Rapid Site-Directed Mutagenesis Using Two-PCR-Generated DNA Fragments Reproducing the Plasmid Template

Abstract

We describe a new rapid and efficient polymerase chain reaction (PCR)-based site-directed mutagenesis method. This procedure is effective with any plasmid and it employs four oligonucleotide primers. One primer contains the desired mutation, the second is oriented in the opposite direction (one of these two primers should be phosphorylated), and the third and fourth should be coding in complementary fashion for a unique restriction site to be introduced in a nonessential region. The method consists of two simultaneous PCR reactions; the PCR products are digested with the enzyme that recognizes the newly introduced unique restriction site and then ligased and used to transform competent bacteria. Additionally, the use of Dpn I facilitates the elimination of template DNA. The newly introduced restriction site is essential for ligation in the correct orientation of the two-PCR products and is further used for mutant screening. Resulting plasmids carry both the new restriction site and the desired mutation. Using this method, more than 20 mutants have already been generated (using two different kinds of templates); all these mutants were sequenced for the desired mutation and transfected into AtT-20 cells and the expressed mutant proteins encoded by the vector were assayed.

Figure 1

Schematic representation of site-directed mutagenesis strategy: primer M′ contains the desired mutation∗, primer M oriented in the opposite direction is phosphorilated, and the primers S and S′ are complementary and code for newly introduced unique restriction site BamH I (urs). The two-PCR products are digested with BamH I and ligased. After Dpn I digestion, the circular DNA carry the desired mutation.

Figure 2

PCR products generated from pLNCX2-SOM and their ligation followed with Dpn I treatment. Lane 1: fragment α, lane 2: fragment β, lane 3: fragment α and β ligased, lane 4: ligation and Dpn I treatment, and lane 5: marker X (Roche diagnostics, Mannheim, Germany). DNA samples were fractionated in 1% agarose gel and stained with ethidium bromide.

Figure 3

Minipreps analysis for mutant screening: clone 4 generates a 1.6-kb DNA fragment with EcoR I and is resistant to BamH I digestion. It may represent residual DNA after Dpn I digestion. Lane5: marker X (Roche diagnostics, Mannheim, Germany). DNA samples were fractionated in 1% agarose gel, stained with ethidium bromide.

Figure 4

Schematic representation of the used vector and primers design to perform the three different kinds of mutations. M1/M′1 for single mutation, M2/M′2 for deletion, M3/M′3 for insertion, and S1 and S′1 are the mutagenesis common primers.

Figure 5

Example of successful deletion and insertion. A: 13 residues were deleted from the N-terminus of human somatostatin cDNA. B: 9 residues coding for bradykinin and a stop codon (30 nucleotides) were introduced at the C-terminus of somatostatin precursor.

Figure 5

Example of successful deletion and insertion. A: 13 residues were deleted from the N-terminus of human somatostatin cDNA. B: 9 residues coding for bradykinin and a stop codon (30 nucleotides) were introduced at the C-terminus of somatostatin precursor.