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Osmotic Shock of Blood Cells as DNA Extraction ProcedureA. Sala, G. Penacino and D. Corach DNA extraction is required for PCR amplifications. There are several methods available to achieve this goal: Pk-SDS, CTAB, and others which do not need posterior organic extraction such as salting out procedures or resin or silica-based purifications protocols (e.g. Chelex-P100, Glass Max or Gene CleanII). However, when multiplicity of target sequences are available, extraction procedure can be replaced by a simple osmotic shock. The aim of this work is to describe an efficient procedure for PCR amplifications of DNA from blood samples without DNA extractions. Fresh blood samples were osmotically shocked with distilled water in ratios ranging from 1/5 to 1/1000. We chose mtDNA as a model system. PCR amplifications were performed with 2 ml of those dilutions without further purification. Reactions included the following pair of primers: L15926/H00580, L159997/H16401 and L048/H408, in order to obtain 1.2 Kb and 400 bp fragments. Two microliters of the first reaction were used as template for nested amplification (primers L048/H408 and L15997/H16401). Evaluation of amplicons were analyzed in 1.5% agarose gels and then sequenced using a dsDNA sequencing procedure. No sequence difference was observed between conventionally extracted DNA and this protocol. This approach is being validated for STR typing in our lab. Go to proceedings home page
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