New England Biolabs, Inc.

Other

Shengxi Guan

Zhenyu Zhu

Aine Quimby

Shuang-Yong Xu

Siu-Hong Chan

Dapeng Sun

Penghua Zhang

Hua Wei

Xuhui Lai

Xianghui Li

Yishu Huang

Zhiyi Sun

Theodore B Davis

Romualdas Vaisvila

Laurence Ettwiller

Lana Saleh

Chunhua Zhang

Nicole Nichols

Janine Borgaro

Thomas C Evans, Jr

Jennifer Ong

Ivan R Correa, Jr

Yan Xu

Nan Dai

Yu Brian Zheng

Thomas C Evans

Yanxia Bei

Lynne Apone

Sean Maguire

Hua Wang

Aine Blanchard

Shuang- Yong Xu

High Fidelity Restriction Endonucleases

Methods and compositions are provided for engineering mutant enzymes with reduced star activity where the mutant enzymes have a fidelity index (FI) In a specified buffer that is greater than the FI of the non-mutated enzyme in the same buffer.

High fidelity restriction endonucleases

Compositions and methods are provided for enzymes with altered properties that involve a systematic approach to mutagenesis and a screening assay that permits selection of the desired proteins. Embodiments of the method are particularly suited for modifying specific properties of restriction endonucleases such as star activity. The compositions includes restriction endonucleases with reduced star activity as defined by an overall fidelity index improvement factor.

Methods and compositions are provided for identifying any of the presence, location and phasing of methylated and/or hydroxymethylated cytosines in nucleic acids including long stretches of DNA. In some embodiments, the method may comprise reacting a first portion (aliquot) of a nucleic acid sample with a dioxygenase and optionally a glucosyltransferase in a reaction mixture containing the nucleic acid followed by a reaction with a cytidine deaminase to detect and optionally mapC in a DNA. Optionally, a second portion can be reacted with glucosyltransferase followed by reaction with a cytidine deaminase to detect and optionally mapC in a DNA.

Compositions and methods for analyzing modified nucleotides

Compositions are provided for 3' adapters and methods of use are provided that include methods requiring a plurality of ligation steps involving a single-stranded target polynucleotide and 3&#x2032; and 5&#x2032; adapters. Embodiments of the 3&#x2032; adapters comprise a cleavable linker positioned between a single-stranded region and a double-stranded region. Upon ligating the 3&#x2032; adapters, the single-stranded region is released by cleaving the cleavable linker.

Polynucleotide adapter design for reduced bias

A method for identifying any of the presence, location and phasing of modified cytosines (C) in long stretches of nucleic acids is provided. In some embodiments, the method may comprise (a) reacting a first portion of a nucleic acid sample containing at least one C and/or at least one modified C with a DNA glucosyltransferase and a cytidine deaminase to produce a first product and/or reacting a second portion of the sample with a dioxygenase, optionally a DNA glucosyltransferase and a cytidine deaminase to produce a second product and; (b) comparing the sequences from the first and optionally the second product obtained in (a), or amplification products thereof, with each other and/or an untreated reference sequence to determine which Cs in the initial nucleic acid fragment are modified. A modified TET methylcytosine dioxygenase with improved efficiency compared to unmodified TET2 at converting methylcytosine to carboxymethylcytosine is also provided.

Methods and compositions are provided for engineering mutant enzymes with reduced star activity where the mutant enzymes have a fidelity index (FI) in a specified buffer that is greater than the FI of the non-mutated enzyme in the same buffer.

A method for identifying the location and phasing of modified cytosines (C) in long stretches of nucleic acids is provided. In some embodiments, the method may comprise (a) reacting a first portion of a nucleic acid sample containing at least one C and/or at least one modified C with a DNA glucosyltransferase and a cytidine deaminase to produce a first product and optionally reacting a second portion of the sample with a dioxygenase and a cytidine deaminase to produce a second product and; (b) comparing the sequences from the first and optionally the second product obtained in (a), or amplification products thereof, with each other and/or an untreated reference sequence to determine which Cs in the initial nucleic acid fragment are modified. A modified TET methylcytosine dioxygenase that is more efficient at converting methylcytosine to carboxymethylcytosine is also provided.

Provided herein in some embodiments is a non-naturally occurring variant of a wild type restriction enzyme defined by SEQ ID NO: 20, wherein the variant has at least a 2 fold increase in cleavage at 5-&#x3b2; glucosylhydroxymethylcytosine (5&#x3b2;ghmC) compared with methylcytosine relative to the wild type enzyme. Methods for examining hydroxymethylation of a DNA sample using the variant enzyme are also provided.

Compositions and methods for identifying hydroxymethylcytosine in a DNA

Compositions and methods for performing a template-switching reaction are provided that may include reducing or eliminating concatemerization of the template-switching oligonucleotide (TSO). In some embodiments, the composition may comprise: a reverse transcriptase; a TSO that includes a recognition sequence for a site-specific double strand nucleic acid cleaving enzyme, wherein the TSO has at its 3' end at least one nucleotide capable of hybridizing to at least one or more non-templated nucleotides added to a templated cDNA strand by the reverse transcriptase; and a site-specific double strand nucleic acid cleaving enzyme that cleaves the TSO at the recognition sequence.

Methods and compositions for preventing concatemerization during template-switching

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Stoneham, MA, United States of America

Shengxi Guan