New England Biolabs, Inc.

Other

Vladimir Potapov

Jennifer Ong

Shaorong Chong

Gregory Lohman

George Tzertzinis

Kuo-Chan Hung

Haruichi Asahara

John M Pryor

Richard D Morgan

Nathan Tanner

Yinhua Zhang

Yanxia Bei

Katharina Bilotti

Rebecca Kucera

Thomas C Evans, Jr

Aine Quimby

Laurence Ettwiller

Andrew F Gardner

Lixin Chen

Eric J Cantor

Ashley Luck

Kelly M Zatopek

Heidi Crosby

Andrew F Garner

Thomas Evans

A composition and its uses and additionally a kit are provided. The composition is a synthetic self-complementary oligonucleotide that has a double-stranded region and a loop, wherein the double-stranded region contains a binding sequence for PaqCI. Additionally, the oligonucleotide includes unligatable 3' and 5′ ends that cannot be cleaved by PaqCI. This oligonucleotide composition has been combined with PaqCI or a variant of PaqCI Type IIS restriction endonuclease in a reaction mixture, where the reaction mixture includes PaqCI or variant that can further be combined with a ligase and optionally a deadenylase, crowding molecule such as PEG and/or a repair enzyme such as Endo MS. The kit includes the oligonucleotide and Type IIS restriction endonuclease in the same or different containers.

Ordered assembly of multiple DNA fragments

Ordered assembly of large numbers of fragments into a single large DNA have been improved in both frequency and fidelity of the assembled product. This has been achieved by novel compositions and methods that are utilized in a computer system that integrates comprehensive ligation data from multiple sources to provide optimized synthetic overhangs or overhangs from restriction endonuclease cleavage on DNA fragments for assembly by ligation. Intragenic cut sites are avoided by the use of a novel restriction endonuclease which recognizes 7 nucleotides (bases) and cuts DNA to create 4-base overhangs with the help of a synthetic activator oligonucleotide. Variations in ligation preferences by different ligases provide extra precision in assembly reactions. The use of the improved methods are exemplified by the successful assembly from 52 fragments of a viral genome and also a 52 fragment ordered assembly of a bacteria operon.

Compositions and methods for improved in vitro assembly of polynucleotides

A bacteriophage RNA polymerase variant is provided. In some embodiments, the variant may have increased thermostability relative to the corresponding wild type bacteriophage RNA polymerase and/or wild type T7 RNA polymerase. Compositions, kits and methods that employ the variant are also provided.

Thermostable variants of T7 RNA polymerase

The present disclosure relates, according to some embodiments, to systems, apparatus, compositions, methods, and workflows that include DNase I variants with desirable properties including, for example, salt tolerance. A DNase I variant, in some embodiments, may have an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, and/or at least 98% identical to SEQ ID NO:1 and may be identical to SEQ ID NO:1 at one or more positions selected from the group of positions corresponding to L29, A35, D87, Q88, S94, P103, T108, P121, P132, A135, D145, E161, G172, P190, H208, and A224 of SEQ ID NO:1.

DNase I variants, compositions, methods, and kits

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

Variants of the bacteriophage B103 DNA polymerase are described herein. The variant has improved properties, that include when compared to wild-type Phi29 DNA polymerase, at least one of the following: increased thermostability, improved reaction rate for DNA amplification, reduced background and a reduction of bias. Methods of using the DNA polymerase variant are also described herein.

Variant DNA polymerases having improved properties and method for improved isothermal amplification of a target DNA

Mutant bacteriophage DNA ligases that have increased tolerance to salt and/or heat is provided. Methods, compositions and kits that employ the same are also provided.

DNA ligase variants

Growing community of inventors

Auburndale, MA, United States of America

Vladimir Potapov