Table 2: Summary of the effect of optimisation on signal-to-noise ratios and on cost of the multiplex oligonucleotide ligation-PCR assay.

MOL-PCR stepOptimised parameterEffect of optimisation
Signal-to-noise ratiosCost of the assay

Preparatory workDNA isolation+++

Multiplex oligonucleotide ligationProbe concentration+++
DNA volume+
Taq DNA ligase+++a
Initial denaturation+

PCRLigation product volume
Taq DNA polymerase+++b
Cycles+

Hybridisation and analysis on MAGPIXPCR product volume+
Microspheres per reaction++++c
SAPE concentration++++d
Reporter dye++

++: major effect; +: minor effect; —: no effect; MOL-PCR: multiplex oligonucleotide ligation-PCR. aFactor 2-3 for the cost of the Taq DNA ligase used; bFactor 2 for the Taq DNA polymerase used; cFactor 3.3 for the amount of microspheres per reaction used; dFactor 2.5 for the amount of SAPE per reaction used.