A Method for Parallel Assembly of Multiple Segments of DNA

Abstract

The invention relates to a method for parallel assembly of multi-segment DNA. Choose to insert the circular or linear unit vector into the circular host or linear host to form a gene carrier, and then recover the circular or linear vector to obtain the recombined DNA genetic material. Its characteristic is that it can realize parallel and simultaneous operation of multiple pieces of DNA, thereby improving the efficiency of DNA assembly to form a new DNA structure, and preventing DNA pollution and disproportionation, ensuring the quality of assembled DNA, so that it can achieve the design purpose. The time required to assemble multiple pieces of DNA is approximately proportional to log(N). The assembly operation is carried out outside or inside the cell, so that multiple DNA molecules can be spliced ​​in any order to form a new DNA structure. It is suitable for application in the field of genetic engineering to obtain new organisms through genetic recombination.

Description

technical field [0001] The invention relates to gene recombination technology in the field of genetic engineering, in particular to a method for parallel assembly of multiple segments of DNA. Background technique [0002] All the forms of life that humans have come into contact with at present require a complete cell structure to exert all the characteristics of an organism. In cells, DNA is the main genetic material, DNA is transcribed to form RNA, RNA is translated to produce protein, and RNA and protein realize biological functions in biological cells. Therefore, the most essential modification of organisms is the modification of DNA. The ability to transform DNA determines the ability of human beings to transform and even create creatures. [0003] After 2005, technologies related to synthetic biology need to develop appropriate large-scale gene assembly technology to meet the development and needs of genetic engineering and metabolic engineering. [0004] "Recombina...

AI Technical Summary

Problems solved by technology

Long fragments of DNA often contain multiple restriction endonuclease sites and are difficult to manipulate

[0006] 2. Restriction endonuclease sites need to be operated in vitro, and long fragments of DNA are difficult to operate in vitro

[0007] 3. When it is necessary to avoid restriction enzyme cutting sites, it is difficult to parallelize the operation

Disadvantages of this method: 1. The cost of synthetic DNA is relatively high

2. It cannot avoid the problem that large fragments of DNA are difficult to operate in vitro

Disadvantages of this method: 1. The problem of containing this type of restriction site in the natural sequence cannot be avoided

2. The number of fragments assembled at one time should generally not exceed 10, otherwise the connection efficiency is very low

3. It is difficult to operate in vitro for large fragments

Disadvantages of this method: 1. Due to the complete use of homologous recombination, erroneous recombination will occur when there are repeated sequences in the reassembled fragments

2. The reorganization efficiency of this method is not high, and few people actually use it

Disadvantages of this method: 1. It is a sequential assembly system and cannot be parallelized

Disadvantages of this method: 1. Because it involves the PCR process, it is easy to introduce mutations

2. Not suitable for assembling long fragments

Disadvantages of this method: 1. Due to the increase in the length required for recombination, for natural fragments, it is difficult to obtain recombined fragments of more than 80 bases in one PCR, so this method is mainly used for full chemical synthesis of genomes

However, the cost of fully chemically synthesized genomes is too high, resulting in a very limited range of applications

2. During the assembly process, it is necessary to use a vector with a higher copy number for intermediate cloning. It is not suitable to assemble fragments exceeding 50Kbp in E. coli

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