Inexpensive and safe DNA gel electrophoresis using household materials.

Gel electrophoresis is the single most important molecular biology technique and it is central to life sciences research, but it is often too expensive for the secondary science classroom or homeschoolers. A simple safe low-cost procedure is described here that uses household materials to construct and run DNA gel electrophoresis. Plastic containers are fitted with aluminum foil electrodes and 9-V batteries to run food-grade agar-agar gels using aquarium pH buffers and then stained with gentian violet. This activity was tested in a high school biology classroom with significantly positive responses on postactivity reflective surveys. The electrophoresis activity addresses several Life Science Content Standard C criteria, including aspects of cell biology, genetics, and evolution. It also can be used to teach aspects of motion and force in the physical science classroom.

Development of real-time PCR assays for detection of the Streptococcus milleri group from cystic fibrosis clinical specimens by targeting the cpn60 and 16S rRNA genes.

Cystic fibrosis (CF) is a multiorgan disease, with the majority of mortalities resulting from pulmonary failure due to repeated pulmonary exacerbations. Recently, members of the Streptococcus anginosus group (S. anginosus, S. constellatus, and S. intermedius), herein referred to as the "Streptococcus milleri group" (SMG) have been implicated as important etiological pathogens contributing to pulmonary exacerbations in CF patients. This is partly due to better microbiological detection of the SMG species through the development of a novel specific medium termed "McKay agar." McKay agar demonstrated that SMG has been an underreported respiratory pathogen contributing to lung exacerbations. Our aim was to develop a real-time PCR assay to expedite the detection of SMG within diagnostic samples. The cpn60 gene was chosen as a target, with all three members amplified using a single hybridization probe set. SMG strain analysis showed that speciation based on melting curve analysis allowed for the majority of the S. constellatus (96%), S. intermedius (94%), and S. anginosus (60%) strains to be correctly identified. To increase specificity for S. anginosus, two 16S rRNA real-time PCR assays were developed targeting the 16S rRNA gene. The 16s_SA assay is specific for S. anginosus (100%), while the 16s_SCI assay is specific for S. constellatus and S. intermedius (100%). These assays can detect <10 genome equivalents in pure culture and >10(4) genome equivalents in sputum samples, making this a great tool for assessment of the presence of SMG in complex polymicrobial samples. Novel molecular methods were developed providing detection ability for SMG, an emerging opportunistic pathogen.