title: Assessment of five soil DNA extraction methods and a rapid laboratory-developed method for quality soil DNA extraction for 16S rDNA-based amplification and library construction.
abstract: Extraction of DNA from soil samples using standard methods often results in low yield and poor quality making them unsuitable for community analysis through polymerase chain reaction (PCR) due to the formation of chimeric products with smaller template DNAs and the presence of humic substances. The present study focused on the assessment of five different methods for metagenomic DNA isolation from soil samples on the basis of processing time, purity, DNA yield, suitability for PCR, restriction digestion and mDNA library construction. A simple and rapid alkali lysis based on indirect DNA extraction from soil was developed which could remove 90% of humic substances without shearing the DNA and permits the rapid and efficient isolation of high quality DNA without the requirement of hexadecyltrimethylammonium bromide and phenol cleanup. The size of DNA fragment in the crude extracts was >23 kb and yield 0.5-5 μg/g of soil. mDNA purification using Sephadex G-50 resin yielded high concentration of DNA from soil samples, which has been successfully used for 16S rDNA based amplification of a 1500 bp DNA fragment with 27F and 1492R universal primers followed by restriction digestion and mDNA library construction.
authors.affiliation: Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, 784028, Assam, India. Electronic address: kalpsaga@gmail.com.
authors.forename: Kalpana
authors.initials: K
authors.lastname: Sagar
authors.affiliation: Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, 784028, Assam, India.
authors.forename: Salam Pradeep
authors.initials: SP
authors.lastname: Singh
authors.affiliation: Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, 784028, Assam, India.
authors.forename: Kapil Kumar
authors.initials: KK
authors.lastname: Goutam
authors.affiliation: Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, 784028, Assam, India.
authors.forename: Bolin Kumar
authors.initials: BK
authors.lastname: Konwar
mesh_terms: D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification*; D015723:Gene Library; D006812:Humic Substances / Q000032:analysis; D008828:Microbiological Techniques / Q000379:methods* / Q000592:standards*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012987:Soil / Q000737:chemistry; D012988:Soil Microbiology
chemical_list: D004269:DNA, Bacterial; D006812:Humic Substances; D012336:RNA, Ribosomal, 16S; D012987:Soil
keywords: Chimeric; Humic substances; Metagenomic DNA