Capillary electrophoresis single-strand conformation polymorphism analysis for monitoring soil bacteria.
The ability to effectively monitor a microbial community is necessary to design and implement remediation strategies for contaminated soil. Single-strand conformation polymorphism (SSCP), a technique which separates DNA fragments based on their sequence, was used to analyze amplified 16S rRNA gene fragments of 12 common soil bacteria. Separation was performed using capillary electrophoresis (CE), as opposed to other common gel techniques, to eliminate the need for band analysis on gel matrices. Four different universal bacterial primer sets were used for DNA amplification: 341-534, P11-P13, Er10-Er11, and Er14-Er15 corresponding to the V3, V8, V2, and V4 regions, respectively. The forward strand of each primer was labeled with 6-carboxy fluorescein fluorescent dye. Analyses were performed on the Applied Biosystems 310 genetic analyzer using GeneScan Analysis Software version 3.5. The best results were obtained using primer 341-534, in which 6 of the 12 bacteria could be distinguished. By combining primer sets 341-534 and Er10-Er11, all 12 of the bacteria could be separated, indicating various degrees of polymorphism within the selected primer regions. When performing simultaneous amplification and analysis of all 12 species some preferential amplification occurred, as not all peaks could be observed. However, SSCP profiles obtained for pure bacterial cultures show the potential of CE-SSCP for bacterial community analysis.
King
Stephanie
S
Department of Chemistry and Biochemistry, 171 Clippinger Laboratories, Ohio University, Athens, OH 45701, USA.
McCord
Bruce R
BR
Riefler
R Guy
RG
D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D019075:Electrophoresis, Capillary; D006090:Gram-Negative Bacteria / Q000235:genetics* / Q000302:isolation & purification; D006094:Gram-Positive Bacteria / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D012988:Soil Microbiology
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S