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Construction method of single enzyme digestion vector

A construction method and carrier technology, applied in the field of DNA recombination, can solve the problems of increasing the difficulty of operation steps for nucleic acid degradation, reducing the efficiency of single-enzyme digestion and connection, and long connection time, so as to improve the efficiency of single-enzyme digestion and connection, improve the efficiency of single-enzyme The effect of cutting connection efficiency and saving manpower

Active Publication Date: 2014-09-24
CHINA AGRI UNIV
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main problems that limit the application of single enzyme digestion technology are low connection efficiency and long connection time
This is because the 4 sticky ends are the same after digestion, the vector is very prone to self-ligation, and the target gene will be randomly integrated into the vector in two different orientations. These two factors greatly reduce the efficiency of single-digestion ligation. Only about 5-10% of double digestion efficiency
Researchers have tried many methods to improve the efficiency of single enzyme digestion, such as carrier 5, end dephosphorylation, etc., but the efficiency is not high, and it increases the difficulty of the operation steps and the chance of nucleic acid degradation

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Construction of pBC1-HIOMT mammary gland-specific expression vector

[0055] The pBC1 mammary gland-specific expression vector was purchased from Invitrogen. The structure of the vector is relatively simple, with only one ampicillin resistance gene and no multiple cloning site (MCS). There are only 3 available restriction sites, XhoI, NotI, and SalI, on the entire vector, and it is required that the target gene must be inserted into the XhoI site for normal expression ( figure 1 ). Therefore, only the single enzyme digestion method can be selected when constructing the vector.

[0056] 1. Cloning of the full-length HIOMT gene with XhoI restriction site

[0057] (1) Design of HIOMT full-length cloning primers

[0058] We designed upstream and downstream primers at the 5' and 3' non-coding regions of the HIOMT gene respectively, and introduced the recognition site CTCGAG of the XhoI restriction endonuclease at the 5' end of the upstream and downstream primer...

Embodiment 2

[0104] Example 2 Optimization of the method for constructing a single-enzyme-cut vector

[0105] In this example, 400 samples were subjected to PCR at different ligation times at 16°C, and the positive rates are shown in Table 1:

[0106] Table 1 The influence of different connection time on the positive rate

[0107] connection time 60min 90min 4h 16h positive rate 5.20％ 8.33％ 10.41％ 3.12％

[0108] It can be seen from Table 1 that when the connection time is 4 hours, the positive rate reaches 10.41%, and when the connection time is 90 minutes, the positive rate reaches 8.33%. When the connection time is 90 minutes, the connection efficiency has been significantly improved. Taking time cost and connection efficiency into consideration, the present invention selects 90 minutes as the optimal connection time.

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Abstract

The invention provides a construction method of a single enzyme digestion vector. The construction method comprises the following steps: 1) selecting single enzyme digestion sites; 2) introducing the enzyme digestion sites to the two ends of a target gene; 3) performing enzyme digestion and electrophoretic gel cut recovery on the vector and the target gene; 4) performing temperature gradient pretreatment on the mixed solution of the target gene and the vector; 5) connecting the vector and the target gene by use of solution I for connecting a T vector. The invention provides a new method for constructing the single enzyme digestion vector, and the enzyme digestion connection efficiency is greatly improved, and meanwhile, the connection time is greatly shortened. Due to the use of the method, the single enzyme digestion connection efficiency is remarkably improved, the connection time is only about 90min, the human and material resources are greatly saved and the operation difficulty of the single enzyme digestion is reduced.

Description

technical field [0001] The invention relates to DNA recombination technology, in particular to a method for constructing a single-enzyme-cut vector. Background technique [0002] The construction of the expression vector is a key step in transgenic technology. The main process is to recombine the target gene expression cassette with the vector through a series of enzyme digestion and ligation reactions, and then transfect the recombined vector into the host cell to realize the expression of the target gene in the host cell. expression. At present, enzyme digestion technology is mainly divided into double enzyme digestion and single enzyme digestion. [0003] Double digestion refers to finding two suitable restriction sites on the vector sequence during vector construction, and introducing corresponding restriction sites upstream and downstream of the target gene; Two different cohesive ends are formed; finally, through the ligation reaction, the target gene can be inserted...

Claims

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Application Information

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IPC IPC(8): C12N15/66
Inventor 刘国世何长久朱宽峰孔瑾汪锋田秀芝宋玉坤
Owner CHINA AGRI UNIV
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