The presence of mitochondrial DNA pseudogenes in the nuclear genome has implications for primer and probe design. Approximately 0.016% of the nuclear genome is nonfunctional mitochondrial DNA (Woischnik and Moraes, 2002). This issue is more pronounced with real-time PCR amplicons as they are short in length and this increases the possibility of pseudogenic amplification. To ensure that the primers you have designed are amplifying a product of genuine mitochondrial amplification it is useful not only to check that the primers do not anneal to the nuclear genome by in silico methods (using BLAST) but to also amplify extracted DNA from rho0 cells. Rho0 cells are devoid of mtDNA therefore no amplification products should be seen.
There are specific features of mtDNA which can interfere with assay design. Strand asymmetry, the uneven distribution of purine and pyrimi-dine nucleotides across the heavy and light strands can give rise to large regions with undesirable primer characteristics. The presence of C-tract length variants and the highly polymorphic nature of the D-loop, make this region particularly difficult for robust assay design. It is suggested that investigators avoid this region unless they have good D-loop characterization for the cohort of samples they are investigating and are aware of these variations. Web based systems, such as MFold (Zuker, 2003), determine the three dimensional structure of a target DNA sequence. By understanding the structure of the region of interest, primers can be selectively placed in accessible locations. As with any PCR based assay, good primer design initially will save time and money in the long term.
Primer and probe design is covered by Wang and Seed in Chapter 5 of this book with additional considerations given in Chapter 7. The considerations when designing a mtDNA assay are the same as those required when designing any real-time assay. Most real-time PCR machines are supplied with primer design software in some format, however there are good programs freely available on the web to aid in amplicon selection.
When designing primers for a DNA binding dye real-time reaction the investigator can apply the same design parameters as for any normal PCR reaction. Amplicon size is less restrictive with binding dye reactions as the polymerase does not have to displace a probe. Amplicons in the size range 100 bp to 250 bp can be readily amplified using short, two step amplification protocols (denaturing then combined annealing/extension) with no visible effect on efficiency. With longer amplicons it may be necessary to resort to three step PCR amplification (denaturing, annealing and then extension). Wherever possible primers should be selected which have no self binding, loop or dimer formation. Where this is not possible we suggest that primers with dG binding values greater than -2 kCal/mol are avoided. From experience, primers adhering to this restriction give more reproducible results.
In probe-based assays the polymerase uses its 5' endonuclease activity to displace a bound probe, this slows the extension of the amplicon. It is with that in mind that probe based assays are not recommended to exceed
150 bp, amplicons of less than 100 bp are recommended. Restrictions on primer design follow similar parameters to those applied to primers for SYBR® Green assays. These criteria should also be applied to the probe, ensuring that no binding occurs between either primer or the probe.
When designing multiplex reactions the consideration of primer/probe interaction becomes of greater importance. For example, in a singleplex TaqMan® probe reaction there are three oligonucleotides that can interact with each other. With a four color multiplex there are now 12 oligonu-cleotides to optimize in the same reaction, posing a particular challenge during assay development.
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