( and sequence analysis of gel...)Monitoring Bacterial Genetic Diversity in a Freshwater Lake Using TTGE and DNA Sequence Analysis

Ammonia-oxidizing bacteria mediate the nitrification stage of the nitrogen cycle, hence their activity is of ecological importance, but the influence of environmental parameters upon their community structure is largely unknown.¹ Described here is the combined application of TTGE and sequence analysis of gel-resolved 16S rDNA PCR products amplified from freshwater Nitrosospira spp. collected throughout a twelve month period in an attempt to observe temporal genetic variation within this functionally important microbial group.

Materials and Methods
Genomic DNA, extracted from lakewater samples collected at monthly intervals from January to December, was PCR-amplified to generate 240 bp GC-clamped fragments of the 16S rRNA gene spanning the variable V3 region from ammonia-oxidizing bacteria belonging to the genus Nitrosospira.^2,3 Approximately 200 ng of PCR product were mixed with 2x bromophenol blue loading dye and electrophoresed in a 6% polyacrylamide gel (acrylamide/bis 37.5:1) containing 7 M urea, 20% formamide, 2% glycerol in 1.25x TAE buffer (50 mM Tris-Cl, 25 mM acetic acid, 1.25 mM EDTA, pH 8.0) using the DCode system at 100 V for 16 hr across a temperature range of 4055 C and a temperature ramp of 1 C/hr. After electrophoresis, gels were equilibrated in 1.25x TAE buffer for 15 min., stained in ethidium bromide (25 mg/ml in TAE) for 20 min. and washed in TAE before viewing and photographing under UV light (302 nm). Various individual DNA bands were cut from the gel with a
'"/>

Source: