Table 1.

Melting temperature determination for different probe designs

	Measured θmb
Probea	HP:QO	HP:target perfect- match	HP:target 1 mismatchc	HP:target 2 or 3 mismatchesd
HP20	32	61	52	n/a
HP20:QO12 (1:1)	39	61	52	n/a
HP20:QO12 (1:3)	42	61	52	n/a
HP20:QO14 (1:1)	46	61	52	n/a
HP20:QO14 (1:3)	48	61	52	n/a
HP20:QO16 (1:1)	52	61	n/b	n/a
HP20:QO16 (1:3)	55	61	n/b	n/a
HP25	21	62	57	49
HP25:QO12 (1:1)	38	63	55	50
HP25:QO12 (1:3)	43	62	57	49
HP25:QO14 (1:1)	47	62	55	49
HP25:QO14 (1:3)	49	63	56	n/d
HP25:QO16 (1:1)	52	63	55	n/b
HP25:QO16 (1:3)	55	63	55	n/b
HP31	∼20	68	63	51
HP31:QO12 (1:1)	39	67	62	51
HP31:QO12 (1:3)	43	67	62	51
HP31:QO14 (1:1)	46	67	64	51
HP31:QO14 (1:3)	49	67	63	n/d
HP31:QO16 (1:1)	52	67	63	n/d
HP31:QO16 (1:3)	55	67	63	n/d

^aProbes tested in melting analysis were either hybridization probe alone or partially double-stranded probes formed with hybridization probe (HP) and quencher oligo (QO). The double-stranded probes are named as HP:QO (ratio of [HP]/[QO]). Concentration for HP20, HP25 and HP31 was set at 81 nM.

^bθ_m (melting temperature) was calculated as the temperature where maximal absolute first-derivative of fluorescence change over temperature (|dfl/dT|) was observed.

^cComplement DNA that formed the HP:target duplex contained a single mismatch at the 12th position starting from the 5′ end of HP.

^dComplement DNA that formed the HP:target duplex contained three mismatches at the 12th, 18th and 27th positions starting from the 5′ end of HP. Thus there were two mismatches for HP20 and HP25 and three mismatches for HP31.

n/a: not tested. n/d: tested but θ_m not determined because the melting profile showed a bi-phasic curve where first derivative method cannot accurately calculate θ_m. n/b: probe did not fully bind to target even at the lowest temperature tested. Therefore, accurate estimation of melting temperature for HP:target was not possible.