The slope of the curve represents a measure of the efficiency of PCR amplification. The use of the fluorescent dye SYBR Green I allowed detection of small amounts of amplified DNA at low PCR cycle numbers, eliminating the use of radioactivity. Quantitation of PCR amplified DNA bands on stained agarose gels was performed by digital image analysis using NIH-Image with our modified gel plotting macros. The conventional plotting of product versus cycles needs more data per cycle titration for reliable quantitative analysis. Quantitation by PCR cycle titration strongly depended on the exact evaluation of the efficiency of the reaction.
Small derivations cause a huge error in the extrapolated results.
This plotting method is applicable for any kind of cycle titrations, even with labelled primer or radioactive labelled probes. Analysis of data will be more correctly due to a shorter extrapolation scale of the data. We could show that the efficiency of PCR did not alter for a wide range of dilutions for one specific PCR set at one time. The variables needed for quantitation can be easily determined. A large number of samples can be analysed with only 3-4 PCR reactions for each DNA sample.