Optimal Conditions and Validation of Single-strand Conformation Polymorphism Technology for the Analysis of Microbial Communities in Activated Sludge

Abstract:

Single-Strand Conformation Polymorphism(SSCP) is an effect method for investigating environ-ment microbial genetic polymorphism, with its characterization of rapidness, simplicity, and sensitivity. However, many factors can influence the results of SSCP in the analysis of complex environment samples, and its optimization is highly needed. In this paper, optimal PCR-SSCP conditions were discussed based on PAGE concentration, formamide deionized in denaturing loading buffer, electrophoresis time and tempera-ture. The resluts showed that the optimal conditions were as follows: 16S rDNA V1~V3 was selected as the targeted gene, the ratio of acrylamide to N, N-dimethylacrylamide in 12% polyacrylamide gel electrophore-sis(PAGE)gel was 49:1, the ratio of formamide deionized in denaturing loading buffer was 1:3, running the SSCP gel at 300 V for 18 h (under 4℃). Aside from this, the validations using samples from a simultaneous desulfurification and denitrification bioreactor were conducted under this optimal conditions.