Quantitative Assessment of Heteroplasmy of Mitochondrial Genome: Perspectives in Diagnostics and Methodological Pitfalls

Table 2

The basic methods suitable for quantitative measurement of mtDNA heteroplasmy based on qPCR with fluorescence detection.

	Principle of operation	Advantages	Disadvantages
Nonspecific methods
Intercalating dyes (SYBR Green I and others)	The fluorescence signal increases due to an intercalating dye binding to double-stranded DNA	High level of signal, the same approach to the design of various experiments	False positive results are possible due to primer-dimers or nonspecific amplicons, so these methods are not immediately suitable for diagnostic purposes
Amplifluor	Allele-specific PCR reveals a hairpin structure of fluorescently labeled oligonucleotides
Lux	The fluorescence signal changes when the fluorescently labeled primer is included in the PCR product
DzyNA-PCR	Binding of the probe with the PCR products generates restriction enzyme recognition site
Specific methods
TaqMan	The dye and the quencher are separated by 5′-exonuclease activity of Taq-polymerase	High signal level, simplicity of design, and synthesis	Problems with the discrimination of some sequences
Molecular beacons	Dye and quencher are separated after binding of the probe to the amplicon and disclosure of the hairpin	Good signal-to-noise ratio	The low level signal due to reaction kinetics
Eclipse	Similar to TaqMan, but is resistant to exonuclease activity of the polymerase; a signal is generated after binding of the probe to the amplicon	High signal level, simplicity of design, and synthesis	Poor signal-to-noise ratio, problems with the discrimination of some sequences
Hyb probes	Two dyes are converging after binding of the probes to the amplicon, the signal is generating by the FRET mechanism	Undefined	Poor signal-to-noise ratio, not suitable for all devices, the reaction requires a trimolecular interaction
Scorpions	The probe-primer is included in the PCR product, the signal is generated after disclosure of the hairpin and binding of the free end of the probe to the amplicon	Good signal-to-noise ratio, monomolecular reaction	Relatively expensive synthesis