Introduction

The ultimate goal of many genetic analysis methods is to discover the full identity of DNA segments for which the sequence is only partially known. The first step often entails the selective hybridization of a synthetic oligonucleotide probe to the target DNA molecule under investigation. Successful annealing of the probe and target results in the formation of a structure called a DNA duplex, which can either be detected directly or alternatively and can act as a substrate for further enzymatic and/or chemical manipulations (1,2). In either case, the end product is frequently detected using one of a number of fluorescence detection methods (3).

An effective way of generating fluorescence signals is through a phenomenon called fluorescence resonance energy transfer (FRET) (4). FRET systems

From: Methods in Molecular Biology, vol. 335: Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols Edited by: V. V. Didenko © Humana Press Inc., Totowa, NJ

consist of pairs of matched fluorophores in which the emission spectrum of a donor fluorophore overlaps the excitation spectrum of the acceptor fluorophore. The arrangement in hybridization-based assays could be, for example, having the donor attached to the target DNA and an acceptor attached to the oligonucleotide probe. Before hybridization, the donor fluorophore will emit fluorescence if illuminated with the appropriate wavelength of light. Upon hybridization, however, the donor and acceptor are brought close together by formation of the DNA duplex. If positioned sufficiently close, i.e., within the Förster radius of 10-100 Ä (5,6), the donor fluorophore can resonantly transfer some of its excitation energy to the acceptor fluorophore. The acceptor fluorophore, in turn, can dissipate the energy by emitting fluorescence. An increase of fluorescence signals at the emission wavelength of the acceptor is a positive indication of energy transfer, the presence of energy transfer indicates probe-target hybridization, and the hybridization event indicates that the probe and target have complementary sequences. Because DNA hybridization is dependent on temperature as well as on sequence complementarity, a more thorough analysis would be achieved by generating a melting curve in which the DNA hybridization status is measured over a range of temperatures.

DNA melting curves can be created using a variation of FRET termed induced FRET (iFRET) (7). The key defining feature of iFRET is the use of a dye that is specific for double-stranded DNA as the donor fluorophore. Instead of attaching a donor covalently to the target, the donor fluorophore is added to the solution containing the DNA target and oligonucleotide probe. After probetarget hybridization, the donor fluorophore can interact with the DNA duplex and be excited (see Fig. 1). Energy transfer to the acceptor labeled probe is evident as a dramatic increase in acceptor fluorescence. To generate a melting curve, the sample can be heated slowly from low to high temperature while continually monitoring the fluorescence of the acceptor. When the melting temperature (Tm) of the probe-target duplex is reached and surpassed, the probe and target will disassociate, releasing the donor fluorophore back into solution. This results in the loss of energy transfer which is marked by a large drop in fluorescence in the melting curve. To simplify interpretation of the results, the negative derivative of the fluorescence as function of temperature can be plotted resulting in a graph where the peak represents the Tm of the probe-target duplex.

In the current procedure, the target DNA is produced by polymerase chain reaction (PCR) and subsequently immobilized to the well of a microtiter plate. The immobilization is made possible by the addition of a 5' biotin to one of the PCR primers, and by the use of a microtiter plate coated with streptavidin. Capture of the biotinylated PCR product to the streptavidin-coated plate is highly efficient and serves to simplify many of the steps outlined in the remainder of the procedure.

Fig. 1. The chemical components and basic principle for induced fluorescence resonance energy transfer (iFRET) detection of DNA hybridization status. The necessary components include a DNA target, a complementary probe bearing an acceptor molecule such as 6-caboxy-X-rhodamine, and a hybridization solution containing SYBR Green I (the iFRET donor). In the nonhybridized state, each component is separate and effective energy transfer is prevented. After hybridization, the donor dye binds to the probe-target duplex, which then enables energy transfer to the acceptor attached to the probe. Because the hybridization reaction is reversible, the hybridization status can be determined by monitoring the fluorescence output of the acceptor.

Fig. 1. The chemical components and basic principle for induced fluorescence resonance energy transfer (iFRET) detection of DNA hybridization status. The necessary components include a DNA target, a complementary probe bearing an acceptor molecule such as 6-caboxy-X-rhodamine, and a hybridization solution containing SYBR Green I (the iFRET donor). In the nonhybridized state, each component is separate and effective energy transfer is prevented. After hybridization, the donor dye binds to the probe-target duplex, which then enables energy transfer to the acceptor attached to the probe. Because the hybridization reaction is reversible, the hybridization status can be determined by monitoring the fluorescence output of the acceptor.

The following protocols describe procedures for creating DNA melting curves using an iFRET detection strategy. The primary advantage of the iFRET system over the simple use of SYBR® Green I alone is the increased specificity gained by the energy transfer reaction. Fluorescence signal from the probetarget hybridization are spectrally separated from background fluorescence produced by SYBR Green I binding to DNA structures other than those of the duplex under investigation. When compared with traditional FRET detection systems as mentioned above, iFRET offers approx 40 times greater fluorescence signal strength and requires the covalent linkage of only one of the FRET-pair dyes (7).

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