Improved purification and PCR amplification of DNA from environmental samples.
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.
Arbeli
Ziv
Z
Departamento de Agronomía, Facultad de Agronomía; Universidad Nacional de Colombia--Sede Bogotá, Bogotá, D.C., Colombia. aziv@unal.edu.co
Fuentes
Cilia L
CL
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
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D006812:Humic Substances; D012336:RNA, Ribosomal, 16S; D011092:Polyethylene Glycols; D004247:DNA