FLUORESCENCE CROSS-CORRELATION - A NEW CONCEPT FOR POLYMERASE-CHAIN-REACTION

In this article we present a new concept for the detection of any spec ifically amplified target DNA sequences in multiple polymerase chain r eactions (PCR) based on fluorescence correlation spectroscopy (FCS). T he accumulation of double-stranded target DNA is monitored by the cros s-correlated fluorescence signals provided by two amplification primer s which are 5'-tagged with two different kinds of fluorophores (Rhodam ine-Green(TM) and CY5(TM)). Only the amplified target DNA sequence car rying both primers is observed. Its signal emerges from the background of non-incorporated or non-specifically incorporated primers. Down to 10-25 initial copy numbers of the template in the PCR compartment DNA can presently be detected. No external or internal standards are requ ired for determining the size and the amplified copy number of specifi c DNA. The PCR amplification process is started with all ingredients i n a single compartment (e.g, of a microtiter plate), in which amplific ation and measurement are performed. This eliminates the need for post -PCR purification steps. The homogeneous one-tube approach does not de pend on fluorescence energy transfer between the fluorogenic dyes. Thu s, it does not interfere with the enzymatic amplification reaction of PCR and allows the continued use of different conditions for amplifyin g DNA. The results exemplified by PCR-amplified 217-bp and 389-bp targ et DNA sequences demonstrate that the analysis based on two-color fluo rescence cross-correlation is a powerful method for simplifying the id entification of targets in PCR for medical use. For this purpose, an i nstrument optimized for two-color excitation and detection of two-colo r emission has been developed, incorporating the principle of confocal arrangement. (C) 1998 Elsevier Science B.V. All rights reserved.