Guidelines for Optimisation of a Multiplex Oligonucleotide Ligation-PCR for Characterisation of Microbial Pathogens in a Microsphere Suspension Array

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 step	Optimised parameter	Effect of optimisation
		Signal-to-noise ratios	Cost of the assay
Preparatory work	DNA isolation	++	+
Multiplex oligonucleotide ligation	Probe concentration	++	+
	DNA volume	+	—
	Taq DNA ligase	+	++^a
	Initial denaturation	+	—
PCR	Ligation product volume	—	—
	Taq DNA polymerase	+	++^b
	Cycles	+	—
Hybridisation and analysis on MAGPIX	PCR 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.