Detection of single-base mutation by affinity capillary electrophoresis using a DNA-polyacrylamide conjugate.
We have developed an affinity capillary electrophoresis (ACE) method for detection of gene point mutations using a DNA-polyacrylamide conjugate as a pseudostationary affinity phase. In this study, the target DNA was prepared by mixing two PCR products: the wild type of K-ras gene and its codon 12 point mutant. The ligand DNA was designed to be complementary to codons 11 and 12 of the wild type. The target DNA was denatured by the addition of formamide and by heating at 95 degrees C for 5 min, and then electrophoretically separated by difference in affinity to the pseudoimmobilized ligand DNA. The method successfully separated a mixture of the wild-type DNA and each of six codon 12 point mutants by the same ligand DNA. The limit of mutation detection was determined by mixing the wild-type DNA with decreasing concentrations of the mutant DNA. The lowest level of detection was 10% mutant DNA in a background of the wild type. The practicability of this method has been confirmed using a colorectal carcinoma cell line. This study is the first demonstration of detection of gene point mutation in polymerase chain reaction (PCR) products using ACE, and opens up a new possibility of CE-based gene diagnosis.
Sato
Kae
K
Bioengineering Laboratory, RIKEN (The Institute of Physical and Chemical Research), Hiroshima 2-1, Wako, Saitama 351-0198, Japan.
Inoue
Akira
A
Hosokawa
Kazuo
K
Maeda
Mizuo
M
D000180:Acrylic Resins / Q000737:chemistry; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D000099166:Polyacrylamides
D000180:Acrylic Resins; D004247:DNA; D004273:DNA, Neoplasm; D000099166:Polyacrylamides