A modified vector for rapid cloning and sequencing of PCR products

Abstract

▼PCR products are often cloned into a plasmid vector prior to sequencing to obtain high-quality templates and to take advantage of universal sequencing primers. However, this strategy is only useful for obtaining a short single-stranded sequence from each end of an insert. To obtain the sequence of an insert larger than several hundred base pairs, shotgun or nested deletion strategies must also be employed. Here we introduce a modified plasmid vector that is specifically designed for rapid cloning of PCR (and other) products and for constructing nested deletions for sequencing large inserts. The parent plasmid vector pZErO TM -1 (Invitrogen) is an excellent cloning vector because it produces no background of non-recombinant colonies, it does not require expensive X-Gal for color selection, and the linearized ends do not require dephosphorylation (Invitrogen Corporation’s Zero Background Cloning Kit, Version C Instruction Manual). We redesigned half of the polylinker region of pZErOTM-1 to include a recognition site for the blunt-end-producing 8-cutter restriction enzyme, Srf I (Fig. 1). This enzyme will rarely cut within a fragment to be cloned, allowing a bluntended insert such as a PCR product to be ligated into the vector in the presence of both T4 ligase and Srf I in a 1-h reaction (Stratagene pCR-ScriptTM SK(+) Cloning Kit Instruction Manual). High cloning efficiency is assured by a selection procedure following transformation in which only cells containing a recombinant plasmid can grow. In addition to a NotI site already present, we added a third 8-cutter site, AscI, on the opposite side of the Srf I site. We also added a 5’ CG overhang producing a 6-cutter site for ClaI, which our nested deletion strategy requires (Ref. 1). We named this modified vector pZErO-Z. The general strategy for constructing nested deletions is as follows. The cloned DNA in pZErO-Z is amplified with universal primers [M13 reverse and M13 (−40)]. This DNA is partially digested with one or more 4-cutter re-