Analysis of polymerase chain reaction products by high-performance liquid chromatography with fluorimetric detection and its application to DNA diagnosis.
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.
Arakawa
H
H
School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Nakashiro
S
S
Tsuji
A
A
Maeda
M
M
D000483:Alleles; D010584:Bacteriophage phi X 174 / Q000235:genetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004252:DNA Mutational Analysis; D015342:DNA Probes; D004279:DNA, Viral / Q000302:isolation & purification; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D005470:Fluorometry; D011905:Genes, ras; D006801:Humans; D007231:Infant, Newborn; D015997:Neonatal Screening; D009693:Nucleic Acid Hybridization; D010661:Phenylketonurias / Q000175:diagnosis / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length
D015342:DNA Probes; D004279:DNA, Viral; D005456:Fluorescent Dyes; D016650:Fluorescein-5-isothiocyanate