Oligonucleotide fluorescent probes

We appreciate that there are a variety of different types of probe technologies available to the investigator. Detailed information of the different probes can be found in Chapters 1 and 5. For the purpose of this chapter we will simply refer to all types of them under the umbrella term 'probes.'

Probes have two major advantages over DNA binding dyes. First they add an extra layer of specificity to a reaction. The correct binding of both primers and the probe are required to generate a signal. This combination of primer/probe binding makes it less likely to generate a signal from a nonspecific product or from misannealing.

Second, probes facilitate multiplex reactions. Oligonucleotide probes can be labeled with fluorophores which emit at different wavelengths, this means different probes can be used to detect multiple targets within a single reaction tube. Using the machines currently available it is possible to perform a four color multiplex. It should be noted however, that the optimization required to perform this is extensive and expensive for both time and reagents. A large proportion of the genetic analysis performed in the mitochondrial field is carried out at the single-cell level, limiting DNA quantity, pressing the investigator into detecting several targets in a single reaction.

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