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Preparation method and application of Cucumis sativus. L chromosome-6 probe

A chromosome and single-chromosome technology, applied in the field of plant biology, to achieve the effect of avoiding cumbersomeness and interference of repetitive sequences

Inactive Publication Date: 2015-05-06
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0008] The present invention aims at the cumbersomeness and insufficiency of previous methods of microseparating single chromosomes or preparing single chromosome probes based on BAC libraries, and invents a method for preparing cucumber single chromosome probes through genome-specific sequences, which provides a new method for the preparation of chromosome probes The method accelerates the development and application of chromosome probes

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  • Preparation method and application of Cucumis sativus. L chromosome-6 probe
  • Preparation method and application of Cucumis sativus. L chromosome-6 probe
  • Preparation method and application of Cucumis sativus. L chromosome-6 probe

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Embodiment Construction

[0023] (1) Partial sequences were selected from the cucumber genome sequence at intervals of ~100kb, and repeated sequences were compared using RepeatMasker software (http: / / www.repeatmasker.org) and BLAST analysis, and sequences without repeated sequences were screened out.

[0024] (2) Use Primer Premier 5.0 software to design primers. The primer length is set to 20 bp, the Tm value is set to 55-65° C., the GC ratio is set to 40-60%, and the PCR product length is set to 2500-4000 bp. Primers with a Rating value above 75 and without hairpin structure, dimer structure, mismatch and dimer structure crossover were selected for repeat sequence alignment and BLAST analysis, and ideal primers without repeat sequences were screened out.

[0025] (3) Using cucumber genomic DNA as a template, amplify according to the PCR system. PCR reaction system:

[0026] 5×buffer: 4ul

[0027] dNTPs: 1.6ul

[0028] DNA: 1.2ul

[0029] Prime Star: 0.6ul

[0030] dd water: 12ul

[0031] Sense ...

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Abstract

The invention discloses a preparation method and application of a Cucumis sativus. L chromosome-6 probe. According to the method, on the basis of a Cucumis sativus. L genomic sequence, a DNA sequence containing no repetitive sequence is selected through RepeatMasker and Cucumis sativus. L genome database BLAST analysis, and primer design is performed through Primer Premier 5.0. A PCT product covering single chromosomes is obtained through PCR amplification, the PCT product is purified in agarose gel electrophoresis, the concentration is measured with a nucleic acid concentration measurement instrument, then equal amounts of products of all pairs of primers are mixed, and a probe is marked by a nick translation method and is hybridized to a chromosome flake by a FISH (Fluorescence In Situ Hybridization) technology so as to complete the preparation, detection and application of the single-chromosome probe. Through the application of the method, Cucumis sativus. L single chromosomes and a probe of any chromosome region can be obtained quickly, and precise analysis and research on a single chromosome and the morphological structure of any chromosome can be performed.

Description

1. Technical field [0001] The invention discloses a method for preparing a No. 6 chromosome probe by using the cucumber genome sequence, which is beneficial to promoting the application of the cucumber single-chromosome probe in research, and belongs to the field of plant biotechnology. 2. Technical Background [0002] Cucumber (Cucumis sativus L., 2n=2x=14) is one of the most important vegetable crops in the world. Cucumber has a small number of chromosomes, only 14, making it one of the model species for molecular cytogenetic research in recent years. At the same time, research institutions in China and the United States completed the sequencing of the cucumber genome in 2009. The sequencing results showed that the cucumber genome is relatively small, about 370Mb, and the sequencing results have been continuously revised and improved. The current sequencing data is quite accurate. It can be directly used for reference. [0003] Studies related to cytogenetics require the...

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12N15/10
Inventor 娄群峰张振涛张云霞李子昂陈劲枫
Owner NANJING AGRICULTURAL UNIVERSITY
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