ANALYSIS OF POLYMERASE-CHAIN-REACTION PRODUCTS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH FLUOROMETRIC DETECTION AND ITS APPLICATION TODNA DIAGNOSIS

We describe the development of a sensitive high-performance liquid chr omatographic (HPLC) method for polymerase chain reaction (PCR) product s 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. Thi s 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 develop ed a hybridization method analyzed by HPLC. DNA. fragments (149 bp) co ntaining the mutation site (C-->A,G,T) in the N-ras gene were amplifie d by PCR. Fluorescein isothiocyanate (FITC)-labeled DNA probes were al so prepared by PCR using FTTC-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 targ et DNA (149 bp) and a FITC DNA probe. The effects of various factors o n hybridization were examined to establish optimal assay conditions. U nder the conditions determined, a point mutation in PCR products obtai ned from the N-ras gene could be detected specifically by this method. The analysis of PCR products by HPLC may potentially be useful for DN A diagnosis. (C) 1998 Elsevier Science B.V. All rights reserved.