New England Biolabs, Inc.

Ira Schildkraut

Geoffrey G Wilson

Ivan R Correa, Jr

Laurence Ettwiller

Huimin Kong

George Tzertzinis

Madalee G Wulf

Rebecca B Kucera

William E Jack

Lauren Sears Higgins

Nan Dai

Michael A Dalton

Bo Yan

John Buswell

Rebecca Kucera

Julie Forney Menin

Erbay Yigit

Michael Sproviero

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 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

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.

Removal of the guanine cap on the 5' terminus of RNA

The present invention relates to the use of site-specific nucleic acid nicking enzymes to create single-stranded regions in duplex nucleic acids. Such single-stranded regions can take the form of gaps interior to the duplex, or terminal single-stranded regions. Single-stranded termini can be crafted to allow linkage of various elements via base-pairing with elements containing a complementary single-stranded region. This joining is useful, for example, in an ordered, oriented assembly of DNA modules to create cloning or expression vectors. This joining is also useful in attaching detection probes and purifying DNA molecules containing the single-stranded region. Gaps are useful in similar applications, including attaching detection or purification probes.

Use of site-specific nicking endonucleases to create single-stranded regions and applications thereof

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

The present invention provides a novel Type II restriction endonuclease obtainable from Bacillus coagulans. The endonuclease known as Bcg I, recognizes the following nucleotide sequence and has a cleavage point at both ends outside of its recognition sequence: ##STR1## to produce a 34 base pair fragment. Also described is a process for obtaining purified Bcg I from Bacillus coagulans, as well as processes for mapping chromosomal DNA and methods for reducing background in transformants with enzymes such as Bcg I.

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Ipswich, MA, United States of America

Ira Schildkraut