The commercially available restriction enzyme BspHl is blocked by overlapping methylation

Abstract

The restriction endonuclease Bspill. conunercially available from New England Biolabs (Bishop's Stortford, UK), cleaves the site T/CATGA. This restriction enzyme is of particular use in the construction of gene expression cassettes in the vector pKK233 -2 (I) as it is one of the three enzymes along with AflIII and NcoI that has an 'ATG' in its recognition sequence. In this paper we show that BspHI is Dam methylase (2) sensitive and will not cleave DNA that has the overlapping sequ~nce GATC. The oligonucleotides: OS3142 5' -CTAGATCATGAGCGAACTG~ TCGTGAACGTGATCAAAGCTTGGTAC- 3' and OS3143 5'-CAAGCTTTGATCACGTTCACGATCAGTTCGCTCATGAT- 3' were annealed and ligated into XbaI-KpnI digested bluescript SKI1+ (Stratagene, Cambridge, UK}.