Method for building long-fragment target gene through polynucleotide kinase and application of method

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a method for building a long-fragment target gene through polynucleotide kinase and application of the method. The method for building the long-fragment target gene through the polynucleotide kinase can effectively reduce the economic cost generated when the target gene is built and reduce the probability of base mutation. The method is characterized by comprising the steps that the target gene sequence is fragmented according to actual requirements, enzyme cutting site sequences are added to the twoends of each fragment respectively, and then corresponding target product sequence fragments are synthesized; by using the oligonucleotides 5' end of the T4 polynucleotide kinase, the target productis phosphorylated; the phosphorylated target product is annealed to obtain target fragments; a carrier vector is subjected to enzyme cutting, the formed sticky ends correspond to the enzyme cutting site sequences, an enzyme cutting product is recycled, and carrier fragments are obtained; the target fragments are connected with the carrier fragments to obtain the connection product.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for constructing a long fragment target gene by polynucleotide kinase and its application. Background technique [0002] The ligation of DNA fragments is catalyzed by DNA ligase. DNA ligase catalyzes the connection of adjacent bases between DNA fragments with blunt ends or complementary cohesive ends through 3' hydroxyl ends and 5' phosphate ends to form phosphodiester bonds. This reaction is an energy-requiring reaction, usually requiring the addition of ATP or NADH , the ligation efficiency of DNA ligase on cohesive ends is much higher than that on blunt ends. In genetic engineering experiments, the most commonly used ligase is T4 DNA ligase derived from T4 phage. It can be directly ligated for blunt ends or complementary cohesive ends. One fragment with blunt ends and the other with cohesive ends or both fragments with cohesive ends but not pa...

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Problems solved by technology

[0003] When many companies synthesize primers, if the customer has no special requirements, the 5' end of the primer will be dephosphorylated. Generally, the phosphorylated 5' end will be synthesized only when the customer requests it, and a higher fee will be charged. cost, which adds some economic burden to the already expensive sequence synthesis work

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