Abstract: Compositions, methods and related uses are provided relating to cleaving modified DNA. For example, a set of DNA fragments obtainable by enzymatic cleavage of a large DNA is described where at least 50% are similarly sized and have a centrally positioned modified nucleotide. In addition, an enzyme preparation is provided that includes one or more enzymes that recognize a modified nucleotide in a DNA and cleave the DNA at a site that is at a non-random distance from the modified nucleotide. The one or more enzymes are further characterized by an N-terminal conserved domain with greater than 90% amino acid sequence homology to WXD(X)10YXGD. The related uses include creating a methylome, methods of purifying DNA fragments containing a modified nucleotide and diagnostic applications.

Abstract: Specified restriction endonucleases have been characterized for the first time by their amino acid and DNA sequences. These sequences and those with at least 90% identity thereto have been used as probes in sequence similarity analyses to identify sequence matches in a sequence database that corresponds to novel restriction endonucleases or isoschizomers. The sequence similarity analyses includes selecting a positive sequence match from any sequence producing an expectation value of less than or equal to e-02.

Abstract: Methods and compositions are described for detecting hydroxymethylated nucleotides (hmNs) in a polynucleotide preparation with a view to mapping the location of hmNs in a genome, quantifying the occurrence of hmNs at selected loci and correlating the occurrence of hmNs with gene expression and phenotypic traits. Embodiments describe the use of modifying enzymes together with site-specific endonucleases to detect the hmNs.

Abstract: Methods and compositions are provided that relate to obtaining a recombinant DNA and RNA cleaving nuclease. This involves the over-expression of a fusion protein between maltose-binding protein and a truncated nuclease in a soluble form in the cytoplasm of a host cell from which it can be readily extracted.

Abstract: Methods and compositions are described that relate to obtaining concentrated preparations of secreted recombinant proteins. These proteins are expressed in the form of fusion proteins with a chitin-binding domain (CBD). The fusion proteins are capable of being concentrated in the presence of chitin. Also described is: a shuttle vector that includes a modified LAC4 promoter; a chitinase-negative host cell; a CBD capable of eluting from chitin under non-denaturing conditions; and sterilized chitin, which can be optionally magnetized for facilitating recovery of recombinant protein.

Abstract: A nicking endonuclease is described which has an amino acid sequence with at least 70% identity to SEQ ID NO:6 and comprising a mutation at least one of an arginine or glutamic acid corresponding to position 507 and position 546 respectively in SEQ ID NO:6.

Abstract: Methods and compositions are provided for repairing a polynucleotide so that it can be copied with improved fidelity and/or yield in, for example, an amplification reaction. This involves the use of a reaction mixture that includes a ligase and a cofactor selected from NAD+ or ATP and incubating the polynucleotide with the reaction mixture in the absence of Endonuclease VI. The reaction mixture may further contain an AP endonuclease and a polymerase. If used, these enzymes may be selected according to their ability to withstand high temperatures. For example, the reaction mixture may be used prior to a polynucleotide synthesis reaction in which case enzymes that are not thermophilic may be used. The repair reaction is not time sensitive with respect to seconds, minutes or hours of incubation in the enzyme mixture in as much as the repair is effected rapidly and prolonged incubation is not generally adverse.

Abstract: A restriction endonuclease is provided that has been engineered to have a cleavage specificity for a DNA recognition sequence containing a modified nucleotide. Methods for engineering enzymes to cleave DNA containing modified nucleotides at specific sequences are provided.

Abstract: Compositions and methods are provided for selection and enrichment of a target gene from a library of polynucleotide sequences such as might be formed from a genome or by random mutagenesis of a genetic sequence. The selection and enrichment occurs in aqueous droplets formed in an emulsion that compartmentalize individual polynucleotides from the library or a plurality of polynucleotides that may include polynucleotides not derived from the library, transcription and translation reagents and optionally additional chemical and enzyme reagents. The selection and enrichment method utilizes a polynucleotide adaptor which when ligated to the polynucleotide fragment enables amplification to occur in the presence of an adaptor specific primer.

Abstract: Methods and compositions are provided for efficiently removing a guanine cap from the 5? end of an RNA using enzymes. Decapped RNA can be used for cloning, sequencing or other RNA manipulations.

Abstract: Methods are provided for making restriction endonucleases with reduced star activity by one or more targeted mutations to a catalytic site within the restriction endonuclease. Examples of modifications to restriction endonucleases with significant sequence identity with KpnI are provided and reduced star activity demonstrated.

Abstract: Methods and compositions are described that relate to obtaining concentrated preparations of secreted recombinant proteins. These proteins are expressed in the form of fusion proteins with a chitin-binding domain (CBD). The fusion proteins are capable of being concentrated in the presence of chitin. Also described is: a shuttle vector that includes a modified LAC4 promoter; a chitinase-negative host cell; a CBD capable of eluting from chitin under non-denaturing conditions; and sterilized chitin, which can be optionally magnetized for facilitating recovery of recombinant protein.

Abstract: Methods are provided for determining stereochemistry for a component of a composition where the composition includes a glycosaminoglycan (GAG) where the method includes (a) cleaving the GAG with a lyase; (b) separating the products of the cleaved GAG by chromatography in the presence of one or more solvents wherein at least one solvent comprises an organic acid or organic acid derivative in an amount of no more than 50% v/v of solvent; and determining the stereochemistry of the component.

Abstract: Methods and compositions are provided that relate to a composition that includes a modified maltose-binding protein (MBP) which when fused to a protein results in an increase in binding affinity for maltodextrin compared with the wild type MBP fused to the protein, the modified MBP maintaining enhanced solubility, The modification includes a mutation selected from the group consisting of: F68L, I318V, Q326R, V344M, and T372TTTITITTTLGIEGR387 or consist of two or more mutations selected from the group consisting of: F68L, S146T, A313V, I318V, I318A, Q326R, V344M and T372TTTITITTTLGIEGR387 mutants.

Abstract: The present invention relates to compositions including: (1) isolated DNA encoding the StuI restriction endonuclease and isolated DNA encoding cognate and non-cognate methylase; (2) vectors and cells containing the isolated DNA; and (3) methods for producing the StuI restriction endonuclease.

Abstract: Compositions and methods are provided for generating biofuels by fermentation from carbon sources other than glucose using genetically engineered yeast strains. For example, a Saccharomyces strain which is capable of converting glucose to ethanol but not of metabolizing N-acetyl glucosamine is genetically engineered to utilize N-acetyl glucosamine as a nutrient carbon source.

Abstract: Compositions and methods are provided that relate to a modified DNA cleaving enzyme having at least 35% amino acid sequence identity with T7 Endo I. The modified enzyme includes two catalytic centers separated by a ?-bridge where the ?-bridge contains at least one mutation having an effect of altering enzyme cleavage activity compared to the unmodified enzyme. Activities associated with the modified DNA cleaving enzyme that can be modulated in different reaction conditions include at least one of: (a) non-sequence specific nicking activity; (b) cleaving the second strand of a duplex DNA at a preexisting nick site to produce a linear duplex with a single strand overhang; (c) non-sequence specific DNA cleavage; (d) cleaving DNA flanking a mismatch; and (e) cleavage at a cruciform structure in a DNA duplex.

Abstract: single-stranded extension having a desired length and sequence composition. Methods for forming single-stranded extensions include: the use of a cassette containing at least one nicking site and at least one restriction site at a predetermined distance from each other and in a predetermined orientation; or primer-dependent amplification which introduces into a polynucleotide molecule, a modified nucleotide which is excised to create a nick using a nicking agent. The methods and compositions provided can be used to manipulate a DNA sequence including introducing site specific mutations into a polynucleotide molecule and for cloning any polynucleotide molecule or set of joined polynucleotide molecules in a recipient molecule such as a vector of choice.

Abstract: An enzyme preparation is described that includes a non-specific nuclease and a T7 Endo I mutant in a unit ratio of less than 1:200. This enzyme preparation may be used to generate double-stranded DNA fragments of a size suitable for DNA sequencing. The ends of the fragments can be readily modified as necessary to ligate adaptors or individual nucleotides to one strand of the double-stranded DNA fragments.

Abstract: Methods and compositions are provided for repairing a polynucleotide so that it can be synthesized efficiently with improved fidelity and yield in, for example, an amplification reaction. This involves the use of a reaction mixture that includes a ligase and a cofactor selected from NAD+ or ATP and incubating the polynucleotide with the reaction mixture in the absence of Endonuclease VI. The reaction mixture may further contain an AP endonuclease and a polymerase. These enzymes are optionally selected according to their ability to withstand high temperatures so they can be included in an amplification mixture. The reaction mixture may be used prior to a polynucleotide synthesis reaction in which case enzymes that are not thermophilic may be used. The repair reaction is not time sensitive with respect to seconds, minutes or hours of incubation in the enzyme mixture.