Single nucleotide polymorphism detection method by temperature-gradient affinity chromatography using a single-stranded oligo-DNA coupled column.
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.
Katsuda
Tomohisa
T
Graduate School of Science and Technology, Kobe University, 657-8501 Kobe, Japan.
Nishijima
Ken
K
Kamura
Mitsumasa
M
Nishiwada
Yasushi
Y
Katoh
Shigeo
S
D020137:Base Pair Mismatch; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods; D004277:DNA, Single-Stranded / Q000737:chemistry*; D009691:Nucleic Acid Denaturation; D020641:Polymorphism, Single Nucleotide; D013696:Temperature
D004277:DNA, Single-Stranded