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1.
Southeast Asian J Trop Med Public Health ; 1995 ; 26 Suppl 1(): 329-32
Artículo en Inglés | IMSEAR | ID: sea-32437

RESUMEN

Among various methods which have been developed for facilitating the screening of point mutations in human genomic DNA, PCR-Primer Introduced Restriction Analysis (PCR-PIRA) is of particular interest due to its practicality and short procedure allowing detection of point mutations by simple restriction enzyme digestion directly after PCR amplification. However, one limitation of PCR-PIRA method is the absence of restriction sites in the region of detection, thus creation of the recognition site in primers has been introduced. Detection of a point mutation at codon 12 in K-ras oncogene by BstNI requires one base change in the primer sequence so that only the normal but not mutant PCR product will be digested by the enzyme. However, false positive results generated from undigested normal DNA sequence are always obtained. This effect is compounded when it is used to analyse mixed cell populations in paraffin embedded section of cancer cells. Assay of a mutant band generated from normal DNA by densitometric quantitation enabled the determination of background values and thereby eliminated false positive results. Samples with higher ratios between mutant and normal bands than the background one after the first PCR-PIRA would be subjected to the second PCR-PIRA in order to confirm the results. Screening of such mutations in cervical carcinomas from paraffin embedded sections using the above criteria should reduce misinterpretation of PCR-PIRA results.


Asunto(s)
Secuencia de Bases , Línea Celular , Codón , Neoplasias del Colon/genética , Cartilla de ADN , ADN de Neoplasias/aislamiento & purificación , Desoxirribonucleasas de Localización Especificada Tipo II , Genes ras , Humanos , Datos de Secuencia Molecular , Mutación Puntual , Reacción en Cadena de la Polimerasa/métodos , Mapeo Restrictivo , Células Tumorales Cultivadas
2.
Southeast Asian J Trop Med Public Health ; 1994 Sep; 25(3): 430-5
Artículo en Inglés | IMSEAR | ID: sea-31804

RESUMEN

Specific DNA probe hybridization technique is one method of choice for detection of malaria infection. It provides an obvious advantage over conventional microscopy when large numbers of samples are simultaneously monitored. The method was simplified so that preparation and processing of blood specimens were all performed on membrane filters. Background signals generated from blood components were removed by treating samples spotted on the membrane with a series of buffer washes without the necessity of a protease digestion step. Hybridization was monitored using either 32P-or digoxigenin-labeled DNA probe. 849 field samples collected from various malaria endemic areas in Thailand have been evaluated by this protocol and compared with microscopic examination. Sensitivity obtained by this procedure was comparable to that of microscopy at a malaria clinic. The specificities of both types of DNA probes were better than 93%, but digoxigenin-labeled probe performed better than 32P-labeled one when the numbers of parasites were less than 25 per 200 white blood cells.


Asunto(s)
Sondas de ADN , Humanos , Malaria Falciparum/diagnóstico , Microscopía , Hibridación de Ácido Nucleico , Sensibilidad y Especificidad
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