While conventional MLPA is already a highly robust technique for the detection of copy number changes, digitalMLPA takes this a step further. Several factors contribute to the enhanced robustness of digitalMLPA as opposed to conventional MLPA.
- digitalMLPA is less sensitive to contaminants in sample DNA.
- A barcode solution is added to the sample DNA, which stabilizes the DNA and dilutes the sample DNA, reducing the effect of contaminants. In addition, the barcode solution has buffering capacity, preventing depurination during the denaturation step.
- Compared to conventional MLPA, polymerase activity is increased, resulting in a lower sensitivity to polymerase inhibitors. This reduces the effect of certain contaminants. Enhancing polymerase activity is not possible in conventional MLPA, as this would lead to a high number of nonspecific peaks, which, contrary to digitalMLPA, cannot be removed by the analysis software.
- Nonspecific amplicons can be identified and removed by the Coffalyser digitalMLPA software.
- digitalMLPA probemixes can contain up to 1,000 target probes, allowing better coverage of the regions of interest. In comparison, maximally 60 target probes can be included in conventional MLPA probemixes.
- digitalMLPA probemixes can contain more reference probes than conventional MLPA probemixes, which decreases the effect of an aberrant reference probe on the analysis, thereby making the analysis more robust.
- digitalMLPA probemixes contain additional control probes, allowing a broader quality assessment. This includes probes to evaluate sample DNA fragmentation, depurination, denaturation, hybridisation temperature, completeness of the hybridisation reaction, ligation start temperature, ligase & polymerase activity, and ligase inactivation (for details, see the digitalMLPA General Protocol).
- Control probes are used to automatically identify the probemix lot, barcode solution lot and sex of the sample DNA, reducing the chance of mix-ups and sample swaps.
- digitalMLPA probemixes incorporate SNP-specific control probes to help identify contamination of the sample DNA or reagents with DNA from another individual.