New England Biolabs, Inc.

Biohelix Corporation

Huimin Kong

Lauren Sears Higgins

Yan Xu

Michael A Dalton

Yanhong Tong

Myriam Vincent

Ira Schildkraut

Geoffrey G Wilson

Rebecca B Kucera

Keith D Lunnen

John J Pelletier

Rebecca Kucera

Wen Tang

Caroline Besnier

Jason M Aliotta

The invention provides methods and compositions for enhancing the speed and sensitivity of helicase-dependent amplification through the use of an endonuclease.

Endonuclease-enhanced helicase-dependent amplification

Compositions and methods useful in nucleic acid assays are provided. The invention permits detection of test and control nucleic acids. Test nucleic acids can be immobilized at multiple locations, such that amplification of either a test nucleic acid or a control nucleic acid provides a captured nucleic acid in a control capture zone.

Detection of nucleic acid amplification products in the presence of an internal control sequence on an immunochromatographic strip

Methods and a kit are provided for selectively and exponentially amplifying nucleic acids and include the use of a helicase preparation and a DNA polymerase such that the amplification can be performed isothermally.

Helicase-dependent amplification of nucleic acids

Methods and a kit are provided for selectively and exponentially amplifying nucleic acids and include the use of a single strand helicase preparation or a thermostable helicase in the absence of a single strand binding protein and a DNA polymerase such that the amplification can be performed isothermally.

Helicase-dependent amplification of RNA

Helicase dependent amplification of nucleic acids

The present invention relates to methods to engineer nicking endonucleases from existing Type IIs restriction endonucleases, and the production of the engineered nicking endonucleases. Two engineering methods are disclosed. One involves inactivating the dimerization function of a Type IIs restriction enzyme using site-directed mutagenesis approach. The other involves replacing the cleavage domain of a Type IIs restriction enzyme with the cleavage domain from a natural occurring nicking endonuclease, N.BstNBI.

Engineering nicking endonucleases from type IIs restriction endonucleases

The present invention relates to recombinant DNA which encodes the MlyI restriction endonuclease as well as the MlyI and BstNBII methyltransferases and expression of MlyI restriction endonuclease and M.BStNBII in cells containing the recombinant DNA.

Method for cloning and expression of MlyI restriction endonuclease and MlyI methylase and BstNBII methylase in E. coli

The present invention relates to recombinant DNA which encodes the PleI restriction endonuclease as well as PleI and BstNBII methyltransferase and expression of PleI restriction endonuclease and M.BstNBII in cells containing the recombinant DNA.

Method for cloning and expression of PleI restriction endonuclease and PleI and BstNBII methylases in E. coli

The present invention relates to the recombinant DNA which encodes the SwaI restriction endonuclease, modification methylase, and the production of SwaI restriction endonuclease from the recombinant DNA. Related expression vectors, pHKUV5 which features a strong, constitutive UV5 promoter without the Lac repressor binding site and pHKT7 which contains a powerful controllable T7 promoter and a low copy number origin of replication, are also disclosed.

Method for cloning and producing the SwaI restriction endonuclease

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 relates to the recombinant DNA which encodes the DraIII restriction endonuclease modification methylase, and the production of DraIII restriction endonuclease from the recombinant DNA. Related expression vectors, pHKUV5 vector which features a strong, constitutive UV5 promoter without the Lac repressor binding site and pHKT7 vector which contains a powerful controllable T7 promoter and a low copy number origin of replication, are also disclosed.

Method for cloning and producing the DraIII restriction endonuclease

The present invention relates to the recombinant DNA which encodes the SgrAI restriction endonuclease and the MspI modification methylase, and the production of SgrAI restriction endonuclease from the recombinant DNA.

Method for cloning and producing the SgrAI restriction endonuclease

The methylase selection method was used to clone the SnaBI methylase gene (snaBIM) from Sphaerotilus natans (ATCC 15291). An active SnaBI methylase was cloned in E. coli using pSnaBI-2, a pUC19 derivative containing two SnaBI sites. Because methylase and restriction genes are usually located alongside each other in a restriction-modification systems, efforts were made to clone the downstream DNA by inverse PCR. Inverse PCR cloned the missing portion of the SnaBI endonuclease and also identified a control, or C, protein. The two open reading frames snaBIR (ORF1) and snaBIC (ORF2) converged toward the SnaBI methylase gene (ORF). Attempts to establish a snaBIR-recombinant plasmid expressing the SnaBI endonuclease in E.coli modified with SnaBI methylase failed. Overexpression of the SnaBI endonuclease in E. coli required the use of the heterospecific BsaAI methylase.

Method for cloning and producing the SnaBI restriction endonuclease and

The present invention relates to the cloning and purification of a thermostable DNA polymerase, Bst polymerase I from Bacillus stearothermophilus. More specifically, it provides a novel method for producing a truncated Bst polymerase using recombinant DNA and protein fusion techniques.

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Wenham, MA, United States of America

Huimin Kong