Search

A new restriction endonuclease (ENase), SanDI, has been isolated from an unidentified species of Streptomyces. SanDI recognizes the 7-bp interrupted palindrome 5'-GG/GWCCC-3' (W = A or T) and cleaves double-stranded DNA after the second G in the sequence, producing 3-nt long 5' protruding ends. SanDI is a rare-cutting ENase and should therefore be useful for megabase mapping and vector constructions.

A general solid-phase method for the site-directed mutagenesis of double-stranded DNA (dsDNA) is described. Plasmid DNA is linearized using either a restriction endonuclease (ENase) or the RecA-assisted ENase or RecA-AC cleavage method. Alternatively, PCR may be used to generate linear dsDNA. One or both strands of the DNA is biotinylated and attached to a solid support, and the DNA strands are separated using 0.2 M NaOH. An extension oligodeoxyribonucleotide (oligo) and a single or multiple oligo(s) containing the desired mutation(s) are annealed to one of the bound DNA strands and used to initiate the synthesis of a complementary strand by a nonstrand-displacing DNA polymerase. The in vitro synthesized strand incorporating the desired alteration(s) is melted off of the support and recircularized using one of several types of bridging oligos, DNA ligase, and a DNA polymerase and transformed into the host. Greater than 90% mutagenic efficiency has been obtained using this method.

A new restriction endonuclease, SrfI has been isolated from an unidentified species of Streptomyces. SrfI recognizes the 8-bp palindrome, 5'-GCCCGGGC and cleaves double-stranded DNA after the third C in the sequence, producing blunt ends. SrfI is a rare-cutting enzyme and should therefore be useful for megabase mapping.