Nucleic acid purification

摘要

Methods and composition for nucleic acid isolation are provided. In one embodiment, a method is provided for nucleic acid purification from biological samples, such as whole blood samples, extracted with phenol-based denaturing solvents, which does not require phase separation or nucleic acid precipitation. Methods according to the invention may also be used for of small RNAs (e.g., siRNAs or miRNAs) purification and are amenable to automation.

主权项

1. A method for purification of RNA comprising:
(a) contacting a nucleic acid containing sample, which comprises RNA, with a denaturing solvent comprising a chaotropic salt and at least 20% phenol; (b) adding a binding agent to the sample, wherein the binding agent comprises a chaotropic salt, or a lower alcohol or a mixture thereof; (c) contacting the sample with a silica substrate, in the presence of at least 10% phenol, thereby binding RNA to the silica substrate; (d) washing the silica substrate and bound RNA with a wash solution; and (e) eluting the RNA from the silica substrate with an elution buffer, thereby providing purified RNA; wherein, after the addition of the binding agent, the sample does not comprise separate aqueous and organic phases prior to binding the RNA to the silica substrate and wherein: (i) the sample comprises whole blood or blood cells; (ii) the RNA comprises RNA molecules less than 200 nucleotides in length or (iii) the silica substrate comprises magnetic silica beads and wherein one or more of steps (a)-(c) is automated.