CLONING DNA FRAGMENTS BETWEEN 2 ADJACENT OVERLAPPING RESTRICTION SITES USING A POSITIVE STUFFER/

Here we describe a solution to a common problem encountered in recombi nant DNA cloning when directional cloning of a DNA fragment into a pre determined plasmin requires the use of restriction enzymes with adjace nt or overlapping recognition sites. In preparing the double-digested plasmid, only one enzyme will often cut, where as 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 rest riction sites are separated by a positively selectable stuffer with re sistance to neomycin. This approach is particularly useful in cases wh ere the choices of restriction sites are severely limited, for example , when it is necessary to clone an additional piece of DNA into a comp lex vector already containing multiple gene cassettes.