Method of determining nucleotide sequences in cells and of isolating nucleic acids from cells.

摘要

It has been a problem that nucleic acids present in cells have had to be isolated by difficult extraction procedures, using e.g. phenol or a protease, before they can be assayed. The present invention provides an assay method which can be carried out on crude cell lysate, without the need for any such extraction. The method comprises lysing the cells in a lysis buffer comprising a chaotropic agent for disrupting nucleoproteins and which inhibits the nuclease, e.g. a guanidinium salt present in the lysate in a concentration of at leat 3 M at the time of hybridisation, when DNA is being determined treating the lysate to single-strand it, contacting the lysate under hybridisation conditions with a polynucleotide having a nucleotide sequence complementary to a sequence present in the nucleic acid to be determined, and determining the extent of hybridisation, e.g. by a sandwich or competition assay. A similar procedure can be used to extract the nucleic acid from the cell by a hybridisation method and isolate the desired nucleic acid from the resultant hybrid.