An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis.
A rapid direct-extraction method was used to obtain DNA from environmental soil samples. Heat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. The DNA was purified by agarose gel electrophoresis, amplified with 16S rRNA-based primers by use of the polymerase chain reaction, and then digested with the restriction endonuclease PalI. The extraction method was used to obtain DNA from a variety of plants, bacteria, and fungi including Gossypium hirsucum (cotton), Pseudomonas, Bacillus, Streptomyces, and Colletotrichum. Up to 100 micrograms DNA/g (wet weight) of soil and 400 micrograms DNA/g of plant material were recovered. Restriction endonuclease analysis patterns of amplified rDNA from pure microbial cultures and plant species contained three to five different DNA fragments. Amplified rDNA of mixed population DNA extracts from soil samples, digested with the restriction endonuclease PalI, contained 12-20 DNA fragments, appearing as sample "fingerprints." Results from eight environmental soil samples that were analyzed suggest that the amplified rDNA fingerprints can be used to help characterize the genetic and biological diversity of the microbial populations in these samples.
Porteous
L A
LA
Biotechnology Program, United States Environmental Protection Agency, Corvallis, OR 97333.
Armstrong
J L
JL
Seidler
R J
RJ
Watrud
L S
LS
D001483:Base Sequence; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D006146:Guanidines; D017879:Isothiocyanates; D008969:Molecular Sequence Data; D010944:Plants / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D012988:Soil Microbiology
D004269:DNA, Bacterial; D004271:DNA, Fungal; D006146:Guanidines; D017879:Isothiocyanates; C054435:guanidine isothiocyanate