Method for cloning single-stranded oligonucleotides in a plasmid vector.

P Mounts; T C Wu; K Peden

Home/Archive/PubMed

PubMed · 2698200

Method for cloning single-stranded oligonucleotides in a plasmid vector.

P Mounts·T C Wu·K Peden

View original source ↗Export metadata ↓

Abstract

A method for cloning single-stranded oligonucleotides in a plasmid vector has been developed. The method relies on ligation of the oligonucleotide into suitable restriction enzyme sites of the cloning vector such that the site at the 5' end has a 5' overhang [for example, a Bgl II site (A decreases GATCT)], and the site at the 3' end has a 3' overhang [for example, a Sac I site (GAGCT decreases C)]. This arrangement allows the oligonucleotide to anneal to the single-stranded ends of the vector and to be covalently joined by T4 DNA ligase. The complementary strand can be synthesized in vitro to generate a double-stranded plasmid, or the partially single-stranded molecule can be used as a target for site-directed mutagenesis. The subsequent transfer of the oligonucleotide to test plasmids or excision for other manipulations, such as band shift experiments to identify protein binding sites, is facilitated by cloning of the oligonucleotide into a polylinker containing multiple restriction enzyme sites. For this purpose, the plasmid vector, pKP59, which is a 2.0 kB derivative of pBR322 lacking "poison sequences" and containing 16 cloning sites, has been the most satisfactory.

Explore related subjects

Biotechnology Cloning, Molecular Escherichia coli Genetic Vectors Mutation Oligodeoxyribonucleotides Plasmids Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

P Mounts, T C Wu, K Peden. 1989. Method for cloning single-stranded oligonucleotides in a plasmid vector.. https://pubmed.ncbi.nlm.nih.gov/2698200/

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2026

Genomic features, metabolism, and biotechnological applications of Candida tropicalis and other non-albicans Candida species.

The production of bio-based products by yeasts from agroindustrial byproducts is a key strategy for advancing circular bioeconomy. While Saccharomyces species remain the predominant industrial yeasts, their limited ability to assimilate lactose, pentoses, and glycerol, as well as their sensitivity to lignocellulose-derived inhibitors, restricts their efficient application in bioprocesses based on using industrial byproducts as fermentation media. In contrast, several non-albicans Candida species exhibit broad substrate utilization capacities and enhanced tolerance to industrial stresses, making them attractive candidates for the bioconversion of agroindustrial residues. This review critically examines recent advances in the genomic, metabolic, and physiological characterization of promising non-albicans Candida species, including Candida tropicalis, Candida parapsilosis, Candida viswanathii, Candida sojae, and Candida maltosa. Emphasis is given to genome-scale metabolic models, carbon assimilation pathways, stress-response mechanisms, and metabolic engineering approaches aiming at the production of value-added compounds. By identifying current achievements, knowledge gaps, and biotechnological bottlenecks, this review highlights the potential of these yeasts as emerging platforms for sustainable bioprocesses within a circular bioeconomy framework.

Biotechnology↗

PubMed2026

Cell wall-forming chitin synthases in a chytrid fungus.

Chitin is a critical structural component of fungal cell walls, yet our understanding of its synthesis across the kingdom Fungi remains limited. Here, we investigate chitin synthase diversity, transcription and localisation in the aquatic saprotrophic chytrid Rhizoclosmatium globosum, expanding insights into fungal cell wall biology beyond Dikaryan models. We identified 20 chitin synthase genes in the R. globosum genome, mostly canonical Division I and II types with conserved functional motifs. Transcriptomic analysis through zoospore, germling and immature thallus developmental stages revealed stage-specific expression patterns, with active gene diversity correlating with increasing morphological complexity. Using electroporation-based transformation and fluorescent fusion constructs, we indicate expression and localisation of two chitin synthases during cell development. Localisation patterns suggest dynamic redistribution from cytoplasmic dispersion in early encysted cells to concentrated signals at the sporangium wall. Expression in and around the apophysis indicates the importance of these structures in cell maintenance. Our findings highlight functional specialisation among chitin synthases and underscore the importance of cell wall integrity in chytrid development. This work establishes R. globosum as a genetically tractable model for studying chytrid cell biology and contributes to a broader understanding of fungal evolution and cell wall dynamics.

Biotechnology↗

PubMed2011

A versatile adeno-associated virus vector producer cell line method for scalable vector production of different serotypes.

Application of adeno-associated virus (AAV) vector in large animal studies and clinical trials often requires high-titer and high-potency vectors. A number of currently used vector production methods, based on either transient transfection or helper virus infection of cell lines, have their advantages and limitations. We previously developed a 293-cell-based producer cell line method for high-titer and high-potency AAV2 vectors. Similar to several other methods, however, it requires multiple cloning steps for the vector and packaging plasmids and a two-step transfection and selection for stable cell lines. Here we report a simplified method with several key improvements and advantages: (1) a one-step cloning of AAV vector cassette into the serotype-specific packaging plasmid; (2) a single plasmid transfection and selection for stable AAV vector producer cell lines; (3) high vector yields of different serotypes, e.g., AAV2, 8, and 9, upon infection with an E1A/E1B-deleted helper adenovirus; (4) efficient packaging of both single-stranded and double-stranded (self-complementary) AAV vectors; and (5) efficient packaging of large AAV cassettes such as a mini-dystrophin vector (5.0 kb). All cell lines were stable with growth rates identical to the parental 293 cells. The vector yields were consistent among serotypes, with 5 × 10(13) to 8 × 10(13) vector genome particles per Nunc cell factory (equivalent to 40 15-cm plates). The vectors showed high potency for in vitro and in vivo transduction. In conclusion, the simple and versatile AAV producer cell line method can be useful for large scale AAV vector production in preclinical and clinical studies.

Biotechnology↗