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TOPO cloning is a molecular biology cloning technique in which DNA fragments are rapidly joined to plasmid vectors by the enzyme topoisomerase I, without the requirement for DNA ligase.

What are the types of TOPO cloning?

There are three types of TOPO cloning:

  1. Sticky-end TA cloning
  2. Blunt-end cloning
  3. Directional cloning

Sticky-end cloning and blunt-end cloning are bidirectional with inserts able to ligate in either orientation into the vector. Directional cloning ensures inserts ligate in a preferred orientation.

Sticky-end TOPO TA Cloning

Sticky-end TOPO cloning exploits the inherent activity of Taq polymerase, which adds a single deoxyadenosine (A) to the 3′-end of PCR products. Inserts are PCR amplified and then ligated into linearized TOPO TA vectors which have a complementary deoxythymidine (T) overhang.

Learn how to simulate TOPO TA cloning in SnapGene.

Fig 1. Example of a TOPO TA cloning experiment showing the insert with A overhangs and the vector with T overhangs respectively. The product shows the single base overhang in the final product sequence.

Blunt-end TOPO Cloning

For blunt-end TOPO cloning, both the recipient vector and the DNA insert have blunt ends with no overhangs. The vector is digested with a type IIS restriction enzyme that produces CCCTT ends that topoisomerase I can bind to. The ligation of blunt-ended insert fragments with the blunt-ended vector is then mediated by the topoisomerase I.

Learn how to simulate blunt-end TOPO cloning in SnapGene.

Fig 2. Blunt end TOPO cloning showing the vector and insert with no overhangs

Directional TOPO Cloning

For Directional TOPO cloning the insert is added in a selected direction due to a short (4bp) extension added to the 5′ end of the insert during PCR amplification. The extension is complementary to a TOPO extension on the vector enabling it to ligate in the correct direction.

Learn how to simulate directional TOPO cloning in SnapGene.

Fig 3. Directional TOPO cloning showing the 5′ overhang directing orientation

How Does TOPO Cloning Work?

The key to the TOPO cloning method are the properties of the enzyme topoisomerase I from Vaccinia virus that are exploited in order to ligate two DNA molecules together.

Natively the topoisomerase I enzyme, like restriction enzymes, has both a recognition site that it binds to as well as a cleavage site. In the case of Vaccinia virus topoisomerase I this is the sequence 5´-CCCTT-3.

Additionally, the enzyme has ligation activity where it catalyzes the exposed phosphate group attached to the 3’ most T residue after it has cleaved the DNA strand. The enzyme’s function in the cell is to cleave tightly wound DNA sequences, allow them to unwind and then repair the cleavage before releasing the DNA sequence.

TOPO vectors are prepared and topoisomerase I is introduced so that it covalently joins to the linearized vector ends. This vector topoisomerase I complex is distributed by the manufacturer, Thermo Fisher Scientific.

A suitably prepared DNA fragment is then introduced to the vector topoisomerase I complex. The topoisomerase I’s native ligation functionality joins together the vector with the DNA fragment.

Unlike the T4 DNA ligase enzyme that is typically used in restriction enzyme ligation cloning and is carried out on ice for 30-60 minutes, ligation with the Vaccinia virus topoisomerase I is complete in only 5 minutes at room temperature.

Fig 4. A TOPO TA cloning vector reaction. The presence of the topoisomerase enzyme is shown bound to its cleavage site. After ligation is completed the enzyme is shown to dissociate from the ligated molecule. Image is sourced from Thermo Fisher Scientific.

Try simulating a TOPO cloning experiment today with a SnapGene free trial.

What are the Main Strengths of the TOPO Cloning Method?

There are many advantages to using the TOPO cloning method:

  • TOPO cloning is fast, a typical reaction can be performed and be ready for transformation within 5 – 10 minutes
  • The reaction is performed at room temperature
  • It is a restriction enzyme and ligase free cloning method

What are the Main Limitations of the TOPO Cloning Method?

Even with many advantages, there are limitations to TOPO cloning which you must consider:

  • TOPO vectors are usually prepared and supplied by commercial vendors which can tie you to that vendor’s tools and applications and potentially limit your future design choices
  • You have to be particularly careful about whether your cloned product is in-frame and suitable for expression in an expression vector
  • Single base overhangs do not support directional cloning

When should you use TOPO Cloning?

One of the primary reasons to use the TOPO cloning method is because you want to clone quickly.

Additionally, you will have to determine which type is best suited for your experiment. Here are key considerations when making that decision:

Use blunt-ended or TA TOPO cloning methods when you:

  • Want to clone quickly
  • Want to clone for subcloning or sequencing
  • Want to use restriction enzymes
  • Don’t care about the orientation of the ligated insert

Use directional TOPO cloning when you:

  • Want to clone quickly
  • Want to clone for expression of your insert
  • Want to natively express your insert
  • Want to quickly isolate your insert using the vector’s expression tags

Practical Tips for TOPO Cloning

The following are practical tips to consider when performing a TOPO cloning reaction:

  • The primers used to generate the PCR product should be 5’-phosphate free. This ensures that the topoisomerase can correctly join the two DNA fragments.
  • Use design software that allows you to see the DNA sequence and the associated protein sequence. This is especially important for expression cloning.
  • Use a high-specificity DNA polymerase, such as Pfusion to ensure that there are few to no errors in the amplified sequence. Alternatively, you can include a high-fidelity DNA polymerase with Taq polymerase in the reaction mix. This is especially important for expression cloning applications.
  • If performing TA cloning make sure to increase the final extension time so that 3′ terminal A residues are added to the PCR product.
  • Extra salt in the ligation buffer reduces the likelihood of topoisomerase I rebinding to your newly ligated vector-fragment complex and cutting it again.
  • Typically topoisomerase-mediated ligation is complete in five minutes. Only increase that time if you have a very low concentration of insert or you are cloning very large DNA molecules.

Learn more about cloning

Learn about simulating TOPO cloning in SnapGene and other cloning methods: