Abstract: A highly parallel method for gene cloning is presented. PCR products can be isolated using a solid phase and ligated into a positive selection vector. The cloning method has a very high success rate and can be performed entirely by a liquid handling robot with very little human intervention.

Abstract: In one aspect, the invention provides a method for separating a mixture of polynucleotides, such as DNA or RNA, including (a) applying the mixture to a polymeric separation medium having non-polar surfaces, wherein the surfaces are characterized by being substantially free from multivalent cations, such as metal ions, which are free to interfere with polynucleotide separation, and (b) eluting the mixture with a mobile phase containing organic solvent and counter ion agent. In the separation of single-stranded polynucleotides, improved separation is obtained at a temperature effective to fully denature secondary structure within the polynucleotides.

Abstract: A Matched Ion Polynucleotide Chromatography method and system for size-based segregation of a mixture of RNA molecules. The method includes applying the mixture to a polymeric separation medium having non-polar surfaces and eluting the RNA molecules with a mobile phase which includes counterion reagent and an organic component. The preferred surfaces are characterized by being substantially free from multivalent cations which are free to interfere with RNA segregation. The elution is preferably performed at a temperature sufficient to denature the RNA. The method can be used in segregating RNA molecules having lengths in the range of about 100 to 20,000 nucleotides. Improved segregation is obtained using a chromatography column having an ID greater than about 5 mm. Examples of separation media include beads and monolithic columns.

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: A Matched Ion Polynucleotide Chromatography method and system for size-based segregation of a mixture of RNA molecules. The method includes applying the mixture to a reverse phase column containing polymeric beads and eluting, preferably under denaturing conditions, the RNA molecules with a mobile phase which includes counterion reagent and an organic component. The method can be used in segregating RNA molecules having lengths in the range of about 100 to 20,000 nucleotides. Improved segregation is obtained using a chromatography column having an ID greater than about 5 mm.

Abstract: A Matched Ion Polynucleotide Chromatography method and system for size-based segregation of a mixture of RNA molecules. The method includes applying the mixture to a reverse phase column containing polymeric beads and eluting, preferably under denaturing conditions, the RNA molecules with a mobile phase which includes counterion reagent and an organic component. The method can be used in segregating RNA molecules having lengths in the range of about 100 to 20,000 nucleotides. Improved segregation is obtained using a chromatography column having an ID greater than about 5 mm.

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 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.

Abstract: A Matched Ion Polynucleotide Chromatography method and system for size-based segregation of a mixture of RNA molecules. The method includes applying the mixture to a polymeric separation medium having non-polar surfaces and eluting the RNA molecules with a mobile phase which includes counterion reagent and an organic component. The preferred surfaces are characterized by being substantially free from multivalent cations which are free to interfere with RNA segregation. The elution is preferably performed at a temperature sufficient to denature the RNA. The method can be used in segregating RNA molecules having lengths in the range of about 100 to 20,000 nucleotides. Improved segregation is obtained using a chromatography column having an ID greater than about 5 mm. Examples of separation media include beads and monolithic columns.