RESUMO
A modification of PCR-mediated gene synthesis strategy is introduced. This modification enables the synthesis of a gene from oligonucleotides comprising only one of the two strands. Bridging oligonucleotides (approximately 20-mers in length) complementary to the junctions of template strand oligonucleotides and two outer primers are also needed for PCR. A two-step PCR containing a first step of 10 cycles, followed by a second step of 20 cycles, differing only in the annealing conditions was used. A single-step PCR combining the two different cycle conditions could also be used successfully. Optimal conditions for gene synthesis (and amplification) are described. Human and porcine colipase genes (297 and 309 bp, respectively) have been successfully synthesized, cloned into the Invitrogen TA cloning vector and sequenced. There was absolutely no error in the clones that were sequenced.
Assuntos
Genes , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase/métodos , Animais , Sequência de Bases , Biotecnologia , Clonagem Molecular , Colipases/genética , DNA/biossíntese , DNA/genética , Humanos , Dados de Sequência Molecular , SuínosRESUMO
Twelve healthy male subjects received single oral doses of etretinate, ranging from 25 to 100 mg (1 to 4 x 25-mg capsules) in an open-label, four-way randomized crossover design. Plasma concentrations of etretinate and two active metabolites were determined by a specific high-performance liquid chromatographic (HPLC) method. Analysis of variance and orthogonal contrasts were used to assess dose proportionality. Mean (+/- %CV) maximum concentrations after 25- to 100-mg doses were 133 (50), 195 (33), 261 (53), and 446 (65) ng/ml, whereas AUC0-12 values were 581 (46), 1090 (39), 1500 (52), and 2440 (63) ng.hr/ml, respectively. The test for proportionality indicated that Cmax and AUC0-12 increased proportionally with an increase in dose (P greater than 0.05).
