Analysis of polymerase chain reaction-product by capillary electrophoresis with laser-induced fluorescence detection and its application to the diagnosis of medium-chain acyl-coenzyme A dehydrogenase deficiency.
Capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) has been developed to detect polymerase chain reaction (PCR) amplified samples. LIF detection was performed using Thiazole Orange as the fluorescent intercalating dye. This method was ca. 100x as sensitive as that with UV detection. The highly sensitive CGE-LIF was applied to the detection of the most prevalent mutation (lysine329-to-glutamic acid substitution) in medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency. The disorder, which shows an autosomal recessive inheritance, is known to be highly prevalent among Caucasian population and often mimics as Reye-like syndrome or sudden infant death. A DNA fragment containing the mutation site was PCR-amplified with two sets of allele specific oligonucleotide primers, followed by CGE-LIF. The mutant allele produced a 175-base pairs DNA fragment, which the normal allele generated a 202-base pairs DNA fragment. CGE-LIF clearly distinguished these PCR products, facilitating rapid diagnosis of MCAD deficiency.
Arakawa
H
H
School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Uetanaka
K
K
Maeda
M
M
Tsuji
A
A
Matsubara
Y
Y
Narisawa
K
K
D042964:Acyl-CoA Dehydrogenase; D010584:Bacteriophage phi X 174 / Q000235:genetics; D001483:Base Sequence; D002197:Capillary Action; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D017931:DNA Primers; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D044943:Fatty Acid Desaturases / Q000172:deficiency* / Q000235:genetics; D005453:Fluorescence; D006801:Humans; D007834:Lasers; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D004279:DNA, Viral; D004247:DNA; D044943:Fatty Acid Desaturases; D042964:Acyl-CoA Dehydrogenase; D004262:DNA Restriction Enzymes