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Rapid DNA extraction protocol from soil for polymerase chain reaction‐mediated amplification
Author(s) -
Smalla K.,
Cresswell N.,
MendoncaHagler L.C.,
Wolters A.,
Elsas J.D. van
Publication year - 1993
Publication title -
journal of applied bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 0021-8847
DOI - 10.1111/j.1365-2672.1993.tb02999.x
Subject(s) - dna extraction , polymerase chain reaction , dna , chromatography , polymerase chain reaction optimization , lysis , biology , microbiology and biotechnology , pseudomonas fluorescens , lysis buffer , population , dna polymerase , extraction (chemistry) , chemistry , biochemistry , bacteria , multiplex polymerase chain reaction , genetics , demography , sociology , gene
A simple and rapid method of DNA extraction from soil was developed and DNA was made suitable for subsequent efficient amplification by the polymerase chain reaction (PCR). Key features of the extraction and purification were cold lysozyme‐ and SDS‐assisted lysis with either freezing‐thawing or bead beating, cold phenol extraction of the resulting soil suspension, CsCl and KAc precipitation and, finally, spermine‐HCl or glass milk purification of DNA. Crude DNA preparations contained 4–20 μg DNA per g of soil extracted, and at least 50% of this was recovered in the final purified DNA preparations. The resulting DNA was pure enough to be restricted by various enzymes, and was amplifiable at concentrations of up to 20 ng of soil‐derived DNA per 50 μl reaction mix. Amplification of a 683 bp target sequence, pat, was performed with different Taq DNA polymerases. Application of the protocol enabled us to detect target DNA derived from roughly 10 3 introduced Pseudomonas fluorescens (RP4 :: pat ) cfu per g of soil. The fate of an introduced population in the soil could be followed to this limit with PCR‐assisted detection of target DNA. In addition, target DNA was detected in soil 5 months after release, when the introduced organism was no longer detectable on selective agar plates. The extraction and purification protocol applied to various different soil types resulted in DNA of sufficient purity to permit amplification by PCR.