Some Key Enzymes Used in Cloning

Abstract

Synopsis A large number of different enzymes are used in DNA cloning procedures. DNA ligase is used to join two DNA molecules covalently, the key step in constructing a recombinant plasmid from a cloning vector and a DNA insert. The ends of DNA molecules can be modified to allow or prevent ligation by using the enzymes polynucleotide kinase or alkaline phosphatase. Enzymes that catalyze DNA synthesis, including DNA polymerases, reverse transcriptase, and terminal deoxynucleotidyl transferase, find application in many ways in cloning procedures, including to modify DNA ends to control ligation, in the polymerase chain reaction, in DNA sequencing, to produce DNA copies of RNA molecules, and other applications. A variety of nucleases are available that can be used to remove unwanted DNA, to modify DNA ends, and to delete larger portions of a DNA molecule.