T vector mediated method for testing 3' end flanking unknown sequence

A technology of unknown sequence and carrier, applied in the field of bioengineering, can solve the problems of not being able to determine the sequence of the upstream and downstream regulatory elements of the gene, not being able to study the structure and function in depth, and consuming a lot of manpower and material resources, so as to save manpower, operate with fewer restrictions, low cost effect

Inactive Publication Date: 2011-08-03
JIANGNAN UNIV
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  • Application Information

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Problems solved by technology

However, many of the new genes reported now have only obtained partial DNA sequences. When the complete DNA sequence of a gene is not known, it is impossible to study its structure and function in depth. Therefore, it is necessary to obtain the complete sequence from its partially known DNA sequence
The random sequencing method is to obtain the required DNA sequence through a large number of random DNA sequences and sorting by computer. However, this method has a lot of chance and usually consumes a lot of manpower and material resources.
The 3'- and 5'-RACE (3'and 5'Rapid Amplification of cDNA Ends) method is to obtain the complete mRNA sequence of the target gene when the partial mRNA sequence is known, but the sequence of the upstream and downstream regulatory elements of the gene cannot be determined
In addition, the RACE method is cumbersome, the mRNA is extremely unstable, and reverse transcription is particularly susceptible to low abundance and high-level structures, so the effect is not very ideal
The method of using isotope or biotin-labeled probes to hybridize and screen genomic libraries is time-consuming and labor-intensive

Method used

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  • T vector mediated method for testing 3' end flanking unknown sequence
  • T vector mediated method for testing 3' end flanking unknown sequence
  • T vector mediated method for testing 3' end flanking unknown sequence

Examples

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Effect test

Embodiment 1

[0028] Determination of the 3' flanking sequence of Aspergillus usamii E001 cel5A.

[0029] (1) Preparation of DNA double-enzyme digestion products: PstⅠ and BglII were used to perform double-digestion of Aspergillus usami genomic DNA. The following double enzyme digestion system was constructed: 10×H Buffer 2 μl, PstⅠ 1 μl, BglⅡ 1 μl, genomic DNA 10 μl, sterile water 6 μl; react the system in a 37°C water bath for 4 hours. Purify and recover the double-digested product and dissolve it in 20 μl sterile water.

[0030] (2) Complementation of digested products and addition of A at the 3′ end: 20 μl of double digested products, 2.5 μl of 10×Ex Taq Buffer, 0.5 μl of dNTP, 0.25 μl of Ex Taq enzyme, 1.75 μl of sterile water; 72°C reaction 10min. The target product was named cel5A3UT.

[0031] (3) Ligation of cel5A3UT and pUCm-T: 1 μl of 50% PEG4000, 1 μl of 10×T4 DNA Ligase Buffer, 1 μl of pUCm-T, 1 μl of T4 DNA Ligase, 6 μl of cel5A3UT; overnight at 16°C. The target product was...

Embodiment 2

[0036] Determination of the 3' flanking sequence of Aspergillus usamii E001 cell2A.

[0037] (1) Preparation of DNA digestion products: SalI and BamHI were used to perform double enzyme digestion on the genomic DNA of Aspergillus usami. The following enzyme digestion system was constructed: 3 μl of 10×T Buffer, 1 μl of SalⅠ, 1 μl of BamHI, 10 μl of genomic DNA, and 5 μl of sterile water; the system was reacted in a water bath at 37°C for 4 hours. Purify and recover the double-digested product and dissolve it in 20 μl sterile water.

[0038] (2) Completion of digested products and addition of A at the 3′ end: 20 μl of double digested products, 2.5 μl of 10×PCR Buffer, 0.5 μl of dNTP, 0.25 μl of Taq enzyme, 1.75 μl of sterile water; react at 72°C for 10 minutes. The target product was named cel12A3UT.

[0039] (3) Ligation of cel12A3UT and pUCm-T: 1 μl of 50% PEG4000, 1 μl of 10×T4 DNA Ligase Buffer, 1 μl of pUCm-T, 1 μl of T4 DNA Ligase, 6 μl of cel12A3UT; overnight at 16°C. ...

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Abstract

The invention discloses a method for determining a 3' end flanking unknown sequence based on the known DNA sequence by utilizing technologies such as restriction endonuclease cutting, pUCm-T vector, nested polymerase chain reaction (PCR) amplification and the like. In the method, genome DNA is cut by restriction endonuclease, the purified enzyme-cutting product is complemented by Taq enzyme or Ex Taq enzyme, is subjected to 3' end A addition reaction, and is connected with the pUCm-T vector; a segment of sequence on the downstream of a T/A cloning site on the T vector is selected as a 3' end primer through primer design software; and two sequences of the known DNA close to the unknown sequence end are selected as 5' end primers. A pUCm-T vector junctional complex is taken as a template, the nested PCR amplification is carried out through the designed primers, and the amplified segments are subjected to cloning and sequencing. The method has the characteristics of simple operation, wide application range, a few operational constraints, simple and quick result verification, unlimited length of unknown sequence, low cost and the like.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and is a technique based on partially known DNA ( Deoxyribonucleic Acid) sequence to determine its 3' flanking unknown DNA sequence method. Background technique [0002] With the development of bioengineering technology, more and more functional proteins have been reported gradually. In order to achieve high-level expression of functional proteins for later separation and purification and industrial production, people began to study their gene sequences and heterologous expression, thereby promoting the development of genomics. However, many of the new genes reported now have only obtained partial DNA sequences. Without knowing the complete DNA sequence of a gene, it is impossible to study its structure and function in depth. Therefore, it is necessary to obtain the complete sequence from its partially known DNA sequence. The random sequencing method is to obtain the required DNA sequenc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 邬敏辰史红玲高金湖谭中标李剑芳吴静汪俊卿
Owner JIANGNAN UNIV
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