Chromosome localization method of scallop (Chlamys farreri)

A technology for chromosome positioning and scalloping scallops, applied in the field of molecular cytogenetics, can solve the problems of unstable gene positioning effect, difficult to maintain reaction conditions constantly, etc., achieve clear and stable hybridization signal, improve accuracy and specificity. Effect

Inactive Publication Date: 2008-12-17
OCEAN UNIV OF CHINA
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Problems solved by technology

[0003] The purpose of the present invention is to overcome the shortcoming of prior art, provide a kind of chromosome localization method of efficient Chlamys farreri gene, solve when tempera...

Method used

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Embodiment Construction

[0018] Take the chromosomal location of Chlamys farreri ribosomal rRNA gene as example below and describe the inventive method in detail by specific embodiment:

[0019] (1) Chlamys farreri chromosome preparation on glass slides by air-drying method

[0020] First, the trochophore larvae of Chlamys farreri were soaked in 0.01% colchicine at room temperature for 2 hours, then soaked in 0.075M potassium chloride solution for 30 minutes, and then washed with freshly prepared Carnot's fixative (ethanol: glacial acetic acid). Volume ratio = 3:1) fixed 3 times, 15 minutes each time. After soaking in 50% acetic acid solution, the fixed larvae were dissociated into a cell suspension, which was then dropped onto a glass slide at 60°C and air-dried at 60°C.

[0021] (2) According to the rRNA (18S-5.8S-28S) gene sequence to be mapped, online primer design and primer synthesis

[0022] Four primers (BIP, FIP, B3 and F3) were designed with PrimerExplorer software (http: / / primerexplorer.j...

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Abstract

The invention relates to a high-efficiency chromosomal mapping method for chlamys farreri genes. First, the preparation of chlamys farreri chromosome is carried out on a glass slide by the method of air-drying; then online primer design and primer synthesis are carried out according to the sequence of the gene to be mapped; the chromosomal mapping of rRNA gene is carried out; the glass slide supporting the chromosome is treated by utilizing a ligase; then formamide denaturalization treatment is carried out to the chromosome; LAMP reaction mixture is added and LAMP reaction in situ is carried out; after the reaction is finished, the glass slide is cleaned and added with a biotin antibody marked by fluorescence for incubation; finally, a propidium iodide anti-fading solution is used for redyeing the chromosome; a stable and clear hybridization signal is used for determining the position of a target gene on the chromosome. The method of the invention causes chlamys farreri gene to fast obtain a plurality of amplification products; the hybridization signal is clear and stable, thus promoting the correctness and specificity of chromosomal mapping. As for the mapping of a single-copy or low-copy gene, the method also has very great application potential.

Description

Technical field: [0001] The invention relates to a chromosomal gene positioning method—a high-efficiency scallop gene chromosome positioning method, which belongs to the technical field of molecular cytogenetics. Background technique: [0002] Fluorescence in situ hybridization (FISH), primer in situ extension (PRINS) and in situ PCR are commonly used techniques for chromosomal gene localization. At present, the application of FISH and PRINS technology is mainly limited to the location of highly repetitive genes. Compared with FISH and PRINS techniques, in situ PCR technique has obvious advantages in locating single or low-copy genes. However, this technology has not been promoted in practice, mainly because when the temperature and pressure change, it is difficult to constantly maintain the reaction conditions on the surface of the slide where the chromosomes are located, resulting in unstable gene positioning. Loop-mediated constant temperature amplification (LAMP) techn...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/53
Inventor 黄晓婷胡晓丽王师张玲玲孟庆磊王珊包振民胡景杰
Owner OCEAN UNIV OF CHINA
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