Capillary electrophoretic discrimination of single nucleotide polymorphisms using an oligodeoxyribonucleotidepolyacrylamide conjugate as a pseudo-immobilized affinity ligand: optimum ligand length predicted by the melting temperature values.
We developed a weak-affinity separation system for single-nucleotide polymorphisms (SNPs) based on capillary electrophoresis. In this approach, single-stranded DNA (ssDNA)-polyacrylamide (polyAAm) conjugate was used as a pseudo-immobilized affinity ligand to separate the target DNA, cytochrome P450 2C9 (CYP2C9), and its point mutant. The ligand DNA was designed to be complementary to the normal DNA, and the target DNA was electrophoretically separated by the difference in the affinity with the pseudo-immobilized ligand in the capillary. We showed that the separation efficiency was closely associated with the Tm value of double-stranded DNA (dsDNA) consisting of the target and ligand DNA, which depends on the measurement conditions, such as the base number of the ligand DNA and the concentration of Mg2+ in the buffer solution.
Mishima
Keiko
K
Bioengineering Laboratory, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako 351-0198, Japan.
Takarada
Tohru
T
Maeda
Mizuo
M
D000180:Acrylic Resins; D000345:Affinity Labels; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000302:isolation & purification; D065729:Cytochrome P-450 CYP2C9; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D008274:Magnesium; D015394:Molecular Structure; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D013696:Temperature; D000099166:Polyacrylamides
D000180:Acrylic Resins; D000345:Affinity Labels; D001189:Aryl Hydrocarbon Hydroxylases; D065729:Cytochrome P-450 CYP2C9; D008274:Magnesium; D009838:Oligodeoxyribonucleotides; D000099166:Polyacrylamides; C450260:CYP2C9 protein, human