Methods for electronic fluorescent perturbation for analysis and electronic perturbation catalysis for synthesis
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example 1
Ras G Match / Mismatch
[0097] APEX Chip Preparation and Capture Probe Loading—APEX active DNA chips, with 25 microlocation test sites (80 microns in diameter) were coated with streptavidin agarose accordingly. A 2.5% glyoxal agarose (FMC) solution in water was made according to manufacturer's instructions. The stock was equilibrated at 65° C., for 5 minutes. Chips were spin coated at 2.5K rpm for 20 seconds. Another layer was then applied at 10K rpm for 20 seconds. This second “thin layer was composed of a 1:4 mix of 5 mg / ml streptavidin (BM) in 50 mM NaPhosphate, 250 mM NaCl and 2.5% glyoxal agarose.
[0098] The chips were baked at 37° C. for 30 minutes. Streptavidin was coupled to the agarose via Schiff's base reduction in 0.1M NaCNBH3 in 0.3M NaBorate, pH 9.0, for 60 minutes, at room temperature. The remaining aldehydes were capped with 0.1M glycine, for 30 minutes, at room temperature, and finally rinsed in water, dried under N2 and then stored at 4° C.
[0099] The table below gives...
example 2
Ras G and HLA Match / Mismatches
[0101] The APEX chip preparation procedure was the same as Example 1. Capture probe addressing conditions were the same as Example 1. The Ras 415 sequence was electronically addressed to all 5 microlocations in column 1 and Ras 416 addressed to all 5 microlocations in column 2 of the APEX chip. The HLA 241 sequence was addressed to all 5 microlocations in column 4 and HLA 378 was addressed to all 5 microlocations in column 5. The Ras 411 and HLA 253 fluorescent target probes were mixed and passively hybridized to the APEX chip. Electronic dehybridization and stringency was carried out for the Ras system at 1.5 μA / microlocation, DC pulsing for 0.1 sec on, 0.2 sec off, 150 cycles (20 mM NaPhosphate, pH 7.4). Electronic dehybridization and stringency for the HLA system was carried out at 0.6 μA / microlocation, DC pulsing for 0.1 sec on, 0.2 sec off, 150 cycles (20 mM NaPhosphate, pH 7.4). Data collected as reported above. FIG. 6 shows the results for Examp...
example 3
Fluorescent Perturbation Effect with Single Fluorophore
[0102] APEX Chip Preparation and Capture Probe Loading—APEX active DNA chips, with 25 microlocation test sites (80 microns in diameter) were coated with streptavidin agarose accordingly. A 2.5% glyoxal agarose (FMC) solution in water was made according to manufacturer's instructions. The stock was equilibrated at 65° C., for 5 minutes. Chips were spin coated at 2.5K rpm for 20 seconds. Another layer was then applied at 10K rpm for 20 seconds. This second “thin layer was composed of a 1:4 mix of mg / ml streptavidin (BM) in 50 mM NaPhosphate, 250 mM NaCl and 2.5% glyoxal agarose. The chips were baked at 37° C. for 30 minutes. Streptavidin was coupled to the agarose via Schiff's base reduction in 0.1M NaCNBH3 in 0.3M NaBorate, pH 9.0, for 60 minutes, at room temperature. The remaining aldehydes were capped with 0.1M glycine, for 30 minutes, at room temperature, and finally rinsed in water, dried under N2 and then stored at 4° C.
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