title: Improved purification and PCR amplification of DNA from environmental samples.
abstract: Purification and PCR amplification procedures for DNA extracted from environmental samples (soil, compost, and river sediment) were improved by introducing three modifications: precipitation of DNA with 5% polyethylene glycol 8000 (PEG) and 0.6 M NaCl; filtration with a Sepharose 4B-polyvinylpolypyrrolidone (PVPP) spin column; and addition of skim milk (0.3% w/v) to the PCR reaction solution. Humic substances' concentration after precipitation with 5% PEG was 2.57-, 5.3-, and 78.9-fold lower than precipitation with 7.5% PEG, 10% PEG, and isopropanol, respectively. After PEG precipitation, Sepharose, PVPP and the combined (Sepharose-PVPP) column removed 92.3%, 89.5%, and 98%, respectively, of the remaining humic materials. Each of the above-mentioned modifications improved PCR amplification of the 16S rRNA gene. DNA extracted by the proposed protocol is cleaner than DNA extracted by a commercial kit. Nevertheless, the improvement of DNA purification did not improve the detection limit of atrazine degradation gene atzA.
authors.affiliation: Departamento de Agronomía, Facultad de Agronomía; Universidad Nacional de Colombia--Sede Bogotá, Bogotá, D.C., Colombia. aziv@unal.edu.co
authors.forename: Ziv
authors.initials: Z
authors.lastname: Arbeli
authors.forename: Cilia L
authors.initials: CL
authors.lastname: Fuentes
mesh_terms: D000818:Animals; D011232:Chemical Precipitation; D002845:Chromatography; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D004783:Environmental Microbiology; D005798:Genes, Bacterial; D006812:Humic Substances; D008892:Milk; D011092:Polyethylene Glycols; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D006812:Humic Substances; D012336:RNA, Ribosomal, 16S; D011092:Polyethylene Glycols; D004247:DNA