PURIFICATION OF ESCHERICHIA-COLI CHROMOSOMAL SEGMENTS WITHOUT CLONING

Pairs of genomic insertions made with elements carrying any one of sev eral frequently used rare restriction sites allow physical purificatio n of insertion delimited genes. However, native rare restriction sites can, either by causing (i) fragmentation of targeted intervals or (ii ) generation of additional fragments that overlap electrophoretically with targeted ones, place severe limitations on this approach. We pres ent a series of Escherichia coli mini-Tn10 insertions containing the r are-cutting polylinker 2 (RCP2) of rare restriction sites, which inclu des the 18-base-pair I-SceI site (absent from native E. coli sequences ). Pulsed-field gel purification from RCP2 double insertion mutants of both an I-SceI fragment from strain K-12 (containing similar to 90-95 min) and an allelic I-SceI fragment from a pathogenic strain is demon strated. The complete series of RCP2 insertions, containing different antibiotic resistances at intervals of similar to 35 kb in prototype K -12 strain MG1655, allows rapid purification of the genes from any E. coli chromosomal interval as an isolated I-SceI fragment. (C) 1996 Aca demic Press, Inc.