Inhibitory effects of agarose gel and LB medium on DNA sequencing.

The direct sequencing of PCR products, a bacterial colony (plasmid DNA), or phage plaque (λDNA) is a very powerful technique applicable to several molecular biological experiments. In particular, the sequencing of plasmid DNA directly from within bacterial colonies provides a rapid screening method to confirm the orientation of DNA inserts. Similarly, the direct sequencing of λDNA within plaques offers rapid screening of genomic and cDNA libraries. Recently, the use of the BigDye terminator (Applied Biosystems, Foster City, CA, USA) in the direct sequencing of plasmid DNA (2,5,6) and λphage (4,6) has been reported. However, our direct sequencing efforts occasionally failed because of the presence of agarose gel containing LB medium in the sequencing reaction mixtures. PCR-inhibitory components found within blood cells have been characterized (1). To our knowledge, the inhibitory effects of LB medium and agarose on DNA polymerase activity have not been reported. In this study, we present the results of a semiquantitative investigation of the effects of agarose and LB medium on the direct sequencing reaction. LB medium (Table 1) was tested in a standard reagent mixture containing 2 μL 0.4 μg pGEM-3Zf(+), 2 μL 6.4 pmol -21M13 primer, BigDye Terminator Ready Reaction Mix (6 μL; Applied Biosystems), LB medium, and double-distilled water to a total volume 15 μL. PCR was carried out using a GeneAmp® PCR System 9700 (Applied Biosystems) at 94°C for 1 min, followed by 25 cycles of 96°C for 30 s, 50°C for 15 s, and 60°C for 2.5 min. Mixtures were then centrifuged through a Sephadex® G50 spin column (Amersham Biosciences, Piscataway, NJ, USA) using a microcentrifuge at 730× g for 2 min to remove unincorporated dye-labeled terminators. PCR products were then recovered by ethanol/sodium acetate precipitation. Precipitated DNA was resuspended in 20 μL Template Suppression reagent and then subjected to sequence analysis using an ABI PRISM® 310 Genetic Analyzer (Applied Biosystems). Agarose gel (ultraPURE® agarose; Invitrogen, Carlsbad, CA, USA) (Table 2) was tested in a standard reagent mixture containing 2 μL 0.4 μg pGEM3Zf(+), 2 μL 6.4 pmol -21M13 primer, 6 μL BigDye Terminator Ready Reaction Mix, and 3 μL double-distilled water. PCR was carried out using a GeneAmp PCR System 9700 at 94°C for 1 min, followed by 25 cycles of 96°C for 30 s, 50°C for 15 s, and 60°C for 2.5 min. Mixtures were then centrifuged through a Sephadex G50 spin column using a microcentrifuge at 730× g for 2 min to remove unincorporated dye-labeled terminators. PCR products were then recovered by ethanol/sodium acetate precipitation. Precipitated DNA Benchmarks