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Time (s)

Te

mp

era

ture

(°C

)Supplementary Figure 1

Graph of temperatures measured over time in seven different temperature zones during a test run. The power supply was switched off after approximately 1000 s. The set values were 22, 25, 28, 31, 34, 37, and 40 °C.

Supplementary Figure 2Melting curve analysis using the MTAW. TC-p26-W and TC-p26-M capture sequences were spotted on agarose-coated slides as eight subarrays, each fitting into one chamber in the MTAW. The entire array was hybridized with saturating amounts of Cy3-labeled target (W54-T-W, and theslide was mounted in the MTAW device and the subarrays were subjected to washing in different zones with temperatures ranging from 25 to 60 °C at increments of 5 °C. The slide was subsequently washed at low stringency, dried, and scanned.

TC-p26-W: TTTTTTTTTTCCCCCCCCCCGCCACGCTCATCGACAAGCTTGTC

TC-p26-M: TTTTTTTTTTCCCCCCCCC············T············

W54-T-W :

5’-Cy3-GATGACTTCGCAGAGACAAGCTTGTCGATGAGCGTGGCAGCATCTTCAACTGTCGCAATT -3’

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esce

nce TC-p26-W-

TC-p26-M

Tm=39°C

Tm=37°C

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rmal

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Rat

io

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nal

in

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sity

(a.

u.)

Supplementary Figure 3

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Temperature (ºC)

Temperature (ºC)

MT signalWT signal Ratio

CD5

CD8/9

CD17

CD27/28

IVS I+6

IVS I+5

CD24

CD15

CD8

CD27/28 (12/12)

CD27/28 (13/13)

CD27/28 (12/13)

CD24 (13/13)

Melting curves of the wild-type (square) and the mutant (diamond) probe in each probe pair tested.The graphs are based on quantified signal intensity (in arbitrary units, a.u.) of the results shown in the scanning images in Figure 2 (A and B) for the corresponding temperatures. The secondary Y-axes (triangles) show the normalized ratios (see Materials and Methods) calculated from the signals of the wild-type and mutant probes at the indicated temperatures.

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24

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27/2

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15

CD

17

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I +

5

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I +

6

13nt

13nt

12nt

Tm set

Te

mp

era

ture

(d

eg

ree

C)

Calculated Tm

Tc

Observed Tm

Supplementary Figure 4

Summary of melting temperature (Tm) versus temperature for best classification (Tc).Tc values from data presented in Figures 3 and 4, and summarized in Figure 5. Calculated Tm values for the wild-type probe in each probe pair are from Table 1. Observed Tm is the temperature at which a half maximum signal was observed (Supplementary Figure 3).