LITMUS(TM) - MULTIPURPOSE CLONING VECTORS WITH A NOVEL SYSTEM FOR BIDIRECTIONAL IN-VITRO TRANSCRIPTION

We describe the construction and uses of a set of four multipurpose cl oning vectors: LITMUS(TM) 28, 29, 38 and 39. The vectors feature the h igh-copy pUC origin and an M13 origin for single-stranded DNA producti on as well as polylinker sires for most commercially available restric tion enzymes that recognize nondegenerate hexanucleotide sites and yie ld 4-base sticky ends upon cleavage. Sites are arranged without overla ps, to permit linker addition to blunt-ended fragments and unidirectio nal nested deletions and ave within the lacZ alpha gene to facilitate blue-white screening. Finally, the polylinkers are flanked by a pair o f opposing modified T7 promoters to allow in vitro transcription of ei ther strand of a cloned insert with T7 RNA polymerase. selective unidi rectional transcription from one promoter is achieved by cleaving the other at an internal restriction site (AflII or SpeI). Both modlfied p romoters are fully active under standard RNA probe synthesis condition s. In Southern blots of Dirofilaria immitis genomic DNA, an RNA probe prepared from LIT-MUS performed equivalently to the same RNA probe mad e from a wild-type promoter vector and a DNA probe prepared by random priming.