Transposase / transposon-based biotechnology · BEGINNER LESSON
Tn5
Tn5 is a transposase used in sequencing and genetic tools. In these sources, it is described as part of a Tn5 transposome that can cut DNA and attach sequencing adapters at the same time.
What Tn5 is
Tn5 is a transposase, which is an enzyme that moves DNA segments. In these sources, Tn5 is also described as part of a Tn5 transposome: a complex made of a Tn5 protein homodimer, meaning two matching Tn5 proteins, plus DNA cargo. It is used in next-generation sequencing (NGS), a method that reads many DNA molecules in parallel.
[1] AistSeq: An in-house easy-to-purify Tn5-based plasmid sequencing ... [3] Transposon-based genome editing of industrial microorganisms
How it works
The key action of Tn5 is that it can fragment DNA and attach sequencing adapters at the same time. Sequencing adapters are short DNA pieces that let a sequencing machine read the sample. Because one step does both jobs, NGS library preparation can be faster and simpler. The sources also note that Tn5 can target DNA/RNA hybrids, which broadened its use in RNA sequencing (RNA-seq), a method for reading RNA-derived information.
[1] AistSeq: An in-house easy-to-purify Tn5-based plasmid sequencing ...
Where it is used
Tn5 is widely used in library preparation methods such as the Nextera library preparation kit and in ATAC-seq, which uses Tn5 to insert adapters into open chromatin regions. Chromatin is the DNA-protein material that packages DNA in cells, and open chromatin is more accessible to enzymes. The sources also describe Tn5-based plasmid sequencing workflows, where plasmids, which are small circular DNA molecules, can be sequenced at lower cost in high-throughput runs.
[1] AistSeq: An in-house easy-to-purify Tn5-based plasmid sequencing ... [2] Table 1.
Mechanism and related biology
The broader transposon sources describe transposases as enzymes that recognize terminal inverted repeat sequences, which are matching DNA ends, and move DNA without using the host cell’s homologous recombination system, a DNA repair pathway that exchanges similar DNA sequences. They also describe DNA transposons as usually moving by a cut-and-paste mechanism. In this framework, Tn5 is one of the important tools because it is efficient, random in insertion behavior, and broadly useful in bacteria.
[3] Transposon-based genome editing of industrial microorganisms
Limitations
The sources also show limits. Recombinant Tn5 can be hard to produce and purify at laboratory scale because it binds nucleic acids tightly during purification, so extra purification steps may be needed to remove contaminating DNA. More broadly, transposon systems can be host-specific, so their efficiency may drop in non-model microorganisms.
[1] AistSeq: An in-house easy-to-purify Tn5-based plasmid sequencing ... [3] Transposon-based genome editing of industrial microorganisms
SEE THE IDEA
Tn5 in sequencing workflows
An enzyme that acts on DNA and can be assembled with DNA cargo into a transposome
The starting DNA or DNA/RNA hybrid material to be processed
Short sequencing tags are attached while DNA is fragmented
The prepared library can be read by an NGS instrument
Tn5 acts on the sample, creating adapter-tagged fragments that become a sequencing library.
A simple view of how Tn5 links DNA fragmentation with adapter insertion in one workflow.
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Sources and further reading
This general lesson was generated from public source material. It explains a concept; it does not interpret a private paper or provide medical advice.