine-modified polyacrylic acid coated CdSe / ZnS quantum dots, OPA-coated CdSe / ZnS QDs);

[0026] 3) According to the corn fatty aldehyde dehydrogenase gene, design and synthesize a pair of oligonucleotide primers, and connect the 5' end of one of the primers to 5'-NH 2 -(CH 2 ) 6 -TTTTTT, and through amino (-NH 2 ) and the carboxyl group (-COOH) on the quantum dots react chemically and couple to the surface of the quantum dots;

[0027] 4) Add the oligonucleotide primer coupled with quantum dots together with another primer and corn genomic DNA into the polymerase chain reaction system, and obtain a 480bp DNA molecule coupled with quantum dots by polymerase chain reaction ;

[0028] 5) Separate the product obtained by polymerase chain reaction by agarose gel electrophoresis to obtain a single ...

Embodiment 2

[0034] 1) Prepare maize chromatin fibers on a glass slide;

[0035] 2) Synthesis of Octylamine-modified polyacrylic acid coated CdSe / ZnS quantum dots (Octylamine-modified polyacrylic acid coated CdSe / ZnS quantum dots, OPA-coated CdSe / ZnS QDs);

[0036] 3) Design and synthesize a pair of oligonucleotide primers based on the receptor-like protein kinase 2 gene related to maize somatic embryogenesis, and connect the 5' end of one of the primers to 5'-NH 2 -(CH 2 ) 6 -TTTTTT, and through amino (-NH 2 ) and the carboxyl group (-COOH) on the quantum dots react chemically and couple to the surface of the quantum dots;

[0037] 4) Add the quantum dot-coupled oligonucleotide primer together with another primer and corn genomic DNA into the polymerase chain reaction system, and obtain the quantum dot-coupled 520bp DNA molecule through polymerase chain reaction ;

[0038] 5) Separate the product obtained by polymerase chain reaction by agarose gel electrophoresis to obtain a single ...

Embodiment 3

[0044] 1) Prepare parsley metaphase chromosomes on glass slides;

[0045] 2) Synthesis of Octylamine-modified polyacrylic acid coated CdSe / ZnS quantum dots (Octylamine-modified polyacrylic acid coated CdSe / ZnS quantum dots, OPA-coated CdSe / ZnS QDs);

[0046] 3) According to the chalcone synthase gene of parsley, design and synthesize a pair of oligonucleotide primers, and connect the 5' end of one of the primers to 5'-NH 2 -(CH 2 ) 6 -TTTTTT, and through amino (-NH 2 ) and the carboxyl group (-COOH) on the quantum dots react chemically and couple to the surface of the quantum dots;

[0047] 4) Add the oligonucleotide primer coupled with quantum dots together with another primer and parsley genomic DNA into the polymerase chain reaction system, and obtain the 490bp DNA coupled with quantum dots by polymerase chain reaction molecular;

[0048] 5) Separate the product obtained by polymerase chain reaction by agarose gel electrophoresis to obtain a single 490bp DNA molecule c...

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Abstract

The invention relates to a detection method of vegetal single copy genes based on fluorescence in situ hybridization of quantum dots. By preparing quantum dot coupled DNA (deoxyribonucleic acid) as a probe, according to the traditional fluorescence in situ hybridization method, the hybridization detection of vegetal single copy genes is carried out on interphase nucleuses, chromosomes, DNA fibersor the like. In the method provided by the invention, a direct marking method is adopted, thus an antibody detection process is omitted; and because the quantum dots have high fluorescence intensity and can resist photo-bleaching, the sensitivity and resolution of hybridization are improved, and the vegetal single copy genes can be quickly and effectively located. The method provided by the invention can be widely used for drawing physical maps and analyzing the composition, structure, rearrangement, evolutionary relationship and the like of genomes.

Description

technical field [0001] The invention relates to a method for detecting a plant single-copy gene, belonging to the field of molecular cytogenetics. technical background [0002] Fluorescence in situ hybridization (FISH) has been widely used in cytogenetics and genomics research. It can locate specific DNA sequences on interphase nuclei, chromosomes or DNA fibers, and analyze genome composition, structure, Rearrangement and evolutionary relationship, especially with the advent of the genome sequencing era, the drawing of physical maps based on fluorescence in situ hybridization will play an important supplementary and auxiliary role in genome sequencing and map-based cloning research. In the genome, in addition to containing a large number of repetitive sequences, there are also some single-copy sequences. Accurate detection and localization of single-copy sequences in interphase nuclei, chromosomes, or DNA fibers depends on the specificity of hybridization and the sensitivit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 李立家庞代文何世斌黄碧海
Owner WUHAN UNIV
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