Ramirez C.L.,Massachusetts General Hospital |
Ramirez C.L.,Harvard University |
Certo M.T.,University of Washington |
Certo M.T.,Seattle Childrens Research Institute |
And 11 more authors.
Nucleic Acids Research | Year: 2012
Engineered zinc finger nucleases (ZFNs) induce DNA double-strand breaks at specific recognition sequences and can promote efficient introduction of desired insertions, deletions or substitutions at or near the cut site via homology-directed repair (HDR) with a double- and/or single-stranded donor DNA template. However, mutagenic events caused by error-prone non-homologous end-joining (NHEJ)-mediated repair are introduced with equal or higher frequency at the nuclease cleavage site. Furthermore, unintended mutations can also result from NHEJ-mediated repair of off-target nuclease cleavage sites. Here, we describe a simple and general method for converting engineered ZFNs into zinc finger nickases (ZFNickases) by inactivating the catalytic activity of one monomer in a ZFN dimer. ZFNickases show robust strand-specific nicking activity in vitro. In addition, we demonstrate that ZFNickases can stimulate HDR at their nicking site in human cells, albeit at a lower frequency than by the ZFNs from which they were derived. Finally, we find that ZFNickases appear to induce greatly reduced levels of mutagenic NHEJ at their target nicking site. ZFNickases thus provide a promising means for inducing HDR-mediated gene modifications while reducing unwanted mutagenesis caused by error-prone NHEJ. © The Author(s) 2012. Published by Oxford University Press. Source
Paul N.,Trilink Biotechnologies, Inc.
Methods in molecular biology (Clifton, N.J.) | Year: 2010
Hot Start activation approaches are increasingly being used to improve the performance of PCR. Since the inception of Hot Start as a means of blocking DNA polymerase extension at lower temperatures, a number of approaches have been developed that target the essential reaction components such as magnesium ion, DNA polymerase, oligonucleotide primers, and dNTPs. Herein, five different Hot Start activation protocols are presented. The first method presents the use of barriers as a means of segregating key reaction components until a Hot Start activation step. The second and third protocols demonstrate Hot Start approaches to block DNA polymerase activity through the use of anti-DNA polymerase antibodies and accessory proteins, respectively. The fourth and fifth protocols utilize thermolabile chemical modifications to the oligonucleotide primers and dNTPs. The results presented demonstrate that all protocols significantly improve the specificity of traditional thermal cycling protocols. Source
Trilink Biotechnologies, Inc. | Date: 2013-07-03
Provided herein are methods for ligase mediated nucleic acid replication and amplification of oligo- and probes containing substituted ligase components, particularly substituted ligase cofactors, substituted oligo- and probe acceptors, substituted oligo- and probe donors, substituted adenylated oligo- and polynucleotide donor intermediates carrying thermolabile group or groups. The substituted ligase components are not active until Hot Start activation step converts them into unsubstituted or natural ligase components, which fully support ligase reaction. The described methods are readily applied to ligation-based assays, especially utilizing Ligase Chain Reaction (LCR), for detection of a nucleic acid sequence where the use of the substituted ligase components improves an overall efficiency of LCR, increase discrimination between matched and mismatched templates and reduces or eliminates appearance of false positive signal. Furthermore, the use of the substituted ligase components reduces or eliminates the false positive signal originated from the template independent and blunt-ended ligation.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 749.96K | Year: 2008
DESCRIPTION (provided by applicant): The polymerase chain reaction (PCR) is a powerful technique used to amplify a DNA sequence of interest. Continual advancements to the technique have included development of real-time PCR technologies and reverse-transc ription PCR. With the advent of these and other technologies that allow for accurate quantitation of a sequence of interest, there are continual needs for improvements to the accuracy of the technique. Herein we propose the further development of a novel H ot Start PCR strategy which may improve the specificity in PCR by reducing the number of undesired amplification products. Although numerous Hot Start PCR technologies have been developed, none of these utilize chemically-modified synthetic deoxynucleoside 5'- triphosphates (dNTPs). The present proposal aims to further develop modified dNTPs as a general solution for Hot Start activation in PCR. It is anticipated that this approach to Hot Start PCR should be amenable to existing PCR technologies, allowing f or use with existing PCR systems, by simple substitution of the unmodified dNTP mix for the corresponding modified dNTP mix. In addition, we propose to further explore the utility of this technology in applications, such as single nucleotide polymorphism ( SNP) detection and microarray analysis. Overall, we propose the development of a novel approach to Hot Start PCR that will offer an added level of specificity to nucleic acid amplification, with the flexibility for use in a number of PCR-based platforms.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 89.00K | Year: 2008
DESCRIPTION (provided by applicant): DNA ligases are more frequently being used as a tool in molecular biology applications that include nucleotide sequence detection, single nucleotide polymorphism (SNP) detection, protein detection, and next generation sequencing by ligation. With the increased demand for DNA ligases in the field of biotechnology, so is the need for improved fidelity of ligation. Although many approaches to improving ligation fidelity have been employed, most involve use of ligases fro m different biological sources, point mutations of key amino acid residues, and modified reaction conditions. Herein, we propose a slightly different approach to improving the stringency of ligation, which employs a set of chemically modified ligation comp onents. In our three-pronged approach, we propose the evaluation of chemically modified variants of the ATP cofactor, the donor probe, and the acceptor probe. The significance of this approach is great because each of these three components makes contacts with different key amino acid contacts within the ligase. It is hoped that subtle chemical alterations to the nucleic acid component of DNA ligase may in turn induce an improvement in the fidelity of ligation. PUBLIC HEALTH RELEVANCE: The field of molecu lar diagnostics is a growing market with a current estimated value of 800 million. One key class of enzymes that are used in these efforts is the DNA dependent DNA ligases. To further improve the accuracy of the DNA joining reaction catalyzed by DNA ligas es, we propose the investigation of chemically modified components.