New hydroxyapatite monolithic column for DNA extraction and its application in the purification of Bacillus subtilis crude lysate.
A hydroxyapatite (Hap) monolithic column with micrometer macropores skeleton structure was prepared by sol-gel technique for efficient DNA extraction. The main extraction mechanism of this monolithic column was attributed to the electrostatic interaction between the phosphate groups of DNA and the calcium ions (C site) of Hap. DNA extraction conditions, such as pH, ion concentration, ion type and loading capacity, on the monolithic column were optimized online by capillary electrophoresis with laser-induced fluorescence detection. Under the optimal condition, a 6 cm length monolithic column provided a capacity of 40 ng DNA with an extraction efficiency of 64+/-6.2% (X+/-RSD). As low concentration of salts were used in the extraction procedure, the purified PBE2 plasmid from the Bacillus subtilis crude lysate could be amplified by polymerase chain reaction. This result illustrated that Hap was a potential matrix for DNA purification from complex biological samples which was compatible with the subsequent genetic analysis in miniature format. Since the preparation of this monolithic column was very simple, it was possible to integrate this novel matrix with chip to allow rapid and efficient DNA purification in microscale. This study provided a new attractive solid-phase support for DNA extraction to meet the miniaturized and automated trends of genetic analysis.
Yu
Shengbing
S
College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, China.
Geng
Jing
J
Zhou
Ping
P
Wang
Jing
J
Chen
Xiangdong
X
Hu
Jiming
J
D001412:Bacillus subtilis / Q000737:chemistry / Q000235:genetics*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D017886:Durapatite; D006863:Hydrogen-Ion Concentration; D010710:Phosphates; D017680:Potassium Compounds; D015203:Reproducibility of Results; D052616:Solid Phase Extraction / Q000295:instrumentation* / Q000379:methods
D004269:DNA, Bacterial; D010710:Phosphates; D017680:Potassium Compounds; D017886:Durapatite; C013216:potassium phosphate