New England Biolabs, Inc.

Temple University

Chudi Guan

Christopher H Taron

Ellen P Guthrie

David Landry

Phillips W Robbins

Rebecca B Kucera

Sharon Wong-Madden

Thomas C Evans, Jr

Barton E Slatko

Lixin Chen

Bo Yan

Sanjay Kumar

Romualdas Vaisvila

Pei-Chung Hsieh

Sharon T Wong-Madden

Rebecca Kucera

Romaldas Vaisvila

Frank N Chang, Administrator

Hiroshi Inouye, Deceased,

Barton Slatko

Providing herein, among other things, are kits, compositions and methods that relate to DNA fragmentation. An embodiment of a composition provides combining: one or more enzymes capable of nick translating activity, a dNTP mix comprising at least one dNTP having a modified base, and at least one modification-sensitive nicking endonuclease that is prevented from nicking DNA if its recognition site contains the modified base. When the composition is added to a sample comprising a double-stranded DNA template that comprises recognition sites for the modification-sensitive nicking endonuclease, a reaction mix was produced which could be incubated for any time period in excess of about 5 minutes to produce fragments of a desired size of the double-stranded DNA template. In this method, the fragments produced include the modified base and, as such, are not re-nicked by the nicking endonuclease.

Method for fragmenting DNA by nick translation

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.

Generation of random double-strand breaks in DNA using enzymes

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 NADor 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.

Repair of nucleic acids for improved amplification

Compositions and methods are provided that relate to a modified DNA cleaving enzyme having at least 35% amino acid sequence identity with TEndo I. The modified enzyme includes two catalytic centers separated by a &#x3b2;-bridge where the &#x3b2;-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.

Polynucleotides encoding modified DNA cleavage enzymes and host cells therefor

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 &#x3b2;-bridge where the &#x3b2;-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.

Modified DNA cleavage enzymes and methods for use

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.

Substantially pure glycosidases capable for cleaving selected glycosidic bonds have been described including glycosidases isolated fromand recombinant glycosidases. Substrate specificity of isolated enzymes have been identified for GlcNac&#x3b2;1-x, Gal&#x3b1;1-3R, Gal&#x3b1;1-6R, Gal&#x3b2;1-3R, Fuc&#x3b1;-2R, Fuc&#x3b1;1-3R, Fuc&#x3b1;1-4R, Man&#x3b1;1-2R, Man&#x3b1;1-3R, Man&#x3b1;1-6R, Man&#x3b2;1-4R, Xyl&#x3b2;1-2R, Glc&#x3b2;1-4R, and Gal&#x3b2;1-4R providing improved capability for selectively cleaving a glycosidic linkage in a carbohydrate substrate and for forming modified carbohydrates.

Isolation and composition of novel glycosidases

Substantially pure glycosidases capable for cleaving selected glycosidic bonds have been described including glycosidases isolated from Xanthomonas and recombinant glycosidases. Substrate specificity of isolated enzymes have been identified for GlcNac&bgr;1-X, Gal&agr;1-3R, Gal&agr;1-6R, Gal&bgr;1-3R, Fuc&agr;-2R, Fuc&agr;1-3R, Fuc&agr;1-4R, Man&agr;1-2R, Man&agr;1-3R, Man&agr;1-6R, Man&bgr;1-4R, Xyl&bgr;1-2R, Glc&bgr;1-4R, and Gal&bgr;1-4R providing improved capability for selectively cleaving a glycosidic linkage in a carbohydrate substrate and for forming modified carbohydrates.

Substantially pure glycosidases capable for cleaving selected glycosidic bonds have been described including glycosidases isolated from Xanthomonas and recombinant glycosidases. Substrate specificity of isolated enzymes have been identified for GlcNac&bgr;1-X, Gal&agr;1-3R, Gal&agr;1-6R, Gal&bgr;1-3R, Fuc&agr;1-2R, Fuc&agr;1-3R, Fuc&agr;1-4R, Man&agr;1-2R, Man&agr;1-3R, Man&agr;1-6R, Man&bgr;1-4R, Xyl&bgr;1-2R, Glc&bgr;1-4R, and Gal&bgr;1-4R providing improved capability for selectively cleaving a glycosidic linkage in a carbohydrate substrate and for forming modified carbohydrates.

Methods and products are provided for producing and/or purifying virtually any hybrid polypeptide molecule employing recombinant DNA techniques. More specifically, a DNA fragment coding for a protein molecule, e.g. a polypeptide or portion thereof, is fused to a DNA fragment coding for a binding protein, such as the gene coding for the maltose binding protein. The fused DNA is inserted into a cloning vector and an appropriate host transformed. Upon expression, a hybrid polypeptide is produced which can be purified by contacting the hybrid polypeptide with a ligand or substrate to which the binding protein has specific affinity, e.g. by affinity chromatography. The hybrid polypeptide so purified may in certain instances be useful in its hybrid form, or it may be cleaved to obtain the protein molecule itself by, for example, linking the DNA fragments coding for the target and binding proteins with a DNA segment which codes for a peptide which is recognized and cut by a proteolytic enzyme, such as Factor Xa. The present invention also relates to certain vectors useful in practicing the above process.

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Wenham, MA, United States of America

Chudi Guan