Analysis of polymerase chain reaction products by high-performance liquid chromatography with fluorimetric detection and its application to DNA diagnosis

• Published in: Journal of chromatography. B, Biomedical sciences and applications Vol. 716; no. 1; pp. 119 - 128
• Main Authors: Arakawa, Hidetoshi, Nakashiro, Shuji, Tsuji, Akio, Maeda, Masako
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 25.09.1998; Elsevier Science
• Subjects: Alleles; Analytical, structural and metabolic biochemistry; Bacteriophage phi X 174 - genetics; Biological and medical sciences; Chromatography, High Pressure Liquid - methods; DNA; DNA Mutational Analysis; DNA Probes; Dna, deoxyribonucleoproteins; DNA, Viral - isolation & purification; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Fluorometry; Fundamental and applied biological sciences. Psychology; Genes, ras; Humans; Hybridization; Infant, Newborn; Neonatal Screening; Nucleic Acid Hybridization; Nucleic acids; Phenylketonurias - diagnosis; Phenylketonurias - genetics; Point Mutation; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymerase chain reaction; DNA; Hybridization; Performance evaluation; Double stranded DNA; Fluorescence detector; Reaction product; HPLC chromatography; Molecular biology; Quantitative analysis
• ISSN: 0378-4347; 1387-2273
• DOI: 10.1016/S0378-4347(98)00283-7

We describe the development of a sensitive high-performance liquid chromatographic (HPLC) method for polymerase chain reaction (PCR) products using bisbenzimide (Hoechst 33258 dye) based fluorimetric detection. The detection limit and specificity for double-strand DNA detection are improved in comparison with HPLC with UV absorbance detection. This HPLC, using a column packed with diethylaminoethyl-bonded non-porous resin particles, was applied to the detection of allele-specific PCR and restriction fragment length polymorphism analysis. We also developed a hybridization method analyzed by HPLC. DNA fragments (149 bp) containing the mutation site (C→A,G,T) in the N-ras gene were amplified by PCR. Fluorescein isothiocyanate (FITC)-labeled DNA probes were also prepared by PCR using FITC-labeled 5′ primer. Analysis of mutation was performed by the separation of a hybrid and non-reactive DNA probe with HPLC with fluorimetric detection after the hybridization of target DNA (149 bp) and a FITC DNA probe. The effects of various factors on hybridization were examined to establish optimal assay conditions. Under the conditions determined, a point mutation in PCR products obtained from the N-ras gene could be detected specifically by this method. The analysis of PCR products by HPLC may potentially be useful for DNA diagnosis.