QUANTITATIVE-ANALYSIS OF DOUBLE-STRANDED DNA AMPLIFIED BY A POLYMERASE CHAIN-REACTION EMPLOYING SURFACE-ENHANCED RAMAN-SPECTROSCOPY

Surface-enhanced Raman spectroscopy (SERS) was utilized for the quanti tative analysis of double-stranded (ds) DNA amplified by a polymerase chain reaction (PCR). 4',6-Diamidino-2-phenylindole dihydrochloride (D API), which intercalates into ds-DNA but does not form a complex with single-stranded (ss) DNA, was added to a DNA solution after amplificat ion by PCR. When the solution was mixed, including ds-DNA-DAPI complex es and free DAPI with silver colloid sol, only free DAPI was adsorbed on the colloid surface. The dye on the colloid gave very intense SERS signals with excitation at 514.5 nm, whereas DAPI engaging in the inte rcalation with ds-DNA did not show any SERS signal. The SERS spectrum of DAPI on the colloid showed a strong band at 1610 cm(-1) due to the C=N stretching mode, and a linear relationship was observed between th e peak intensity of the C=N stretching band and the concentration of f ree DAPI. Therefore one can determine the concentration of free DAPI b y the SERS measurement. The more ds-DNA there is in the solution, the less free DAPI there is. Thus it is possible to quantitatively analyze the ds-DNA amplified by PCR indirectly by using SERS. The correlation coefficient between the peak intensity of the C=N stretching band and the concentration of ds-DNA amplified by PCR was calculated to be 0.9 88 for a concentration range from 0.1 to 1.3 mg/ml. (C) 1998 Optical S ociety of America.