Introduction- Fluorescence Energy Transfer (FRET)
Hybridization probes are composed of a pair of oligonucleotides, designed to hybridize adjacent to each other. Each carries a dye at their facing ends. These fluorophores can interact with a Fluorescence Resonance Energy Transfer (FRET), in which case the shorter wave dye fluorescein, excited by blue light, will transfer its energy to the longer wave dye LightCycler® Red. This fluorophore emits red light, which is detected. Therefore, the signal will only activate when both probes bind adjacent to each other. In an excess of probes, the fluorescent signal will be proportional to the accumulated target sequence.
Basics of Hybridization
Two nucleic acid strands will anneal if their sequences are complementary. The efficiency of hybridization is sequence dependent. An understanding of the molecular fundamentals simplifies the selection of adequate hybridization probes.
The hybridization process is dependent on the target sequence and the probe. Two complementary sequences will not attract each other. Rather, the individual molecules will collide by pure coincidence. If complementary structures meet, they will bind. This of course also holds true for ‘similar’ target sequences. If the flanking bases match, the hybridization process will develop from this initial crystallization point similar to the closing of a zipper.
The bond is reversible. The strength of the hybrid is calculated in thermodynamic dimensions. It can also be viewed as the binding probability. In a kinetic assay the on and off rates would be compared. For practical purposes the melting point Tm will be used as a measurable element. The Tm describes the temperature at which bound and unbound molecules are in equilibrium. The Tm can be determined experimentally in a melting curve analysis.
The probes will be present in excess in order to obtain a high binding rate.
Selection of hybridization probes
In the following text we will discuss some elementary selection criteria. They will differ for the two main applications, quantification and genotyping.
For quantification, it is important to generate a strong signal. The probes can be placed at an ideal position anywhere within the amplicon. When genotyping, the position is restricted to the site of the mutation, "difficult" sequences may not always be avoidable.
Hybridization probes may not be the adequate analysis format for some problems:
- Determination of the number of (long) repetitive elements
- The analysis of variations within repetitive sequences
- A differential quantification in a duplex PCR in the presence of extreme different quantities of the target sequences
- A differential quantification when using identical primers and type specific probe