Construction and features of lambda EMBL3cosW, a lambda replacement vector for detailed analysis of large regions of genomic DNA

Abstract

A phage lambda replacement vector, lambda EMBL3cosW, is described which expedites detailed analysis of large regions of chromosomal DNA. Two features of the vector aid this process. Firstly, the replaceable stuffer in lambda EMBL3cosW is flanked by SP6 and T7 promoters so that end-specific hybridisation probes can be rapidly generated from cloned inserts for identification of sequentially overlapping clones in genomic libraries. Secondly, because all the phage coding sequences in the vector (which are placed to the right of the replaceable stuffer) can be removed from cloned inserts by cleavage with NotI, restriction mapping of cloned inserts using partial digest strategies is greatly facilitated. Other features of the vector are: (1) strategically placed BamHI and XhoI sites for the cloning of genomic DNA partially digested with MboI or Sau3AI by two different methods; (2) SalI and SfiI sites for the isolation of intact cloned inserts; and (3) transcription terminators to insulate vector genes from transcriptional interference from cloned insert DNAs.