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Method for connecting DNA fragments through T4 bacteriophage DNA topoisomerase

A technology of topoisomerase and bacteriophage, applied in the field of biochemistry, can solve the problems of many steps, time-consuming, various cumbersome and other problems

Inactive Publication Date: 2016-12-07
BIOTOOL LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method involves many steps, and is cumbersome and time-consuming.

Method used

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  • Method for connecting DNA fragments through T4 bacteriophage DNA topoisomerase
  • Method for connecting DNA fragments through T4 bacteriophage DNA topoisomerase
  • Method for connecting DNA fragments through T4 bacteriophage DNA topoisomerase

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Embodiment 1

[0014] A method utilizing T4 bacteriophage DNA topoisomerase I to connect DNA fragments, the specific steps are as follows:

[0015] Step 1, design primers: connect two DNA fragments with a length of 1.5kb, primers A, B and primers C, D each amplify a fragment with a length of 1.5kb, wherein primers A and D only contain sequences complementary to the template , primers B and C include linker DNA sequence, T4 phage DNA topoisomerase I recognition site DNA sequence and template paired DNA sequence from 5' to 3' end in turn, and the linker DNA sequence of primers 2 and 3 is reverse complementary. The source, size and primers A, B, C and D of DNA fragments are shown in Table 1

[0016] Table 1 DNA fragments and primers

[0017]

[0018] Bold letters indicate the linker sequence, and the underline indicates the T4 phage DNA topoisomerase I recognition site sequence.

[0019] Step 2, fragment amplification: use the corresponding DNA fragments to be ligated as templates, and per...

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Abstract

The invention discloses a method for connecting DNA fragments through T4 bacteriophage DNA topoisomerase. The method comprises the steps that primers are designed, wherein the primers are designed for at least two DNA fragments to be connected respectively; 2, the fragments are amplified, wherein the corresponding DNA fragments to be connected are used as templates, the primers of the DNA fragments are subjected to PCR amplification to obtain amplified products, and the amplified products are purified; 3, the fragments are connected. According to the DNA fragment connection method, T4 bacteriophage DNA topoisomerase I is used for achieving the functions of enzyme digestion and connection at the same time, the method has the advantages of being rapider and more efficient compared with a traditional method that a restriction enzyme is utilized at first for producing matched tail ends for the DNA fragments, T4 ligase is utilized for connecting fragments after purification, sequences inside the fragments to be connected in the method will not influence connection, and defects of a digestion step in the traditional method are eliminated.

Description

technical field [0001] The invention relates to the field of biochemical technology, in particular to a method for connecting DNA fragments by using T4 bacteriophage DNA topoisomerase I. Background technique [0002] With the development of science and technology, various new technologies gradually appear in people's production and application, and cloning technology is one of them. Cloning refers to the asexual reproduction of organisms through somatic cells, and the population of offspring individuals with identical genotypes formed by asexual reproduction. Usually, biotechnology is used to produce individuals or populations that have exactly the same genes as the original individuals through asexual reproduction. The current conventional gene cloning method is to use T4 DNA ligase (T4 DNA ligase) method to connect the target gene to be cloned with the vector, transform the recirculated recombinant into competent bacterial cells, and then screen and identify the correct c...

Claims

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Application Information

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IPC IPC(8): C12P19/34
CPCC12P19/34
Inventor 张存清李成栋
Owner BIOTOOL LLC
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