RESUMEN
Loop-mediated isothermal DNA amplification (LAMP) is an alternative method for the amplification of DNA sequences. It has been applied primarily for the detection of specific targets. We demonstrate the novel use of LAMP to amplify SSR alleles in a set of rice varieties and show the results to be consistent with analysis performed by PCR. Furthermore, we test the sensitivity of the assay and show it to amplify from near single copy target.
Asunto(s)
Repeticiones de Microsatélite , Técnicas de Amplificación de Ácido Nucleico/métodos , Oryza/genética , Cartilla de ADN/genética , ADN de Plantas/genética , Técnicas de Genotipaje , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Análisis de Secuencia de ADNRESUMEN
In an initial investigation of differential expression of genes caused by cis-acting regulatory elements in rice, the lack of reproducibility led us to question the basic premise of allelic expression imbalance determination: namely that departures of cDNA expression ratios from those observed in genomic DNA provide unequivocal evidence of cis-acting polymorphisms. This paper describes experiments designed to demonstrate that stochastic variation in low copy number of targets in PCR reactions give variable allelic ratios even when starting with the same copy numbers of the two alleles. These significant departures from an expected 1:1 ratio provide an explanation to the lack of reproducibility observed for our cDNA measurements.