Tagmentation combines two steps of DNA preparation into a single reaction. It simultaneously breaks DNA into smaller pieces and attaches adapters, specialized molecular tags, to the ends of those fragments. This streamlined approach has made complex genetic and genomic analyses more accessible and faster, advancing genetic material preparation for various high-throughput applications.
The Tn5 Transposase Enzyme
The molecular machine at the heart of tagmentation is an enzyme known as Tn5 transposase. This enzyme originates from certain bacteria, such as Escherichia coli and Shewanella, where it naturally functions to move segments of DNA, called transposons, from one location in the genome to another. This natural movement is a “cut-and-paste” mechanism, involving the excision of a DNA segment and its insertion elsewhere.
Scientists have engineered a modified version of this enzyme, often called “hyperactive” Tn5 transposase, for more efficient laboratory use. This engineered enzyme is a dimer, consisting of two identical protein units working together. It contains an active site with a specific arrangement of acidic residues, known as the DDE motif, which coordinates metal ions like magnesium to perform its catalytic function.
The Tagmentation Process
The tagmentation process begins by pre-loading the Tn5 transposase enzymes with specific DNA sequences known as adapters. These adapters are short, synthetic DNA molecules designed to serve as molecular tags for subsequent steps in genetic analysis. The combination of the Tn5 enzyme and its attached adapters forms a stable complex called a “transposome”.
When this transposome encounters target DNA, it binds to random locations across the DNA molecule. The Tn5 transposase then simultaneously cuts the DNA double helix and ligates the adapter sequences to the newly created ends. This single, rapid reaction results in DNA fragments already tagged with the necessary adapter sequences, leaving small 9-nucleotide gaps that can be filled in later. This concurrent fragmentation and tagging functions as both scissors and glue simultaneously.
Preparing DNA for Sequencing
One widespread application of tagmentation is in preparing DNA for next-generation sequencing (NGS). Before DNA can be read by sequencing machines, it must be converted into a “sequencing library,” a collection of DNA fragments prepared with specific adapter sequences on their ends. Tagmentation streamlines this library preparation process by combining the fragmentation and adapter ligation steps into a single, quick reaction.
Traditional methods for preparing DNA for sequencing involve multiple, separate steps. These include physically breaking the DNA using methods like sonication or acoustic shearing, followed by enzymatic reactions to repair the ends and then separately ligating adapters. Tagmentation eliminates these discrete steps, reducing the overall time required for library preparation, often completing the initial tagging in as little as 5 minutes. This integrated approach also allows for a lower starting amount of DNA material, requiring nanograms of DNA, making large-scale sequencing projects more efficient and accessible.
Mapping Accessible DNA
Tagmentation also enables a specialized technique called ATAC-seq, an acronym for Assay for Transposase-Accessible Chromatin with sequencing. This method leverages the properties of the Tn5 transposase to map regions of the genome that are “open” or accessible within the cell’s nucleus. DNA within cells is intricately packaged with proteins into a complex structure called chromatin. Some regions of chromatin are tightly wound and “closed,” making the DNA inaccessible, while other regions are more relaxed and “open,” allowing cellular machinery to interact with the DNA.
Because the Tn5 transposome is a relatively large molecular complex, it can only efficiently access and tagment the DNA in these open chromatin regions. By performing tagmentation on intact nuclei, researchers can selectively tag only the accessible DNA fragments. Sequencing these tagged fragments then creates a detailed map of where the chromatin is open across the entire genome, providing insights into active genes and regulatory elements that control gene expression.