An effective method for plant rhizosphere microbial genomic DNA extraction was established. High-purity nucleic acids extracted from soil samples, with considerable yield, could be used for study of soil microorganism molecular ecology. Genomic DNAs were extracted from 12 soil samples of different crop roots. Above 10 μg of genomic DNA with approximately 20-kb fragment length was isolated from 1 g of soil sample. The average value of A260/A230 of the genomic DNA was 1.505, while that of A260/A280 was 1.780. The absorption curves of the full wavelengths of the DNA extracted were consistent with that of pure nucleic acid. DNAs from different dilutions of pollutants were applicable for restriction enzyme digestion analysis, and the lambda DNA in 100-fold diluted soil samples had the same restriction enzyme digestion results as done in ddH2O. Amplicons was produced with the expected molecular size by polymerase chain reaction (PCR).Therefore, the DNA extracted by this method was suitable to be used in analysis of bacterial ecology composition by denaturing gradient gel electrophoresis.
Key words: DNA extraction, DNA quality, PCR amplification, soil microbial community diversity, DGGE.
GM, Genetically modified; Bt, Bacillus thuringiensis; DGGE, denaturing gradient gel electrophoresis; PCR, polymerase chain reaction; EB,ethidium bromide;PVPP,polyvinylpolypyrrolidone; EDTA, ethylene diamine tetra-acetic acid; SDS, sodium dodecyl sulphate.
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