Layer Enhanced Fluorescence DNA Chip Setup

Cluster-Layer Enhanced Fluorescence DNA Chip Setup 

To produce binding possibilities for DNA the chip is covered with poly-lysine (0.1% of poly-lysine in phosphate buffered saline (PBS), pH 7.4). Alternatively it can be coated with BSA (Img/ml in PBS pH 7.4), but this approach introduces the need of further activation. Therefore after removing unbound BSA via washing with MQ water the chip is incubated with amino-silane (10 mg/ml N-(3-Dimethylaminopropyl)-N-Ethyl-carbodiimide (EDC) in PBS pH 7), cross-linking and activating the BSA-surface. 

Immediately after EDC activation amino-modified single stranded DNA probes (5 pmol/Fl in PBS, pH 5.4) are arrayed onto the chip. To enable the EDC chemistry to work the chip is stored in a humidity chamber for 30-60 minutes (preferably at 60°C, but also RT is possible), afterwards washed with MQ water and air-dried. Crosslinking with UV-light can be done to further stabilizing the DNA. 

Hybridisation is executed in 3-5 SSC or Dig Easy hybridisation buffer (1-16 h), if the analyte DNA is labeled with a fluorophore (e.g. a PCR product) the chip is washed (S-S SSC and 0.3 SSC), air-dried and scanned. In many cases the analyte DNA will come unlabelled, then it is bound as above, afterwards a complementary reporter DNA (2 pmol/Fl), which is fluorescently labeled (e.g. Cy3 or Cy5) is hybridized to a free sequence of the analyte DNA in 35 SSC or Dig Easy hybridization buffer for 15 minutes to 3 hours. It should be noted that the hybridization temperature of the second step must be below the one of the first step. Finally the chip is washed (3-5 SSC and 0.3 SSC), air-dried and scanned. 

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