ABSTRACT
Objective: To compare the overall efficiency and cost of the construction and screening of libraries of Pseudomonas aeruginosa arylsulfatase (PAAS) through site-directed saturation mutagenesis with random primers (NNN) and precise primers for PCR. Methods: Site-directed mutagenesis libraries were constructed using the random primers (NNN) of the selected site, an equal-mole mixture of precise primers and a purified precise primer each time for the amplification of the fusion vector of Escherichia coli alkaline phosphatase (ECAP) and PAAS through PCR. The libraries were screened in a high-throughput mode through the assay of activity ratios of PAAS/mutants to ECAP after alkaline lysis of host cells. Three approaches for site-directed saturation mutagenesis were compared for their number of monoclones screened, the number of their expected mutants discovered, overall time consumed, overall cost and other pertinent factors. Results: The site-directed saturation mutagenesis of M72 with random primers for PCR resulted in only 10 among 20 expected mutants after the screening of over 600 monoclones, besides obvious codon bias. In contrast, no obvious codon bias was observed and there were already 18 among 20 expected mutants after the screening of less than 150 monoclones for site-directed saturation mutagenesis of G138 with similar random primers. The site-directed saturation mutagenesis of M72 with an equal-mole mixture of precise primers yielded all of the 19 expected mutants after the screening of less than 190 monoclones; the site-directed saturation mutagenesis of M72 with the purified precise primers for PCR one-by-one gave all of the 19 expected mutants after the screening of just 2 monoclones for every expected mutant. The use of the purified precise primers for PCR one-by-one was thus more favorable for the construction of libraries of site-directed saturation mutagenesis for the minimum number of monoclones screened, the least overall time and cost. In comparison to the use of the purified precise primers for PCR one-by-one, the use of random primers or the equal-mole mixture of precise primers for PCR to generate the libraries tolerated much greater overall cost and longer time. Conclusion: The generation and screening of the site-directed saturation mutagenesis libraries with precise primers for PCR one-by-one were more practical in elucidating the sequence-activity relationship while the use of equal-mole mixture of precise primers for PCR was preferable for the screening of positive mutants during site-directed saturation mutagenesis.
ABSTRACT
Aunque la técnica convencional para el diagnóstico del VIH es la PCR a partir de ADN proviral, la técnica RT-PCR cualitativa podría constituir una herramienta de apoyo en el diagnóstico del VIH en pacientes infectados, ya que una de sus principales ventajas es el uso de muestras de plasma, en lugar de sangre total, lo cual facilitaría su transporte entre las distintas regiones del país. En este estudio se planteó como objetivo evaluar las condiciones óptimas, en cuanto al uso de cebadores y enzima transcriptasa reversa, para síntesis de ADN complementario (RT-PCR) de la región de envoltura del VIH, a partir de muestras de plasma de pacientes VIH positivos. Los resultados obtenidos en este trabajo sugieren que la utilización de cebadores azarosos, en una mezcla de reacción con la enzima RT SuperScript TM (200 U/mL), permite maximizar la eficiencia de amplificación del gen de la envoltura de VIH-1, en comparación con cebadores específicos. Esto permite ofrecer una alternativa metodológica a partir de muestras de plasma, para la detección del VIH en aquellas personas que por diversas razones no pueden trasladarse a la institución de referencia.
Even though PCR is the conventional technique for HIV diagnosis in proviral DNA, the qualitative RT-PCR technique could constitute a support tool for HIV diagnosis in infected patients since one of its main advantages is the use of plasma samples instead of whole blood, which facilitates its transportation from the various regions of the country. The main objective of this study was the evaluation of the optimal conditions regarding use of primers and reverse transcriptase enzyme, for the synthesis of complementary DNA (RT-PCR) of the HIV sheath in plasma samples of HIV positive patients. The results obtained in this study suggest that the use of random primers, in a reactive mixture with the RT SuperScript enzyme TM (200 U/mL), allows maximizing the amplifying efficiency of the HIV-1 sheath gene, as compared with specific primers. This offers a methodological alternative using plasma samples for HIV detection in those persons who due to various reasons cannot travel to the reference institution.
ABSTRACT
A chromosome walking method was improved in this work. The new method was named as nonspecific primer anchored PCR (NPA-PCR). Nested gene specific primers were designed based on the known region and long random primer using degeneracy oligonucleotides for nonspecific anchoring. Annealing temperatures were varied to control the priming. Target sequences were obtained by PCR with random primer and gene-specific primer. Nonspecific sequence with long random primers at both ends formed stem loop structure due to inverted terminal repeats. The method was employed to isolate a gene with newly-isolated actinomycin producing strain Streptomyces setonii Z-L-22. A 0.77 kb fragment of actinomycin synthetase gene cluster was isolated from the strain. The fragments of 1474bp and 701bp were obtained, respectively, at the up and down streams of known fragment through the this method. NCBI Blast analysis showed that the walking sequence and the known sequence were located conjointly in the same cluster gene. It demonstrated that the result was correct and this technique could be useful and efficient for chromosome walking or isolating the gene.