How to Design HiFi DNA Assembly

HiFi DNA Assembly is a popular cloning method that allows joining of multiple DNA fragments without relying on restriction sites, making it ideal for complex constructs. On this page, you will find step by step instructions and screenshots showing how to plan and simulate assemblies virtually in CodonCode Aligner.

The HiFi DNA Assembly Wizard

HiFi DNA Assembly is a popular cloning method for one or multiple fragments. CodonCode Aligner supports designing HiFi DNA Assembly cloning experiments with the HiFi DNA Assembly cloning wizard. This feature provides an easy way to plan and visualize the cloning process in silico. Virtual HiFi DNA Assembly in CodonCode Aligner makes it simple to select vector and fragments, can design matching primers automatically, and enables creation and export of cloning related files and samples.

This guide explains how to design HiFi DNA Assemblies in CodonCode Aligner. The assembly is shown with a single fragment, as well as with multiple insert sequences.

Before simulating the HiFi DNA Assembly, open an existing project in CodonCode Aligner, or create a new project, and add the sequences you want to assemble (for example by dragging the sequence files onto the project window).

To start the cloning wizard:

• You can choose the sequence you want to use as the vector now, or later in the cloning wizard. To choose it now, simply click on the sequence so it is selected.
• Choose Tools → HiFi DNA Assembly... to start the cloning wizard.

This will open the cloning wizard with the sequence you selected as the vector:

Selecting Vector and Fragment

The cloning wizard for HiFi DNA Assembly starts with the Vector tab showing. If you have one sequence selected when starting the HiFi DNA Assembly, this sequence will be pre-selected as the vector. If no sequence was selected, or you would like to use a different one, you can pick the sequence in the drop down box on the top right. Note that only sequences that are in your CodonCode Aligner project can be chosen in the drop down box.

By default the vector sequence is linearized with PCR. The radio buttons below the chosen sequence make it possible to change how the vector is linearized. You can either use PCR, restriction enzymes, or an already linearized vector. For the sake of this guide, we will use PCR as the linearization method.

In this example, we will to replace the reporter mRFP1 with GFP. To select everything but mRFP1 as the vector:

• Click on the feature for mRFP1
• Then click on the the Invert Selection button on the right side of the cloning wizard

Clicking on the feature selects all bases for this feature, and the Invert Selection button flips the selection to everything else but what was selected before:

Alternatively, you can select a region by typing the base numbers inside the Start and End fields on the right side of the cloning wizard; or by clicking on features or enzymes; or by dragging along the map or bases with the mouse.

The overview at the bottom of the cloning wizard now shows a selection for the vector (as opposed to not having a selection for the fragment yet).

To select the DNA fragment to replace the mRFP1 segment, switch to the Fragment tab, using the button at the top of the cloning wizard. Then it works just as when selecting the vector: Use the drop down box to choose the sample that contains the insert you want to use, and select the region that covers the insert area.
In this case, select the feature for GFP from the cloning vector pBCiG2 that is in the project:

Note that although the Fragment tab is very similar to the Vector tab, there are a few differences, which are all in the selection area on the right side of the wizard:

• Fragments can be added, deleted and ordered. Since HiFi DNA Assembly can be done with multiple fragments, this fragment is called Fragment 1. We will look at how to simulate a HiFi DNA Assembly with multiple fragments in below.
• Fragments can only be used directly as a linearized fragment, or used for PCR (using restriction digests is only available for vector fragments). The option to use a fragment directly can only be used if a linear sequence is selected, but not if a circular plasmid or vector sequence is selected. When using linear DNA directly in HiFi DNA Assembly, note that either the vector or the fragment can be linear, but not both.
• The direction of the fragment can be reversed by using the Fragment direction buttons on the right side of the cloning wizard.

Primer Design for HiFi DNA Assembly

To create primers for the HiFi DNA Assembly go to the Primers tab.
When you first switch to the Primers tab, and vector and fragment are defined, you will see a dialog prompting you to pick primers:

The dialog shows a summary of the regions to amplify; allows you to set a target melting temperature (Tm) for the primers; and to choose how the primers should overlap. For the overlapping ends you can either choose to use a specific number of bases only, or set a range of bases with a certain Tm. Finally you can select if an overlap should be added to both, the vector and the insert primers, or just one of the two. Please note that which overlap is available may be limited by your earlier choices (for example using a vector that is linearized with restriction enzymes will mean creating primers for the fragment that have an overlap to the vector in order for the HiFi DNA Assembly to work).

The general recommendations to design overlapping primers for HiFi DNA Assembly is to use a 15 - 25 base overlap length with a Tm of 48℃ or higher. For reactions with many fragments or long fragments, increase the primer overlap for better success. Most of the time overlaps do not exceed 40 base pairs.

Click on the Pick Primers button to automatically create the cloning primers that match your chosen settings:

Now that all elements (vector, fragment, and primers) have been chosen and are correct, the cloning wizard shows a cloning product in the overview at the bottom and a green flag at the bottom right of the wizard.
The primers, as well as the primer names, can be changed manually in the table that displays the primers. For example if you do want to add spacers, click inside the primer sequence to edit it. Any sequences that do neither overlap the vector nor the fragment are displayed in blue. If you have primers for insert and vector and manually change one primer, CodonCode Aligner will ask if you would like to update the matching primer and automatically do it for you.

Cloning Product | Creating & Exporting Cloning Samples and Files

Once you are happy with the chosen cloning primers, proceed to the Product tab:

Here you can double check your cloning product and choose which samples and files to create for downstream analysis. In the section on the top right of the cloning wizard, you can choose to create samples for the primers, linearized fragments, and vector. If you choose to create these samples, they will be added to a folder in your CodonCode Aligner project along with the cloning product. You can also export any of these sequences as one or multiple files, which can be useful for ordering primers for example.

Finally, give your cloning product a sensible name at the bottom right, and click on the Clone button. This will create a HiFi DNA Assembly folder with your cloning product and any sequences you chose to create in your CodonCode Aligner project.

You can either close the cloning wizard at this point, or you can leave it open, for example to repeat the cloning experiment with changes, like different primer picking parameters, or another insert sequence.

Cloning Results in your CodonCode Aligner Project

The results from a virtual cloning workflow are added inside a new folder in your CodonCode Aligner project:

A folder called HiFi_DNA_Assembly was added to the project and contains the cloning product and all files we chose to create (in this case these are the primer sequences and the linearized vector and fragment).

HiFi DNA Assembly with Multiple Fragments

HiFi DNA Assembly can also be simulated with up to 15 fragments in CodonCode Aligner. The fragments are added in the Fragment tab using the Add button at the top right:

In the design above, I have used the four linear sequences from the HiFi DNA Assembly.ccap example project to create virtual cloning product. The origin sequence was used as the vector, and the regulator, reporter, and marker sequences were added as the three fragments.

When multiple fragments are added, they are shown in the overview at the bottom in different colors.
Navigating between the different fragment can be done either by using the drop down menu at the top right selecting a fragment directly, or via the arrow buttons next to the drop down menu to cycle through all fragments.

You can add a single fragment by clicking on the Add button, or use the drop down menu in the Add button to add several fragments at once.
Next to the Add button are the Delete and Order buttons. Use the Delete button to delete the fragment that is showing, and the Order button to change the order in which the fragments are assembled:

To change the fragment order, select the fragment(s) in the dialog and use arrow buttons on the right side of the dialog to move them up or down. Changes take effect when you click on Set Order.