Abstract: The present invention describes a method for generating a chromatographic DNA sequencing ladder useful for phasing a chromatographic separation of fragments derived from the DNA sequence, e.g., a DNA footprinting reaction. The chromatographic separation is preferably accomplished using ion pairing reverse phase high performance liquid chromatography under conditions that are substantially free of multivalent cations that are free to interfere with polynucleotide separations.

Abstract: The present invention describes a method for characterizing a DNA-protein complex that entails treating a DNA-protein complex with a DNA cleavage reagent. The bound protein blocks DNA cleavage in a region of the DNA molecule where the protein is bound, so that the DNA is only cleaved in regions that are not blocked by the bound protein. Ion pairing reverse phase high performance liquid chromatography is used to separate and detect the cleaved DNA, thereby identifying regions of the DNA where cleavage has occurred. The absence of cleavage events in a region of the DNA indicates that the protein bound to that region. In a preferred embodiment, the cleavage reagent is hydroxyl radical, the DNA is fluorescently labeled, and the chromatographic separation is carried out under conditions that are substantially free of multivalent cations that are free to interfere with polynucleotide separations.

Abstract: The instant invention provides a non-HPLC chromatographic method for purifying a target polynucleotide comprising the steps of: applying the target polynucleotide to a separation medium having a non-polar separation surface in the presence of a counterion agent, whereby the polynucleotide is bound to the separation medium; eluting the target polynucleotide from the separation medium by passing through the separation medium an elution solution containing a concentration of organic solvent sufficient to elute the target polynucleotide from the separation medium; and collecting the eluted target polynucleotide. The separation medium can be supported in any of a variety of containers, non-limiting preferred examples of which include spin columns and vacuum trays. The invention is particularly useful for the separation of RNA and single and double stranded DNA.

Abstract: The present invention describes a method for identifying bases in an RNA sequence that are relatively inaccessible to solvent as the result of intramolecular and/or intermolecular interactions. Embodiments of the invention can be used to evaluate the three-dimensional structure of an RNA molecule or the interactions between an RNA molecule and a molecule capable of binding to RNA, such as another nucleic acid or an RNA-binding protein. The invention involves contacting an RNA molecule with a cleavage reagent capable of partially hydrolyzing said RNA molecule, wherein said partial hydrolysis is attenuated in a region of said RNA molecule that is relatively inaccessible to solvent; and separating and detecting the cleaved RNA by IP-RP-HPLC, wherein the absence of cleavage events in a region of the RNA indicates that said region is relatively inaccessible to solvent. The separation medium is preferably substantially free of multivalent cations capable of interfering with polynucleotide separations.

Abstract: The present invention describes a method for identifying bases in an RNA sequence that are relatively inaccessible to solvent as the result of intramolecular and/or intermolecular interactions. Embodiments of the invention can be used to evaluate the three-dimensional structure of an RNA molecule or the interactions between an RNA molecule and a molecule capable of binding to RNA, such as another nucleic acid or an RNA-binding protein. The invention involves contacting an RNA molecule with a cleavage reagent capable of partially hydrolyzing said RNA molecule, wherein said partial hydrolysis is attenuated in a region of said RNA molecule that is relatively inaccessible to solvent; and separating and detecting the cleaved RNA by IP-RP-HPLC, wherein the absence of cleavage events in a region of the RNA indicates that said region is relatively inaccessible to solvent. The separation medium is preferably substantially free of multivalent cations capable of interfering with polynucleotide separations.

Abstract: A method for generating single stranded DNA (ssDNA) directly from double stranded PCR (dsPCR) products is described.