Method for synthesizing gene with complex sequence

A synthetic method and gene technology, applied in the field of bioengineering, can solve problems such as difficulty in gene synthesis with complex sequences

Active Publication Date: 2018-05-01
GENSCRIPT NANJING
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Problems solved by technology

[0007] The purpose of the present invention is to solve the problem of difficulty in synthesizing genes with complex se

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  • Method for synthesizing gene with complex sequence
  • Method for synthesizing gene with complex sequence
  • Method for synthesizing gene with complex sequence

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Embodiment Construction

[0044] In order to further understand the method described in the present invention, the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0045] The gene synthesis process of the present embodiment is as follows:

[0046] The target sequence synthesized in this embodiment is a high GC gene with a GC content of 72.3% and a length of 3459 bp. The gene also has more than 100 repeat sequences of 10bp-23bp forward, reverse and complementary strands, which is a typical sequence complex gene. Its sequence is shown in SEQ ID NO.1 in the sequence listing.

[0047] The gene synthesis process of the present embodiment is as follows:

[0048]Step 1: Artificially segment the target gene into 3 fragments of 1208bp, 1211bp, and 1048bp, labeled A1, A2 and A3. Among them, between the 3' end of A1 and the 5' end of A2; between the 3' end of A2 and the 5' end of A3, there is an overlapping region of 4 bases (the overlapping reg...

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Abstract

The invention discloses a method for synthesizing a gene with a complex sequence. The method comprises: 1) designing a series of 30-150 bp single-stranded oligonucleotide sequences containing the positive strand sequence and the negative strand sequence of a gene by using a gene segmentation and single-stranded oligonucleotide design rule; 2) selecting the single-stranded oligonucleotide fragmentsobtained in the step 1), and separately annealing each pair of the positive single-stranded oligonucleotide fragment and the negative single-stranded oligonucleotide fragment by using the annealing procedure designed by the present invention according to the complementary pairing principle of the positive strand and the negative strand to form double-stranded oligonucleotide fragments, loading onto a target vector, sequencing, and verifying; and 3) based on the double-stranded oligonucleotide fragments obtained in the step 2), by using endonuclease capable of externally digesting a recognition sequence, carrying out 1-3 cycles of in vitro linking assemblies, cloning, and verifying so as to complete the synthesis assembly of the 100bp-20kb gene with the complex sequence. With the method ofthe present invention, the complex sequence gene incapable of being synthesized or being difficultly synthesize by the traditional method can be effectively synthesized.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for synthesizing genes with complex sequences. technical background [0002] The artificial synthesis of genes based on DNA sequences provides the basis for the development of modern molecular biology and synthetic biology. Using gene synthesis technology, researchers can arbitrarily edit or copy naturally occurring genes and create genes that do not exist in nature according to specific needs. Artificially synthesized genes are widely used in fields such as agriculture, food engineering, medicine, new materials and new energy. [0003] Currently reported gene synthesis methods mainly include an assembly method based on polymerase chain reaction (Polymerase Chain Reaction, PCR) and a ligation assembly method based on ligase. Both methods need to use chemical synthesis methods, such as solid-phase synthesis, phosphodiester method, phosphotriester method, phosphite triester...

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/10C12Q2521/301C12Q2521/501C12Q2525/131C12Q2525/191Y02A50/30
Inventor 黄小罗翟春华张丽华柳振宇
Owner GENSCRIPT NANJING
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