Inserting DNA fragments into vectors from "summary" of Molecular Cloning by Joseph Sambrook,David William Russell
To introduce foreign DNA into a vector, both the vector and the DNA must be cleaved with the same restriction enzyme. This results in complementary sticky ends that can base pair with each other. The vector and DNA are then mixed together in the presence of DNA ligase, which catalyzes the formation of covalent bonds between the two molecules. This process is known as ligation. After ligation, the recombinant DNA molecules are introduced into host cells, usually bacteria. The bacteria then replicate the recombinant DNA along with their own DNA, producing many copies of the inserted DNA fragment. This allows for the amplification of the DNA fragment, making it easier to study or manipulate. Several methods can be used to introduce the recombinant DNA into host cells, including transformation, transfection, and electroporation. Each method has its advantages and disadvantages, depending on the type of host cell and the size of the DNA fragment being inserted. Once inside the host cell, the recombinant DNA can be further manipulated or studied. For example, the expression of a gene encoded by the inserted DNA fragment can be induced, allowing researchers to study the function of that gene. Alternatively, the DNA fragment can be sequenced to determine its nucleotide sequence, providing valuable information about the gene it encodes. In summary, inserting DNA fragments into vectors is a fundamental technique in molecular biology that allows researchers to study and manipulate DNA. By combining foreign DNA with a vector and introducing it into host cells, researchers can amplify the DNA fragment, study its function, and gain valuable insights into the genetic material of an organism.
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