Application of CE for determination of DNA base composition.
DNA base composition expressed as mol% of guanine plus cytosine (% GC) or GC content is a key parameter of bacterial taxonomy and genomic analyses. Direct chemical determination methods such as HPLC as well as indirect methods based on physical properties of deoxyribonucleic acid (DNA), melting point (T(m)), and buoyant density (B(d)) have been conventionally applied to determine the GC content. However, these methods require relatively large amounts of sample DNA, time, and labor. We have developed a protocol to determine the GC content by fine separation of nucleosides with CZE. Genomic DNAs with known GC content from 23 bacterial strains were determined by CE at the optimized conditions of 27 degrees C, 20 kV in 50 mM of NaHCO(3) (pH 9.0) and 70 mM SDS added. Nucleosides from <1 microg of DNA hydrolyzed with nuclease-P1 and bacterial alkaline phosphatase were separated in a 75 microm wide and 80 cm long silica capillary. The nucleoside peak areas were determined at 254 nm in less than 12 min. The CE-based determination of GC content requires only small amounts of DNA, and thus should be applicable to environmental genomics (metagenomics), as >90% of environmental micro-organisms are nonculturable and produce only small amounts of genomic DNA.
Hua
Ngoc-Phuc
NP
Graduate School of Biosphere Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima, Japan.
Naganuma
Takeshi
T
D001482:Base Composition; D003596:Cytosine / Q000032:analysis*; D004269:DNA, Bacterial / Q000032:analysis*; D019075:Electrophoresis, Capillary; D006147:Guanine / Q000032:analysis*
D004269:DNA, Bacterial; D006147:Guanine; D003596:Cytosine