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Engineering New Nicking Endonucleases

Award Information
Agency: Department of Health and Human Services
Branch: National Institutes of Health
Contract: N/A
Agency Tracking Number: 2R44GM060057-02
Amount: $0.00
Phase: Phase I
Program: SBIR
Solicitation Topic Code: N/A
Solicitation Number: N/A
Timeline
Solicitation Year: N/A
Award Year: 2001
Award Start Date (Proposal Award Date): N/A
Award End Date (Contract End Date): N/A
Small Business Information
32 TOZER RD
BEVERLY, MA 01915
United States
DUNS: N/A
HUBZone Owned: No
Woman Owned: No
Socially and Economically Disadvantaged: No
Principal Investigator
 HUIMIN KONG
 () -
Business Contact
Phone: (978) 927-5054
Email: IRELAND@NEB.COM
Research Institution
N/A
Abstract

Restriction endonucleases are essential tools in genetic engineering, cloning and DNA diagnostics. N.BstNBI is a natural occurring nicking endonuclease which only breaks one strand of double-stranded DNA, and it is the only nicking enzyme that is commercially available. The proposed research is about engineering Type IIs endonucleases into nicking enzymes. In Phase I research, we have cloned and characterized the nicking endonuclease N.BstNBI and two related type IIs endonucleases, PleI and MlyI. Our results showed that type IIs endonucleases PleI and MlyI carry out double-stranded cleavage in a two-step process, in which the DNA is first nicked and then further cleaved on second strand by another enzyme molecule via dimerization. In the case of N.BstNBI, it is the dimerization function that was probably affected by mutation, resulting in a unique endonuclease which nicks DNA. We have successfully converted MlyI into a nicking endonuclease by disrupting its dimerization function. Thus, we have demonstrated that it is possible to convert Type IIs endonucleases into nicking enzymes by mutations. We will apply the techniques used in Phase I as well as additional strategies to convert more Type IIs endonucleases into new nicking enzymes. PROPOSED COMMERCIAL APPLICATIONS: Potentially can lead to the conversion of type IIs restriction endonucleases into nicking enzymes which have great applications in DNA amplification technology (SDA, RCA) and research (DNA replication, repair, etc.).

* Information listed above is at the time of submission. *

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