An effective method of DNA extraction for bioleaching bacteria from acid mine drainage.
An effective and versatile method for microorganism lysis and direct extraction of DNA from bioleaching bacteria was developed using pure cultures and an acid mine drainage (AMD) sediment sample. In the described method, microorganisms are treated at three different incubation temperatures: boiling water incubation for 6-10 min, followed by 60 +/- 5 degrees C for 30 min, then 72 degrees C for 30 min. The extracted DNA is purified using a phenol/chloroform/alcohol mixture and precipitated in absolute alcohol. The 16S ribosomal RNA (rRNA) and gyrB genes of the pure cultures were amplified using the polymerase chain reaction (PCR) and differentiated using repetitive intergenic DNA sequences amplification (Rep-PCR). For the AMD sediment sample, the 16S rRNA and gyrB genes of the amplicons were digested with Hin6I and MspI, and the restriction fragment length polymorphism analysis patterns were used as a fingerprint to discern community diversity. The results indicated that this method is a versatile, reproducible, effective, and rapid technique for routine DNA extraction from bioleaching bacteria. The low cost of this method also makes it attractive for large-scale studies.
Zeng
Leping
L
Xiang-Ya School of Medicine, Central South University, Changsha, China.
Huang
Jufang
J
Zhang
Yanfei
Y
Qiu
Guanzhou
G
Tong
Jianbin
J
Chen
Dan
D
Zhou
Jin
J
Luo
Xuegang
X
D042763:Acidithiobacillus / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics; D016172:DNA Fingerprinting; D027081:DNA Gyrase / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000235:genetics / Q000302:isolation & purification; D005821:Genetic Techniques / Q000191:economics; D019015:Geologic Sediments / Q000382:microbiology*; D008906:Mining; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000302:isolation & purification; D014871:Water Microbiology
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D027081:DNA Gyrase