Guanosine gel for sequence-dependent separation of polymorphic ssDNA.
The separation of fluorescent-labeled ssDNA fragments of equal length based on differences in sequence was achieved through the use of guanosine gels (G-gels) formed by guanosine-5'-monophosphate (GMP) in capillary gel electrokinetic chromatography (CGEKC) with LIF detection. Baseline resolution was achieved for homodimers and homopentamers of A, T, and C. G-gel CGEKC provided better resolution than CZE, MEKC, or a sieving gel in CGE. Resolution improved with increasing GMP, indicating that the interaction is linked to structural organization of the G-gel. Fluorescence intensity and anisotropy show that the order of interaction with G-gels is T>C>A. We then investigated four conformationally similar, polymorphic 76-mers with A/G substitutions that are utilized in forensic DNA typing. Resolution was achieved by CGEKC but not CZE or CGE. In CGEKC, the negatively charged G-gels and oligonucleotides electromigrate toward the positive inlet while being driven by EOF to the negative outlet. The net forward velocity is the greatest for oligonucleotides most closely associated with the slower, more cumbersome G-gel network. For the 76-mers, resolution increases with increasing difference in guanosine content between strands and, for a given difference, with increasing total guanosines in the strands.
Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
William S
WS
Case
Keren D
KD
Glinert
Sam
S
LaBarge
Linda B
LB
McGown
D001482:Base Composition; D001483:Base Sequence; D053801:Capillary Electrochromatography; D004277:DNA, Single-Stranded / Q000032:analysis*; D005456:Fluorescent Dyes; D006157:Guanosine Monophosphate / Q000737:chemistry*; D008969:Molecular Sequence Data
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D006157:Guanosine Monophosphate