New England Biolabs, Inc.

Other

Laurence Ettwiller

Shengxi Guan

Theodore B Davis

Romualdas Vaisvila

Lana Saleh

Zhiyi Sun

Ivan R Correa, Jr

Ira Schildkraut

George Tzertzinis

Madalee G Wulf

Shuang-Yong Xu

Thomas C Evans, Jr

Jennifer Ong

Vladimir Potapov

Andrew F Gardner

Nan Dai

Lixin Chen

Bo Yan

John Buswell

Michael Sproviero

Kelly M Zatopek

Erbay Yigit

Andrew F Garner

Thomas Evans

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

Providing herein, among other things, is a method comprising incubating a double-stranded nucleic acid having a nick with a nick translating activity, a ligase, and a nucleotide mix comprising at least one modified nucleotide, to generate a product comprising a patch of a newly synthesized strand of a duplex nucleic acid containing a plurality of modified nucleoside monophosphates that are at or adjacent to the site of the nick. In some embodiments, the method may be used to map damaged nucleoside monophosphates in a nucleic acid. Compositions and kits for use in performing the method are also provided.

Mapping the location, type and strand of damaged and/or mismatched nucleotides in double-stranded DNA

A method of labeling, and optionally enriching, for a population of target RNA molecules in a mixture of RNAs is provided. In some embodiments, the method may comprise (a) adding a label to the 5' end of 5&#x2032;-diphosphorylated or 5&#x2032;-triphosphorylated target RNA molecules in a sample by incubating the sample with labeled GTP and a capping enzyme; and (b) optionally enriching for target RNA comprising the affinity tag-labeled GMP using an affinity matrix that binds to the affinity tag. The label may be an oligonucleotide, which may further comprise an affinity group attached either internally or at 5&#x2032; or 3&#x2032; end of the oligonucleotide where the oligonucleotide label may be added directly, or indirectly via a reaction with a reactive group to the target RNA.

Methods for labeling a population of RNA molecules

Provided herein is a method for making an cDNA library, comprising adding an affinity tag-labeled GMP to the 5' end of targeted RNA species in a sample by optionally decapping followed by incubating the sample with an affinity tag-labeled GTP and a capping enzyme, enriching for RNA comprising the affinity tag-labeled GMP using an affinity matrix that binds to the affinity tag, reverse transcribing the enriched RNA to produce a population of cDNAs, and adding a tail to the 3&#x2032; end of the population of cDNAs using a terminal transferase, to produce an cDNA library.

Enrichment and sequencing of RNA species

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.

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.

A method of enriching for a population of RNA molecules in a mixture of RNAs is provided. In some embodiments, the method may comprise (a) adding an affinity tag to the 5' end of 5&#x2032;-diphosphorylated or 5&#x2032;-triphosphorylated RNA molecules in a sample by incubating the sample with an affinity tag-labeled GTP and a capping enzyme; and (b) enriching for RNA comprising the affinity tag-labeled GMP using an affinity matrix that binds to the affinity tag.

Methods for enriching for a population of RNA molecules

The present invention relates to isolating DNA coding for DNA polymerase I from Thermomicrobium roseum, expressing the T. roseum DNA polymerase I gene in E. coli and purifying the recombinant T. roseum DNA polymerase I from E. coli cell extract.

Method for cloning and producing Thermomicrobium roseum DNA polymerase I

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Ipswich, MA, United States of America

Laurence Ettwiller