Specificity of Methylation Assays in Cancer Research: A Guideline for Designing Primers and Probes

Table 1

The main characteristics for primer/probe designing in DNA methylation profiling techniques based on bisulfite conversion.

Primer/Probe	Main characteristics
MIP primer	(i) No CpG sites within the sequence.
	(ii) Including an adequate number of “C”s (no-CpG) in the sequence.
	(iii) Spanning a maximal number of CpG sites in the amplicon.
	(iv) Long length primer (25–30 mer).
	(v) Amplicon size maximum 500 bp.
MSP primer	(i) Containing as much CpG sites as possible especially at 3^′-end of the primer.
	(ii) Considering the same CpG sites in the primer sequence for methylated DNA and unmethylated DAN primers.
	(iii) Similar Tm values for both the methylated DNA and unmethylated DAN primers.
	(iv) Amplicon size maximum 500 bp.
Probe	(i) Including CpG sites to maximize specificity.
	(ii) Including several “C”s (no-CpG) in the sequence.
	(iii) Probes length 15–30 mer.
	(iv) Amplicon size 50–150 bp (max 300 bp).