New England Biolabs, Inc.

Rebecca B Kucera

Donald G Comb

Francine B Perler

William E Jack

Thomas C Evans, Jr

Chudi Guan

Romualdas Vaisvila

Sanjay Kumar

Shuang-Yong Xu

Richard D Morgan

Huimin Kong

Pei-Chung Hsieh

Andrew F Gardner

Lauren Sears Higgins

Elisabeth A Raleigh

Ira Schildkraut

Michael A Dalton

Barton E Slatko

Lixin Chen

Daniel F Heiter

Toby E Claus

Juan Pan

The present disclosure relates, according to some embodiments, to telomere resolvases (e.g., TelA variants) having a turnover number over 1.

TelA variants, compositions, and methods

Compositions and methods are provided for enhancing enzymatic ligation between nucleic acid fragments that relies on one or more small molecule enhancers having a size of less than 1000 daltons. For example, enhancement of ligation efficiencies are observed for double-stranded nucleic acid fragments that are blunt-ended, have a single nucleotide overhang at the ligation end, or have staggered ends compared to ligation under similar conditions in the absence of the one or more small molecule ligation enhancer. The use of small molecule enhancers for ligating nucleic acids results in an increased number of transformed host cells after transformation with the ligated molecules. This enhancement can be observed with chemically transformed host cells and with host cells transformed by electroporation.

Ligation enhancement

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

A method for cloning restriction-modification system is provided whereby the target modification methylase is produced and confers full protection during all growth phases in which the cognate restriction enzyme is present. The method is employed in the cloning of the MseI restriction-modification system.

Method for cloning and producing the Msel restriction endonuclease

The present invention relates to recombinant DNA which encodes a novel nicking endonuclease, N.BstNBI, and the production of N.BstNBI restriction endonuclease from the recombinant DNA utilizing PleI modification methylase. Related expression vectors, as well as the application of N.BstNBI in non-thio strand displacement amplification, is disclosed also.

Cloning and producing the N.BstNBI nicking endonuclease

Recombinant DNA polymerases from archaebacteria as well as isolated DNA coding for such polymerases are provided. The isolated DNA is obtained by use or DNA or antibody probes prepared from the DNA encoding T. litoralis DNA polymerase and the T. litoralis DNA polymerase respectively. Also provided are methods for producing recombinant archaebacteria thermostable DNA polymerase and methods for enhancing the expression of such polymerases by identifying, locating and removing introns from within the DNA coding for such DNA polymerases.

Recombinant thermostable DNA polymerase from archaebacteria

Recombinant DNA polymerases from archaebacteria as well as isolated DNA coding for such polymerases are provided. The isolated DNA is obtained by use of DNA or antibody probes prepared from the DNA encoding T. litoralis DNA polymerase and the T. litoralis DNA polymerase respectively. Also provided are methods for producing recombinant archaebacteria thermostable DNA polymerase and methods for enhancing the expression of such polymerases by identifying, locating and removing introns from within the DNA coding for such DNA polymerases.

Recombinant DNA polymeraset from archaebacteria as well as isolated DNA coding for such polymeraset are provided. The isolated DNA is obtained by use of DNA or antibody probet prepared from the DNA encoding T. litoralis DNA polymerate and the T. litoralis DNA polymerate respectively. Also provided are meshocs for producing recombinant archaebacteria thermostable DNA polymerase and methods for enhancing the expression of such polymeraset by identifying, locating and removing introns from within the DNA coding for such DNA polymerases.

There is provided an extremely thermostable enzyme obtainable from Thermococcus litoralis. The thermostable enzyme has a molecular weight of about 90,000-95,000 daltons, a half-life of about 60 minutes at 100.degree. C. in the absence of stabilizer, and a half-life of about 95 minutes at 100.degree. C. in the presence of stabilizer, such as octoxynol (TRITON X-100) or bovine serum albumin. The thermostable enzyme possesses a 3'-5' proofreading exonuclease activity. The thermostable enzyme may be native or recombinant and may be used for second-strand cDNA synthesis in cDNA cloning, DNA sequencing, and DNA amplification.

Growing community of inventors

Hamilton, MA, United States of America

Rebecca B Kucera