Dataset Information

Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase.

ABSTRACT:

Background

The subcloning of a DNA fragment from an entry vector into a destination vector is a routinely performed task in molecular biology labs.

Results

We here present a novel benchtop procedure to achieve rapid recombination into any destination vector of choice with the sole requirement of an endonuclease recognition site. The method relies on a specifically designed entry vector and the combined action of type II and type IIs endonucleases with ligase. The formulation leads to accumulation of a single stable cloning product representing the desired insert carrying destination vector.

Conclusion

The described method provides a fast single step procedure for routine subcloning from an entry vector into a series of destination vectors with the same restriction enzyme recognition site.

SUBMITTER: Fromme T

PROVIDER: S-EPMC2241830 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase.

Fromme Tobias T Klingenspor Martin M

Journal of biological engineering 20071126

<h4>Background</h4>The subcloning of a DNA fragment from an entry vector into a destination vector is a routinely performed task in molecular biology labs.<h4>Results</h4>We here present a novel benchtop procedure to achieve rapid recombination into any destination vector of choice with the sole requirement of an endonuclease recognition site. The method relies on a specifically designed entry vector and the combined action of type II and type IIs endonucleases with ligase. The formulation leads ...[more]

PMID: 18271995

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Project description:IntroductionAspergillus fumigatus (Asp) infections constitute a major cause of morbidity and mortality in patients following allogeneic hematopoietic stem cell transplantation (HSCT). In the context of insufficient host immunity, antifungal drugs show only limited efficacy. Faster and increased T-cell reconstitution correlated with a favorable outcome and a cell-based therapy approach strongly indicated successful clearance of fungal infections. Nevertheless, complex and cost- or time-intensive protocols hampered their implementation into clinical application.MethodsTo facilitate the clinical-scale manufacturing process of Aspergillus fumigatus-specific T cells (ATCs) and to enable immediate (within 24 hours) and sustained (12 days later) treatment of patients with invasive aspergillosis (IA), we adapted and combined two complementary good manufacturing practice (GMP)-compliant approaches, i) the direct magnetic enrichment of Interferon-gamma (IFN-γ) secreting ATCs using the small-scale Cytokine Secretion Assay (CSA) and ii) a short-term in vitro T-cell culture expansion (STE), respectively. We further compared stimulation with two standardized and commercially available products: Asp-lysate and a pool of overlapping peptides derived from different Asp-proteins (PepMix).ResultsFor the fast CSA-based approach we detected IFN-γ+ ATCs after Asp-lysate- as well as PepMix-stimulation but with a significantly higher enrichment efficiency for stimulation with the Asp-lysate when compared to the PepMix. In contrast, the STE approach resulted in comparably high ATC expansion rates by using Asp-lysate or PepMix. Independent of the stimulus, predominantly CD4+ helper T cells with a central-memory phenotype were expanded while CD8+ T cells mainly showed an effector-memory phenotype. ATCs were highly functional and cytotoxic as determined by secretion of granzyme-B and IFN-γ.DiscussionFor patients with IA, the immediate adoptive transfer of IFN-γ+ ATCs followed by the administration of short-term in vitro expanded ATCs from the same donor, might be a promising therapeutic option to improve the clinical outcome.

Project description:More than 3000 type II restriction endonucleases have been discovered. They recognize short, usually palindromic, sequences of 4-8 bp and, in the presence of Mg(2+), cleave the DNA within or in close proximity to the recognition sequence. The orthodox type II enzymes are homodimers which recognize palindromic sites. Depending on particular features subtypes are classified. All structures of restriction enzymes show a common structural core comprising four beta-strands and one alpha-helix. Furthermore, two families of enzymes can be distinguished which are structurally very similar (EcoRI-like enzymes and EcoRV-like enzymes). Like other DNA binding proteins, restriction enzymes are capable of non-specific DNA binding, which is the prerequisite for efficient target site location by facilitated diffusion. Non-specific binding usually does not involve interactions with the bases but only with the DNA backbone. In contrast, specific binding is characterized by an intimate interplay between direct (interaction with the bases) and indirect (interaction with the backbone) readout. Typically approximately 15-20 hydrogen bonds are formed between a dimeric restriction enzyme and the bases of the recognition sequence, in addition to numerous van der Waals contacts to the bases and hydrogen bonds to the backbone, which may also be water mediated. The recognition process triggers large conformational changes of the enzyme and the DNA, which lead to the activation of the catalytic centers. In many restriction enzymes the catalytic centers, one in each subunit, are represented by the PD. D/EXK motif, in which the two carboxylates are responsible for Mg(2+) binding, the essential cofactor for the great majority of enzymes. The precise mechanism of cleavage has not yet been established for any enzyme, the main uncertainty concerns the number of Mg(2+) ions directly involved in cleavage. Cleavage in the two strands usually occurs in a concerted fashion and leads to inversion of configuration at the phosphorus. The products of the reaction are DNA fragments with a 3'-OH and a 5'-phosphate.

Dataset Information

Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase.

Background

Results

Conclusion

Publications

Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase.

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