New England Biolabs, Inc.

Other

Richard D Morgan

Qing Xu

Richard J Roberts

Zhiyuh Chang

Christopher J Noren

Devon R Byrd

Jay Patti

Vladimir Potapov

Gregory Lohman

John M Pryor

Toby E Claus

Katharina Bilotti

Rebecca Kucera

Tanya Bhatia

Geoffrey G Wilson

Theodore B Davis

Romualdas Vaisvila

Keith D Lunnen

Rebecca B Kucera

Elisabeth A Raleigh

Jack S Benner, Ii

Paul Fergus Walsh

Timothy Meixsell

Lindsay Lovasco

Bing Zhou

Celine Nguefeu Nkenfou

Scott A Westmont

Fana B Mersha

Russ Edelburg

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

Specified restriction endonucleases have been characterized for the first time by their amino acid and DNA sequences. These sequences and those with at least 90% identity thereto have been used as probes in sequence similarity analyses to identify sequence matches in a sequence database that corresponds to novel restriction endonucleases or isoschizomers. The sequence similarity analyses includes selecting a positive sequence match from any sequence producing an expectation value of less than or equal to e&#x2212;02.

Restriction endonucleases, DNA encoding these endonucleases and methods for indentifying new endonucleases with the same or varied specificity

Methods and compositions are provided for creating a binding protein that recognizes a rationally chosen recognition sequence in which a first amino acid has been substituted for a second amino acid using site-directed mutagenesis of a member protein of a set of proteins at an identified position or positions correlated with recognition of a chosen specified target module in the recognition sequence. A system is provided for automating the storage and manipulation of the correlations between positions and types of amino acid residues in the binding protein with specific modules at specified positions in the target recognition sequence and for designing and creating proteins with novel specificities.

Synthetic binding proteins

A protein is described that has an amino acid sequence characterized by at least 90% sequence identity with SEQ ID NO: 24, the protein being capable of recognizing a sequence consisting of 5'-GCCGAG-3&#x2032; within the double-stranded DNA and cleaving the substrate predominantly at 21/19 nucleotides from the recognition site. A method is also described that utilizes the protein for creating a DNA tag for use as a unique identifier for paired end sequencing of DNA or serial analysis of gene expression.

Recombinant type II restriction endonuclease, NmeAIII, and a process for producing the same

Specified restriction endonucleases have been characterized for the first time by their amino acid and DNA sequences. These sequences and those with at least 90% identity thereto have been used as probes in sequence similarity analyses to identify sequence matches in a sequence database that corresponds to novel restriction endonucleases or isoschizomers. The sequence similarity analyses includes selecting a positive sequence match from any sequence producing an expectation value of less than or equal to e-02.

Restriction endonucleases, DNA encoding these endonucleases and methods for identifying new endonucleases with the same or varied specificity

A novel restriction endonuclease has been identified fromspecies 2144 (NEB#1398) which can cleave at nt sequence 5'-CAANNNNNGTGG-3&#x2032; (SEQ ID NO:13) in double-stranded DNA molecules.

CspCI restriction endonuclease

A percussion instrument lift comprising two end-lifts connected by a support rod. Each end-lift comprises a frame, a lifting frame coupled to the frame, and a lift extending between the frame and the lifting frame. Two platforms are adjustably mounted to the lifting frame of the end-lift. The platforms can be transversely slid on the lifting frame so as to accommodate an instrument of varying widths. A fastener can secure the positioning of the platforms with respect to the lifting frame.

Percussion instrument lift

In accordance with the present invention, there is provided a novel type II restriction endonuclease, obtainable fromM82B, hereinafter referred to as 'CstMI', which endonuclease: 

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In accordance with the present invention, there is provided a DNA (deoxyribonucleic acid) fragment which encodes the MmeI type II restriction endonuclease enzyme. This one polypeptide possesses two related enzymatic functions; namely an endonuclease activity which recognizes the DNA sequence 5'-TCC(Pu)AC-3&#x2032; and cleaves as indicated by the arrows: and a second enzymatic activity that recognizes the same DNA sequence, 5&#x2032;-TCC(Pu)AC-3&#x2032;, but modifies this sequence by the addition of a methyl group to prevent cleavage by the MmeI endonuclease activity.

Recombinant type II restriction endonucleases, MmeI and related endonucleases and methods for producing the same

In accordance with the present invention, there is provided a novel restriction endonuclease obtainable from36A (NEB#1384), hereinafter referred to as 'BtgZI', which endonuclease: 

A method is provided for identifying a restriction endonuclease, which includes the steps of (a) screening a target DNA sequence for the presence of known methylase sequence motifs, (b) identifying any open reading frames which lie close to the methylase sequence motifs screened in step (a), and (c) assaying the protein products of these open reading frames for restriction endonuclease activity. Methods for identifying isoschizomers of known restriction endonucleases, which isoschizomers possess a desired physical property, such as thermostability, are also provided by the present invention, as are several novel restriction endonucleases isolated from, MjaIII and MjaIV. Additionally, a gene was identified that encoded a previously observed endonuclease activity, designated MjaII. Also provided by the present invention are vectors suitable for cloning a DNA sequence encoding a cytotoxic protein, via independent transcription promotors which may be selectively controlled by several conditions. A method for producing these cytotoxic proteins using such vectors is also provided, as are stable clones of PacI and NlaIII.

Method for screening restriction endonucleases

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

A method is provided for identifying a restriction endonuclease that includes: screening a target DNA sequence for the presence of known methylase sequence motifs, identifying any open reading frames which lie close to the screened methylase sequence motif and assaying the protein products of the open reading frames for restriction endonuclease activity.

A method is provided for identifying a restriction endonuclease, which includes the steps of (a) screening a target DNA sequence for the presence of known methylase sequence motifs, (b) identifying any open reading frames which lie close to the methylase sequence motifs screened in step (a), and (c) assaying the protein products of these open reading frames for restriction endonuclease activity. Methods for identifying isoschizomers of known restriction endonucleases, which isoschizomers possess a desired physical property, such as thermostability, are also provided by the present invention, as are several novel restriction endonucleases isolated from , MjaIII and MjaIV. Additionally, a gene was identified that encoded a previously observed endonuclease activity, designated MjaII. Also provided by the present invention are vectors suitable for cloning a DNA sequence encoding a cytotoxic protein, via independent transcription promotors which may be selectively controlled by several conditions. A method for producing these cytotoxic proteins using such vectors is also provided, as are stable clones of PacI and NlaIII.

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Middleton, MA, United States of America

Richard D Morgan