摘要:
The invention relates to methods for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule. The method involves cleaving a double-stranded nucleic acid molecule containing a mismatch with a mismatch-specific endonuclease which cleaves on the 3′ side of the mismatch, and preserving the integrity of the variant nucleotide by ligating a double-stranded linker with a 3′-overhang to said variant nucleotide. Because the variant nucleotide is immediately adjacent to the linker, PCR and/or sequence-by-synthesis analysis can be readily carried out.
摘要:
The invention relates to methods for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule. The method involves cleaving a double-stranded nucleic acid molecule containing a mismatch with a mismatch-specific endonuclease which cleaves on the 3′ side of the mismatch, and preserving the integrity of the variant nucleotide by ligating a double-stranded linker with a 3′-overhang to said variant nucleotide. Because the variant nucleotide is immediately adjacent to the linker, PCR and/or sequence-by-synthesis analysis can be readily carried out.
摘要:
Nonporous beads having an average diameter of about 0.5-100 microns are suitable for chromatographic separation of mixtures of polynucleotides when the beads comprise a nonporous particle which are coated with a polymer or which have substantially all surface substrate groups endcapped with a non-polar hydrocarbon or substituted hydrocarbon group. The beads provide efficient separation of polynucleotides using Matched Ion Polynucleotide Chromatography.
摘要:
Nonporous beads having an average diameter of about 0.5-100 microns are suitable for chromatographic separation of mixtures of polynucleotides when the beads comprise a nonporous particle which are coated with a polymer or which have substantially all surface substrate groups endcapped with a non-polar hydrocarbon or substituted hydrocarbon group. The beads provide efficient separation of polynucleotides using Matched Ion Polynucleotide Chromatography.
摘要:
An apparatus for effecting base pair length separations of DNA fragments by matched ion paired chromatography comprising a separation column containing separation media having non-polar DNA separation surfaces, separation solution supply means, and a separation solution conduit communicating with the separation column and the separation solution supply means, and a cleaning solution valve means positioned in the separation solution conduit for injecting cleaning solution into the separation solution conduit. A process for cleaning the non-polar DNA separation surfaces in the apparatus comprising interrupting the flow of separation solvent with a block of cleaning solution injected into the flow of separation solution passing to the column, the cleaning solution containing agent which removes accumulated residues from the non-polar surface. The cleaning solution can have an alkaline pH and contain a chelating agent such as EDTA.
摘要:
Non-polar polymeric separation media, such as beads or monoliths, are suitable for chromatographic separation of mixtures of polynucleotides when the surfaces of the media are unsubstituted or substituted with a hydrocarbon group having from one to 1,000,000 carbons and when the surfaces are substantially free from mutivalent cation contamination. The polymeric media provide efficient separation of polynucleotides using Matched Ion Polynucleotide Chromatography. Methods for maintaining and storing the polymeric media include treatment with multivalent cation binding agents.
摘要:
The present invention is directed to improved methods for detection of mutations in DNA using Denaturing Matched Ion Polynucleotide Chromatography (DMIPC). The invention includes the following aspects: analysis of PCR amplification products to identify factors that affect PCR replication fidelity; design of PCR primers; selection of an optimal temperature for performing DMIPC; selection of the mobile phase composition for gradient elution; methods for column preparation and maintenance; and methods for preparing polynucleotide samples prior to chromatographic analysis.
摘要:
The disclosure describes an ambient or low pressure device for separating polynucleotide fragments from a mixture of polynucleotide fragments comprises a tube having an upper solution input chamber, a lower eluant receiving chamber, and a fixed unit of separation media supported therein. The separation media has nonpolar separation surfaces which are free from multivalent cations which would react with counterion to form an insoluble polar coating on the surface of the separation media.
摘要:
A method for removing a target DNA fragment having a predetermined base-pair length from a mixture of DNA fragments comprises the following steps. A mixture of DNA fragments which may contain the target DNA fragments is applied to a separation column containing media having a nonpolar, nonporous surface, the mixture of DNA fragments being in a first solvent mixture containing a counterion and a DNA binding concentration of driving solvent in a cosolvent. The target DNA fragments are separated from the media by contacting it with a second solvent solution containing a counterion and a concentration of driving solvent in cosolvent which has been predetermined to remove DNA fragments having the target DNA fragment base pair length from the media. The target DNA fragments can be collected and optionally amplified. When the method is being applied to collect a putative fragment, if present, no DNA fragments having the base pair length of the target DNA could be present in the mixture. Alternatively, DNA fragments having the base pair length of the target DNA are present in the mixture. The disclosure also describes an ambient or low pressure device for separating polynucleotide fragments from a mixture of polynucleotide fragments comprises a tube having an upper solution input chamber, a lower eluant receiving chamber, and a fixed unit of separation media supported therein. The separation media has nonpolar separation surfaces which are free from multivalent cations which would react with counterion to form an insoluble polar coating on the surface of the separation media.
摘要:
Covalently bound non-polar tags are used to increase the retention times of double stranded polynucleotides on Matched Ion Polynucleotide Chromatography (MIPC) columns. In doing so, separations of DNA mixture components is improved. Additionally, when the non-polar tags are fluorophores, detection limits are also greatly reduced. Strategically tagged primers are used in conduction with PCR to produce DNA fragments having specifically tagged strands. This improves mutation detection by MIPC in several ways. Separations are improved, detection sensitivity is enhanced, and non-stoichiometric addition of wild type DNA prior to hybridization is now possible since only tagged fragments will be observed with a fluorescence detector. Non-polar tags are also used as a novel alternative to G-C clamping during MIPC under partially denaturing conditions. Reversible DNA binding dyes, such as DNA intercalator dyes and DNA groove binding dyes, are used to reduce the detection limit of polynucleotides separated by MIPC.