No abstract

In this manuscript, we discuss the use of photoactivated polycarbonate (PC) for purification of dye-labeled terminator sequencing fragments using solid-phase reversible immobilization (SPRI) prior to gel electrophoretic sorting of these DNAs. An immobilization bed for the DNA purification was produced by exposing a posted microchannel to UV radiation, which induced a surface photooxidation reaction, resulting in the production of carboxylate groups. The immobilization microchannel contained microposts to increase the loading level of DNAs to improve signal intensity without the need for preconcentration. By suspending the sequencing cocktail in an immobilization buffer (TEG/ethanol), the DNA fragments demonstrated a high affinity for this carboxylated surface. The loading density of DNAs to this activated surface was found to be 3.9 pmol cm(-2). The captured DNA could be subsequently released from the surface by incubation with ddH2O. SPRI cleanup of dye-terminator sequencing fragments using the photoactivated PC chip and slab gel electrophoresis produced a read length comparable to the conventional SPRI format, which utilized carboxylated magnetic beads and a magnetic field. The read length for the PC-SPRI format was found to be 620 bases with a calling accuracy of 98.9%. The PC-SPRI cleanup format was also integrated to a capillary gel electrophoresis (CGE) system. The PC-SPRI method was shown to effectively remove excess dye terminator from the CGE tract, but yielded lower plate numbers, as compared to a direct injection method with purification accomplished off-chip. The loss in efficiency was found to result primarily from the extended injection time associated with the microchip purification method.

The levels of uridine diphosphate galactose (UDPGal) and uridine diphosphate glucose (UDPGlc) have been determined in liver autopsy samples, erythrocytes and cultured skin fibroblasts from galactosaemic patients and compared to non-galactosaemic controls. In patients with undetectable erythrocyte galactose-1-phosphate uridyltransferase (transferase) activity, the levels of UDPGal were substantially lower than in controls. In patients with detectable transferase activity, even though in less than 1% of normal values, both UDPGal and UDPGlc levels were in the normal range. Incubation of erythrocytes from both galactosaemic patients and normal individuals with 10 mmol/L uridine increased UDPGal and UDPGlc levels several-fold, both in the presence or absence of galactose in the incubation medium. We hypothesize that a deficit of UDPGal is responsible for the late onset clinical manifestations in galactosaemia which include ovarian failure, speech defect and neurological abnormalities. We suggest that uridine administration may be of therapeutic value in raising the intracellular concentrations of UDPGal. We conclude that the transferase reaction, however small in activity, is essential for optimal UDPGal formation.