Specific sub sequence for separating Ac/Ds flanking sequence and separation method thereof

A technology of linker sequence and flanking sequence, applied in the field of specific linker sequence for separating Ac/Ds flanking sequence and separation thereof

Inactive Publication Date: 2014-04-30
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The latter method is a relatively high-efficiency method, but due to the limitation of the number of high-efficiency endonucleases used, there are still some transposable lines that cannot obtain the flanking sequence of Ac / Ds by this method

Method used

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  • Specific sub sequence for separating Ac/Ds flanking sequence and separation method thereof
  • Specific sub sequence for separating Ac/Ds flanking sequence and separation method thereof
  • Specific sub sequence for separating Ac/Ds flanking sequence and separation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1. Acquisition of linkers: According to the sequence of the blunt-end linkers of Clontech's genomewalking kit, the present invention designs linker sequences suitable for producing 5' protruding ends, wherein linker 1 is quoted from the instructions of Clontech's genomewalking kit, which is suitable for different Linker 2 of the restriction endonuclease was designed by us based on the sequence of linker 1.

[0029]

[0030] Dissolve the adapter 1 and adapter 2 synthesized by the company with sterile water to a final concentration of 200 μM, and prepare the adapter solution in a 0.5ml centrifuge tube according to the following formula:

[0031] Adapter 1 (200μM) 10μl

[0032]Adapter 2 (200μM) 10μl

[0033] 60mM Tris-HCl pH7.5, 150mM NaCl 10μl

[0034] The prepared solution was incubated in a 94°C water bath for 2 minutes, and then naturally cooled to room temperature in a water bath to anneal the two single-stranded DNAs to form a partially double-stranded linker. ...

Embodiment 2

[0045] Example 2: Separation of specific bands co-segregated with mutant phenotypes

[0046] In the isolated population of the corn kernel mutant shu00067, wild-type homozygous plants (+ / +) and wild-type heterozygous plants (dek / +) were selected to extract DNA, and the genomic DNA was digested with SalI, using the method used in Example 2 Ac / Ds flanking sequences were isolated and a band was identified that co-segregated with the dek mutant phenotype ( image 3 ). Primers were designed by sequencing the flanking sequences of this PCR band, and the bands were only amplified in mutant grains (- / -) and wild-type heterozygous (+ / -) with Ac-terminal-specific primers and flanking sequence-specific primers , while no corresponding product was amplified in wild-type control W22 and wild-type homozygous plants ( Figure 4 ), the results indicated that the assigned flanking sequences were genuine and co-segregated with the dek mutant phenotype.

[0047] Shanghai University

[0048]...

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Abstract

The invention relates to a specific sub sequence for separating an Ac/Ds transposon flanking sequence in a corn Ac/Ds transposition mutant and a separation method thereof. The sub sequence is base sequences shown in SEQ ID NO:1 to SEQ ID NO:4. The Ac/Ds flanking sequence in a genome can be well amplified and obtained by using an Ac specific primer and a specific primer of the sub sequence after these sub sequences are connected with corresponding Ac/Ds transposon deoxyribonucleic acid (DNA) subjected to restrictive incision enzyme digestion. The detection method is simple to operate, and important technical means is provided for large-scale obtaining of the Ac flanking sequence of the Ac mutant and separation of the gene of an Ac insertion mutant.

Description

technical field [0001] The invention relates to a specific linker sequence and a separation method for separating the Ac / Ds transposon flanking sequence in a maize Ac / Ds transposition mutant. Background technique [0002] Corn is the world's largest food crop and the main food source in the world. And maize has long been a genetic model plant. The sequence of the maize B73 genome has been completed, which will accelerate scientific research in maize. At least 32,000 genes are estimated to exist in maize, and the cloning and functional analysis of these genes has become the next major challenge. Among the methods of gene cloning, insertion mutation research is a very effective method. It mainly includes T-DNA insertion mutation and transposon insertion mutation. Due to the limitations of transgenic technology, it is difficult to apply T-DNA insertion method for insertion mutation analysis in maize. However, the existence of endogenous transposons in maize is a good tool ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10
Inventor 王飞赵志伟郭圣明徐大彬
Owner SHANGHAI UNIV
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