Application of a new hybrid organic-inorganic monolithic column for efficient deoxyribonucleic acid purification.
A new hybrid organic-inorganic monolithic column for efficient deoxyribonucleic acid (DNA) extraction was prepared in situ by polymerization of N-(beta-aminoethyl)-gamma-aminopropyltriethoxysilane (AEAPTES) and tetraethoxysilane (TEOS). The main extraction mechanism was based on the Coulombic force between DNA and the amino silica hybrid monolithic column. DNA extraction conditions, such as pH, ion concentration and type, and loading capacity, were optimized online by capillary electrophoresis with laser-induced fluorescence detection. Under optimal condition, a 6.0-cm monolithic column provided a capacity of 48 ng DNA with an extraction efficiency of 74+/-6.3% (X+/-RSD). The DNA extraction process on this monolithic column was carried out in a totally aqueous system for the successful purification of DNA and removal of proteins. The PBE2 plasmid could be extracted from Bacillus subtilis (B. subtilis) crude lysate within 25 min, and the purified DNA was suitable for the amplification of a target fragment by polymerase chain reaction. This study demonstrates a new attractive solid-phase support for DNA extraction to meet the increasingly miniaturized and automated trends of genetic analyses.
Yu
Shengbing
S
College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China.
Geng
Jing
J
Zhou
Ping
P
Wang
Jing
J
Feng
Airong
A
Chen
Xiangdong
X
Tong
Hua
H
Hu
Jiming
J
D004247:DNA / Q000302:isolation & purification*; D007287:Inorganic Chemicals / Q000737:chemistry*; D009930:Organic Chemicals / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results
D007287:Inorganic Chemicals; D009930:Organic Chemicals; D004247:DNA