Table 1. Thermal stability study and 5′-end selectivity.

Chemistry	5′-end	Derivatised glass surface
		ATS			BTS
		Loading densitya fmol/mm2	5′-specifica fmol/mm2	Thermal stabilityb	Loading densitya fmol/mm2	5′-specifica fmol/mm2	Thermal stabilityb
EDC	P	9 4	2 2	40 6%	14 1	4 4	60 5%
	OH	7 1			9 1
	DMT	8 2				10 4
s-MBS	SH	25 5	22 4	56 5%	28 5	24 4	52 3%
	OH	3 1			5 0
	DMT	3 1			2 1
s-SIAB	SH	47 21	32 21	51 6%	43 14	26 4	53 8%
	OH	10 4			15 4
	DMT	20 15			18 10
s-SMCC	SH	nd	nd	45 8%	nd	nd	40 3%
s-GMBS	SH	nd	nd	45 6%	nd	nd	45 4%
s-MPB	SH	nd	nd	50 8%	nd	nd	48 6%

Oligonucleotide, up₁. with 5′-SH, 5′-OH, 5′-P, 5′-NH₂ or 5′-DMT were coupled to glass which was derivatised with ATS or BTS according to the type of chemistry used.

^aThe loading density of bound oligonucleotides (fmol/mm²) was determined by hybridisation with a radioactively labelled complementary oligonucleotide, Rup₁. As a control, a non-complementary radioactively labelled oligonucleotide (Rup₂) was used. The loading density of up₁ specifically attached via the 5′-end is calculated as the difference between the density obtained with 5′-SH or 5′-P oligonucleotides and the average of densities obtained with the control oligonucleotides (5′-OH and 5′-DMT).

^bThe thermal stability parameter was calculated as a percentage of attached oligonucleotides remaining after the PCR experiment. Oligonucleotides were hybridised using a complementary fluorescently labelled oligonucleotide (5′-FITC Rup₁) before and after PCR treatment and the fluorescence signal measured using the epi-fluorescence micrsocope. A non-complementary labelled oligonucleotide was used to check the specificity of the hybridisation (5′-FITC Rup₂).

nd, not determined.