QUANTIFICATION OF LOW-COPY TRANSCRIPTS BY CONTINUOUS SYBR(R) GREEN-I MONITORING DURING AMPLIFICATION

Continuous fluorescence observation of amplifying DNA allows rapid and accurate quantification of initial transcript copy number A simple an d generic method for monitoring product synthesis with the double-stra nded DNA dye, SYBR(R) Green I provides initial template copy number es timation limited only by stochastic effects. To reach this degree of s ensitivity, two methods were used. First, specific products generally have a higher melting temperature than nonspecific products, and there fore, specific product formation was monitored by fluorescence acquisi tion at temperatures at which only specific products are double-strand ed. Second, anti-Tag antibodies were used to reduce nonspecific produc t generation. The log-linear portion of the fluorescence vs. cycle plo t was extended to determine a fractional cycle number at which a thres hold fluorescence was obtained These fractional cycle numbers were plo tted against the log of starting template copies to give linear standa rd curves from purified PCR products, allowing easy estimation of cDNA unknowns over a 10(6)-fold range. A single template molecule per reac tion could be distinguished from the absence of template, although sto chastic effects increased the variance of concentration estimates belo w 10 copies. Above 10 copies per reaction, typical replicate coefficie nts of variation were 6%-37%, with better precision at higher copy num bers.