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Single nucleotide polymorphism detection method by temperature-gradient affinity chromatography using a single-stranded oligo-DNA coupled column.

Katsuda T, Nishijima K, Kamura M, Nishiwada Y, Katoh S

Graduate School of Science and Technology, Kobe University, 657-8501 Kobe, Japan.

We developed an affinity chromatographic method for simple single nucleotide polymorphism (SNP) detection by use of a single-stranded DNA-coupled column and temperature gradient elution, utilizing the difference in thermal stability between hybridized double-stranded DNAs with and without mismatched base-pairs in the course of temperature gradient elution. We studied experimentally and theoretically the elution behavior of DNAs with and without SNPs in this chromatography and proposed a numerical calculation method based on a thermodynamic dissociation model. The effects of the column volume, flow rate of eluent and heating rate of the column on elution profiles were clarified. For designing DNA ligands, mismatched base-pair positions favorable for detection of SNPs were also explored by use of hybridized DNAs coding a part of the human TP53 gene.

Published 1 August 2006 in J Chromatogr A, 1123(2): 182-8.
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