SYNTHESIS

PentaBase is providing probes and primers worldwide every day. All our oligo synthesises are performed using phosphoramidite chemistry. We use solid fase CPGs of the highest quality to insure a high yield. After end of synthesis the oligos are cleaved from the support and purified. Primers are synthesised with DMT-on. Each oligo is followed using trityl monitoring during synthesis.

See an example of Quality Control of Trityl data

PURIFICATION

All PentaBase probes are purified by reverse-phase HPLC in order to give the purest products. Primers are also purified by reverse-phase HPLC followed by desalting and removal of the final DMT group. Desaltning removes residual by-products from the different processes.

See an example of Quality Control of HPLC data

QUALITY of fluorescence and affinity of probes

Every probe from PentaBase are strictly controlled for quality and fluorescense potential in an affinity test. To do this test we synthesize a complementary oligotarget and run a melting curve from 95^°C to 35^°C. The figure below shows the result of such a test. The red curve shows the probe in presence of it's complementary target and the yellow line shows the probe without a target. Half way down the dip in fluorescence of the red line corresponds to the melting temperature of the duplex. The exact melting temperature of the probe-target duplex is found by calculating the inverted derivative of the curve.

Above is shown an example of the data obtained from an affinity study. PentaBase make such a study on every probe. The height of the signal indicates the "potential" of the fluorophore.  The temperature where a drop in fluorescence is observed is related to the affinity of the probe.