ULTRASENSITIVE PLASMID MAPPING BY HIGH-PERFORMANCE CAPILLARY ELECTROPHORESIS

This paper compares high performance capillary electrophoresis (HPCE) and conventional slab electrophoresis in mapping of four closely relat ed plasmids with three different restriction enzymes. The Plasmids exp ress full length and truncated forms of a growth factor receptor oncog ene product and were digested with HpaII, HaeIII and RsaI. The resulti ng oligonucleotide fragments were under 2000 base pairs in length, a s ize well suited to separation by HPCE with linear polyacrylamide as a sieving matrix. Plasmid mapping is an essential tool in biotechnology both for the design of an expression system and for monitoring the sta bility of the expression system during fermentation. HPCE can yield mu ch higher resolution of oligonucleotides than attainable in convention al agarose gel electrophoretic procedures for plasmid mapping. In the examples described here, the HpaII digests provided the surest identif ication of individual plasmids in the HPCE analysis and could discrimi nate among all four plasmids. In conventional slab electrophoresis, ho wever, the RsaI digests provided the best discrimination, although two of the plasmids in this system yielded essentially identical electrop horetic patterns. Hence the optimal restriction enzyme for plasmid map ping applications with HPCE may differ from that selected on the basis of conventional slab gel analysis, and the former technique can provi de higher discrimination among related plasmids. The advantages of the HPCE format with respect to speed, low sample consumption and resolut ion are described.