Quantitative fluorescence method for continuous measurement of DNA hybridization kinetics using a fluorescent intercalator

Abstract

We present a quantitative fluorescence method for continuous measurement of DNA or RNA hybridization (including renaturation) kinetics using a fluorescent DNA intercalator. The method has high sensitivity and can be used with reaction volumes as small as 1 microliter and amounts of DNA around 1 ng. The method is based on the observations that (i) for the usual hybridization conditions, intercalators such as ethidium bromide bind (intercalate) to double-stranded DNA (dsDNA) but not single-stranded DNA or RNA and (ii) there is a large increase in fluorescence intensity when intercalators such as ethidium bromide bind to dsDNA. In this application, the intercalator can be considered as a quantitative indicator of dsDNA concentration. When a small amount of intercalator is added to a hybridizing solution, the fluorescence intensity of the intercalators increases with increase in dsDNA. The hybridization reaction can thus be monitored by continuously recording fluorescence intensity vs time. Because the amount of intercalator bound to dsDNA is not necessarily proportional to dsDNA concentration, the time-dependent fluorescence intensity graph is not identical to the kinetic graph [dsDNA] vs t. However, the fluorescence intensity vs time graph can easily be converted to the true [dsDNA] vs t graph by means of an experimental calibration graph of fluorescence intensity vs [dsDNA]. This calibration graph is obtained in a separate experiment using samples containing known amounts of dsDNA in the ethidium bromide buffer used in the kinetic measurement. We present results of experimental tests of the intercalator technique using ethidium bromide as an intercalator and DNA from Escherichia coli and lambda-phage and Poly(I)-Poly(C) RNA hybrids. These DNA and RNA samples have Cot1/2 values that cover a range of 10(6). Our experimental results show that (i) the kinetics of hybridization are not significantly perturbed by the intercalator at concentrations where no more than 10% of the binding sites on DNA or RNA hybrids are occupied, (ii) the kinetic graphs obtained by the intercalator fluorescence method and corrected with the calibration graph agree with kinetic graphs obtained by optical absorbance measurements at 260 nm, and (iii) the intercalator technique can be used in the different salt environments often used to increase the velocity of the hybridization reaction and at the hybridization temperatures (35-75 degrees C) normally used to minimize nonspecific hybridization.