ABSTRACT
Single-Strand Conformation Polymorphism(SSCP) is an effect method for investigating environment 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 temperature. 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 electrophoresis(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.
ABSTRACT
Molecular technologies are sensitive, fast, and cheap in the study on microbial ecology. However, these methods do not provide information about morphology, number, and spatial distribution of the microorganisms. In contrast, Fluorescence in situ hybridization (FISH) combines the precision of molecular biology with the visual information from microscope, and permits visualization and identification of individual miciobial cells within their natural habitat. FISH not only allows the detection of slow growing microorganisms, but also of yet-to-be cultured. It is useful for many applications in diagnosis and assessment of the population structure of complex microbial communities, and is a powerful tool for molecular ecology studies in microbiology, In this paper, major techniques and progresses of FTSH were described. Its application in microbial ecology, as well as problems , pitfalls, and perspectives of FISH are discussed.