Efficient directional seamless DNA (deoxyribonucleic acid) segment connecting method

Abstract

The invention belongs to the technical field of gene engineering, in particular to a directional seamless DNA (deoxyribonucleic acid) segment connecting method. According to the method, 5 to 40 basic groups with completely complementary sequences are respectively arranged at the two ends of connecting primers and parts to be connected of DNA segments 2a and 3a to be connected; because the tm value of the primer is increased along with the increase of the GC content and the basic group number, and the active temperature interval of Taq DNA ligase for completing the connecting reaction is between 65 DEG C and 50 DEG C, so the number of the completely complementary basic groups between the connecting primer 1 and the single connecting segment needs to meet the requirement that the tm value between the connecting primer and the single connecting segment is between 50 DEG C and 65 DEG C; the segments are firstly subjected to phosphorylation, or the primer is subjected to phosphorylation, and then, PCR (polymerase chain reaction) amplification is carried out for ensuring the phosphoric acid group existence at the 5' end of the segments to be connected. The sequence position staggering cannot be generated in the connecting process, the serial connection oligomer is not generated, the self ring connection of carriers and segments is avoided, the operation freedom is high, the operation is simple, the period is short, and the price is low.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a high-efficiency directional seamless DNA fragment connection method. Background technique [0002] The connection and recombination of DNA fragments has always been an important experimental method in the field of life sciences. According to specific purposes, two or more DNA fragments can be connected to construct recombinant molecules. Recombinant molecules can be used in many ways. For example, recombinant expression vectors linked with foreign genes can carry foreign genes to host cells for expression; another example is to obtain non-natural hybrid genes through connection, which can express the required hybrid genes. protein. Recombination engineering has been pursuing higher efficiency, higher accuracy and greater connection length. Advances in this field are of great significance to biological operations such as plasmid construction and gene re...

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Problems solved by technology

[0011] Based on the ligation method of T4 DNA ligase, it is difficult to avoid the self-ligation of the carrier, the self-ligation of the fragments, the uncontrollable connection sequence, the uncontrollable direction of the connection, and the formation of oligomers by several molecules in series in the ligation of complementary cohesive ends and blunt ends. Complementary cohesive ends are restricted by enzyme cutting sites. Although blunt ends have a high degree of freedom of operation, the ligation efficiency is very low (only about 1% of the ligation efficiency of cohesive ends); although the connection of non-complementary cohesive ends can solve the problem of orientation problems, but puts more restrictions on the enzyme cleavage sites of vectors and fragments, poor adaptability to various situations, and low degree of freedom of operation; the difficulty they all face is the small number and short length of one-time ligated fragments

[0012] Although the method based on homologous recombination can also solve the above problems, all the fragments used for ligation need to be amplified by DNA polymerase. Even if high-guaranteed DNA polymerase is used, mismatches caused during the amplification process are inevitable. , which will add unpredictable risks to the research; at the same time, it is necessary to order a corresponding number of long-length Oligo oligonucleotide primers for the manufacture of "homology arms" and amplification fragments, which often greatly increases the cost of this method

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