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We chose to build an oligo-based array because it allowed us to circumvent the labor-intensive and error-prone steps of probe amplification and purification.
The steps of probe preparation, ligation, and PCR amplification were performed using the EK1-FAM Probe Kit and the P300-100R Reference Probemix (MRC-Holland, Amsterdam, the Netherlands), following the manufacturer's protocol.
The principal steps of probe hybridization to genomic DNA (2 hours), gap-filling and ligation with probe circularization and target capture (1.5 hours), and linear DNA removal (0.5 hours) are identical to ssLPPs.
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In all measurements, aqueous Ag/AgCl is used as reference electrode Fig. 4 Impedance responses of different steps of probe-DNA attachment.
Complex impedance plot at a DC bias of +0.25 V vs Ag/AgCl in 1 mM [Fe CN 6]3−/4− + 0.1 M aqueous KCl solution for a different steps of probe-DNA attachment directly to the gold surface and b different steps of probe-DNA attachment to VASWCNTs on the gold surface.
Next efforts to improve capture are directed at the critical steps of probe-target hybridization and gap-filling efficiency.
Several pre-filtering steps of probes were performed.
In the first step of probe design, we generated candidate probes from all four organisms for the target gene sequences in a given family using MIT's Primer3 software [10].
Data preprocessing included the following five steps: generation of probe intensities data, data quality control, data transformation and normalisation, gene filtering and statistical analysis.
The fabrication steps of the probe structures have been developed by means of MEMS technologies using silicon-on-insulator (SOI) wafers.
A sliding window with the size of 50 probes and step of 1 probe was then used to calculate the percent of promoters having corresponding posttranslational histone modification.
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