Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification

Continuous fluorescence observation of amplifying DNA allows rapid and accurate quantification of initial transcript copy number. A simple and generic method for monitoring product synthesis with the double-stranded DNA dye, SYBR Green I provides initial template copy number estimation limited only by stochastic effects. To reach this degree of sensitivity, two methods were used. First, specific products generally have a higher melting temperature than nonspecific products, and therefore, specific product formation was monitored by fluorescence acquisition at temperatures at which only specific products are double-stranded. Second, anti-Taq antibodies were used to reduce nonspecific product generation. The log-linear portion of the fluorescence vs. cycle plot was extended to determine a fractional cycle number at which a threshold fluorescence was obtained. These fractional cycle numbers were plotted against the log of starting template copies to give linear standard curves from purified PCR products, allowing easy estimation of cDNA unknowns over a 10(6)-fold range. A single template molecule per reaction could be distinguished from the absence of template, although stochastic effects increased the variance of concentration estimates below 10 copies. Above 10 copies per reaction, typical replicate coefficients of variation were 6%-37%, with better precision at higher copy numbers.