Abstract
We have developed a DNA assembly platform that utilizes the nonspecific, highly variable sequence signatures of type IIs restriction enzymes to assemble a full-length molecular clone of murine hepatitis coronavirus (MHV) strain A59. The approach also allows changes to be engineered into a DNA fragment by designing primers that incorporate the restriction site and the mutations of interest. By adding the type IIs restriction site in the proper orientation, subsequent digestion removes the restriction site and leaves a sticky end comprising the mutation of interest ready to ligate to a second fragment generated in parallel as its complement. In this chapter, we discuss the details of the method to assemble a full-length infectious clone of MHV and then engineer a specific mutation into the clone to demonstrate the power of this unique site-directed "No See'm" mutagenesis approach.