Identification and separation of PCR products based on their GC content by denaturing high-performance liquid chromatography.
We show that denaturing high-performance liquid chromatography is a suitable method for the separation of DNA molecules of similar sizes but with different GC contents. A mixture of homologous molecules coming from different bacterial species may be obtained when PCR with degenerate primers is used for the amplification of a specific gene from an environmental sample. We have observed that, by selecting an appropriate temperature for the partial denaturation of the molecules, we are able to separate them according to the GC content of each DNA product. This allows us to determine if one or several types of molecules are amplified in the course of a PCR reaction. In the latter case it is possible, even with minority products, to isolate them by collecting the eluted volumes, followed by cloning, sequencing or reamplifying them by PCR, depending on the DNA concentration. We have applied this analysis to the amplification of a fragment of the ribA gene in the bacterial endosymbionts of insects, obtaining a high correlation coefficient (0.978) between retention time and the GC content of the molecules.
Belda
Eugeni
E
Departament de Genètica, Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València, Apartat 22085, 46071 Valencia, Spain.
Sentandreu
Vicente
V
Silva
Francisco J
FJ
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003562:Cytidine / Q000032:analysis*; D004247:DNA / Q000737:chemistry*; D006151:Guanosine / Q000032:analysis*; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*
D006151:Guanosine; D003562:Cytidine; D004247:DNA