Cloning DNA Fragments Between Two Adjacent/Overlapping Restriction Sites Using a “Positive Stuffer”

Here we describe a solution to a common problem encountered in recombinant DNA cloning when directional cloning of a DNA fragment into a predetermined plasmid requires the use of restriction enzymes with adjacent or overlapping recognition sites. In preparing the double-digested plasmid, only one enzyme will often cut, whereas the second will not because of the lack of a sufficiently long stretch of double-stranded DNA at its recognition site. The problem can be solved by construction of a "user-friendly" intermediary plasmid in which the desired restriction sites are separated by a positively selectable stuffer with resistance to neomycin. This approach is particularly useful in cases where the choices of restriction sites are severely limited, for example, when it is necessary to clone an additional piece of DNA into a complex vector already containing multiple gene cassettes.