A one-tube, two-step polymerase chain reaction-based site-directed mutagenesis method with simple identification of the mutated product

Abstract

Numerous PCRand mutagenic primer-based methods have been published during the past decade [1–5]. To utilize one of these protocols one has to design and apply three or four primers including one or two primer(s) with the necessary mutation. Purification of intermediate reaction products and sequencing of the mutated gene make some of these methods complicated and time consuming. Here, we present a PCR-based method where a nucleotide substitution in any cloned sequence can be produced in one reaction tube using one mutagenic and one universal (vector-specific) primer. The procedure is based on the megaprimer technique [1]. However, the sequence with the required substitution is obtained by using only one mutagenic primer and without purifying the megaprimers. Our one-tube, two-step PCR procedure involves two plasmid templates T1 and T2. Both templates have the same target sequence, but in the direction opposite to that of the chosen universal primer binding site (T7, M13 Forward, or M13 Reverse sequences). T1 and T2 can be of different (e.g., pET and pBluescript KS) or same (such as pGEM-T (Promega) or pCR-Blunt (Invitrogen)) origins. In the case of blunt-end or ‘‘TA’’ cloning procedures of PCR products, clones with both orientations can easily be found. Fig. 1 shows the orientation of the cloned inserts and the universal primer binding site of templates T1 and T2. The mutagenic and the appropriate vector-specific (universal) primers amplify an intermediate duplex DNA during the first