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Method for applying adhesive tail end joints to flanking sequence separation

A technology of flanking sequences and sticky ends, applied in the field of sticky end adapters based on isotailase design, can solve the problems of high cost, incompatibility of connection efficiency and versatility

Inactive Publication Date: 2012-08-15
HUNAN HYBRID RICE RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The technical problem to be solved by the present invention is to overcome the incompatibility of connection efficiency and versatility and high cost in the prior art, and provide a sticky end adapter with high connection efficiency, high versatility and low cost for flanking sequence separation Methods

Method used

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  • Method for applying adhesive tail end joints to flanking sequence separation
  • Method for applying adhesive tail end joints to flanking sequence separation
  • Method for applying adhesive tail end joints to flanking sequence separation

Examples

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Embodiment 1

[0055] Example 1: Sticky end adapters are applied to the isolation of flanking sequences of rice pCK vector transgenes to obtain the insertion sites of foreign genes in the rice genome

[0056] The pCK transgene vector has two T-DNA regions, one of which contains the cDNA sequences of two C4 photosynthase genes (PEPC / PPDK), and each target gene is composed of rice Cab (Chlorophyll a / b binding protein, capture chlorophyll a / b-binding protein) promoter and terminated by 35s and NOS terminators, respectively. A T-DNA region contains the hygromycin phospho-transferase (HPT) gene as a selectable marker gene. After the rice was transformed, the transgenic lines without the HPT gene were selected for multigenerational propagation, and the transgene insertion site was analyzed after the homozygosity was stabilized. Proceed as follows:

[0057] 1. Extract the genomic DNA of transgenic rice;

[0058] 2. Analyze the distribution of restriction sites of the pCK vector, select BamHI...

Embodiment 2

[0073] Example 2: Sticky end joints applied to wheat PEBP Isolation of gene flanking sequences to obtain the promoter sequence of the gene

[0074] PEBP The gene encodes phosphatidylethanolamine-binding protein (Phsphatidylethanolamine-binding protein, PEBP), which is a cold-regulated protein that can change the fluidity of the plasma membrane of the cell and is also closely related to ion channels. The currently published wheat genome sequencing map is still in the form of a draft, and the complete wheat genome sequencing map will be completed in the next few years. This draft does not yet provide direct and effective sequences for most genomic studies of wheat. Therefore, we cannot base wheat's PEBP The gene sequence directly finds the sequence of the upstream promoter, and its promoter sequence can only be obtained through the technology of flanking sequence separation.

[0075] This embodiment includes the following steps:

[0076] 1. Extract the genomic DNA of whe...

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Abstract

A method for applying adhesive tail end joints to flanking sequence separation includes following steps: 1) extracting a genome DNA (deoxyribonucleic acid) to be detected; 2) selecting isocaudarner and designing and synthesizing corresponding joints; 3) digesting the genome by the isocaudarner to form DNA segments with identical adhesive tail ends; 4) detecting digestion quality in an electrophoresis manner, quantifying the concentration of the DNA segments, connecting the joints with digestion products of the genome, and forming each DNA segment with 'the joint, the digestion segment and the other joint' sequentially; 5) purifying the DNA segments with 'the joints, the digestion segments and the other joints' sequentially, and performing PCR (polymerase chain reaction); 6) recycling the segments obtained from the step 3) and sequencing; 7) analyzing a sequencing result and obtaining an unknown flanking sequence of a known sequence; and 8) checking the unknown flanking sequence. The method for flanking sequence separation is high in connection efficiency and generality and low in cost.

Description

technical field [0001] The present invention relates to a method for separating flank sequences, in particular to a method for applying a sticky end linker designed based on isotailase to the separation of flank sequences. Background technique [0002] The methods currently applied to the isolation of flanking sequences mainly include SON-PCR, TAIL-PCR and linker-PCR. SON-PCR and TAIL-PCR are PCR methods invented based on the principle of thermal asymmetry. Due to the randomness of the second strand synthesis and the fact that most transgenic lines are not single-copy, these two methods cause confusion in primer matching, resulting in useful Fragments are not efficient. The linker-PCR method can greatly improve the acquisition of effective fragments. Three methods for obtaining flanking sequences by linker-PCR have been published, but there are some shortcomings. PCR amplification in the later stage; although some methods use sticky end adapters to improve the ligation eff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 袁定阳段美娟余东谭炎宁孙志忠袁光杰袁贵龙孙学武刘瑞芬赵炳然袁隆平
Owner HUNAN HYBRID RICE RES CENT
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