The development of in vitro transposition technologies have provided many powerful tools for the molecular genetics research laboratory. In this chapter we describe some of these tools with a focus on the Tn5 transposition system. Tn5 technologies are particularly useful because the Tn5 transposition system has simple requirements, is efficient, random in target recognition, and robust. In particular we will describe the use of in vitro Tn5 transposition in transposon tagging and in the generation of nested deletions. We will also describe a unique in vitro/in vivo technology in which Tn5 inserts can be generated in a wide spectrum of bacterial species through the electroporation of preformed tranposase-transposon DNA complexes.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1385/1-59259-755-6:083 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Sample multiplexing is a common approach to reduce experimental cost and technical batch effect. Here, we present a protocol that for the first time allows the pooling of single nuclei from multiple biological samples prior to performing simultaneous single nuclei RNA-seq and ATAC-seq, which we term ltiplexed ltiome (MuMu). We describe steps for assembling the custom Tn5 transposome, performing the transposition reaction, nuclei pooling, sequencing library preparation, and sequencing data pre-processing.
View Article and Find Full Text PDFDual-Plasmid Mini-Tn5 System to Stably Integrate Multicopy of Target Genes in .
ACS Synth Biol
November 2024
State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong 266237, People's Republic of China.
The efficiency of valuable metabolite production by engineered microorganisms underscores the importance of stable and controllable gene expression. While plasmid-based methods offer flexibility, integrating genes into host chromosomes can establish stability without selection pressure. However, achieving site-directed multicopy integration presents challenges, including site selection and stability.
View Article and Find Full Text PDFOpenTn5: Open-Source Resource for Robust and Scalable Tn5 Transposase Purification and Characterization.
bioRxiv
July 2024
Laboratory of Genome Architecture and Dynamics, The Rockefeller University, New York, NY.
Tagmentation combines DNA fragmentation and sequencing adapter addition by leveraging the transposition activity of the bacterial cut-and-paste Tn5 transposase, to enable efficient sequencing library preparation. Here we present an open-source protocol for the generation of multi-purpose hyperactive Tn5 transposase, including its benchmarking in CUT&Tag, bulk and single-cell ATAC-seq. The OpenTn5 protocol yields multi-milligram quantities of pG-Tn5 protein per liter of culture, sufficient for thousands of tagmentation reactions and the enzyme retains activity in storage for more than a year.
View Article and Find Full Text PDFMeasuring B Cell Chromatin Accessibility by One-Step ATAC-seq.
Methods Mol Biol
July 2024
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA.
The Assay for Transposase Accessible Chromatin (ATAC)-seq protocol is optimized to generate global maps of accessible chromatin using limited cell inputs. The Tn5 transposase tagmentation reaction simultaneously fragments and tags the accessible DNA with Illumina Nextera sequencing adapters. Fragmented and adapter tagged DNA is then purified and PCR amplified with dual indexing primers to generate a size-specific sequencing library.
View Article and Find Full Text PDFLow-input PacBio sequencing generates high-quality individual fly genomes and characterizes mutational processes.
Nat Commun
July 2024
Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
Article Synopsis
- Long-read sequencing with PacBio is a game-changer in genomics, effectively managing repetitive sequences but traditionally requiring a lot of DNA, which is a challenge for small organisms.
- Researchers developed a new method called LILAP, which allows for low-input (100 ng) and cost-effective PacBio library preparation using a one-tube process that doesn’t require DNA amplification.
- Testing LILAP on Drosophila melanogaster resulted in almost complete genomes and new insights into mutation processes, suggesting potential for broader applications in studying small organisms and their symbionts.
Want AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!