Application of the Recom - bination of β - Lactamase Gene to PCR - Based Site - Directed Mutagenesis

Suppression reagent according to the instruction manual and then subjected to sequence analysis using an ABI PRISM310 Genetic Analyzer. In an effort to establish optimal conditions for the direct sequencing of plasmid DNA within bacterial colonies or λDNA within single-phage plaques, a semi-quantitative investigation of the inhibitory effects of LB medium and agarose gel was carried out. It was found that LB medium concentrations greater than 13.3% inhibited the sequencing reaction (Table 1). It was found that agarose gel concentrations greater than 0.2% inhibited the sequencing reaction (Table 2). While Gibb and Wong (3) found that PCR amplification of the βglobin gene was inhibited by 0.033% agar, Setterquist and Smith (7) reported the successful amplification of the HIV1 gag gene using 0.05% agarose-encapsulated PCR reagents. It is difficult to make comparisons because agarose is a purified substance isolated from agar. Therefore, perhaps less agar is required to inhibit DNA polymerase reactions, yet it is proposed that reactions with DNA polymerases would be inhibited at agarose concentrations greater than 0.2%. At these concentrations, agarose would exist in a partly set gel state, thus making for suboptimal reaction conditions. Additionally, we investigated the effect of LB medium in the presence of agarose. We found that amounts greater than 2 μL 1.5% agarose in the presence of LB medium (final concentrations, 11.8% LB medium and 0.17% agarose) inhibited the sequencing reaction. We conclude that the direct sequencing of plasmid DNA within bacterial colonies (5) and of λDNA within single-phage plaques (re-cycle-sequencing) (4) would be more successful if the LB medium and agarose concentrations were less than 20% and 0.15%, respectively.