Optical thermal cycler for use as a fluorimetric plate reader to estimate DNA concentrations.

Recent developments in PCR techniques provide the opportunity to quantify low copy numbers of specific nucleic acid sequences by quantitative, real-time PCR (1). The use of quantitative, real-time PCR is spreading due to its accuracy and relative simplicity. As a consequence, the number of laboratories equipped with an optical thermal cycler is increasing. The use of an apparatus for more than one single purpose is desirable because this would eliminate the need for purchasing costly extra equipment. For instance, optical thermal cyclers are also being used to perform melting curves in SNP differentiation (2), PCR product differentiation (3), and G+C mol % content estimates in microorganisms (4). Here we suggest a nonstandard use of an optical thermal cycler as a fluorescent plate reader and its application to the estimation of nucleic acid concentrations. This is achieved by using a dsDNA-specific fluorescent dye, SYBR® Green I (Molecular Probes, Eugene, OR, USA) (5). Estimates of DNA concentration are a necessary step in molecular biology. DNA concentration has been estimated in solution either from its absorbance at 260 nm or fluorescently with the use of specific dyes (6). Fluorescent estimates of DNA concentration offer a higher sensitivity than absorbance measurements so that lower quantities of DNA are needed. The availability of DNA is often a limiting factor, and fluorimetric determinations can be performed with much lower amounts of DNA than absorbance measurements. In this report, we present the use of an optical thermal cycler, the iQ iCycler® thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA), to estimate the concentration of dsDNA. The iQ iCycler is designed to obtain fluorescence emission from selected wells during PCR, although the accompanying software is flexible enough to allow for single temperature cycles. It can be used as a high-throughput alternative for estimating DNA concentrations in a 96-well plate format as well as a few tube measurements. This was achieved by designing a simple program for the software accompanying the iQ iCycler. Measurements were performed using a program at a constant temperature for 1 min at 25oC, four cycles with two steps for 12 s each at 25oC, obtaining fluorescence measurements during the second steps, and a final 1 min at 25oC. These steps resulted in three estimates, which were averaged to obtain the final estimate of dsDNA in the solution. Figure 1 outlines the protocol used for these measurements. The dsDNA was measured in a 50-μL solution composed of 1 μL DNA solution at the appropriate dilution, 44 μL TE buffer, pH 8.0 (6), and 5 μL SYBR Green I (1:100 000 final concentration). The use of TE at pH 8.0 maximizes the fluorescence by SYBR Green I because this dye presents its highest fluorescence emission at that pH. In Figure 2, we present a plot of fluorescence measurements for known DNA concentrations of genomic DNA from E. coli K12 (CECT 433) and Pseudomonas aeruginosa PAO1 (CECT 4122). Dilutions of the genomic DNA were tested at DNA concentrations ranging from 10-8 to 0.5 μg/μL. Linear relationships between fluorescence and DNA concentration occurred at a 5-fold range between 0.1 and 10-6 μg/μL. A regression line for the linear range between DNA concen-