Inverse PCR

How do I simulate inverse PCR with a circular plasmid?

You can use Inverse PCR to linearize an entire plasmid or part of a plasmid.

Use Option 1 below to amplify/linearize an entire plasmid, or Option 2 to amplify/linearize a part of a plasmid.

This lesson instructs on how to simulate Inverse PCR and recircularization by blunt end ligation.

An equivalent procedure, using overhang-based fusion/recircularization (Gibson or NEBuilder methods) can also be simulated in SnapGene. See Circularize a Fragment by Gibson Assembly® and Circularize a Fragment by NEBBuilder® HiFi DNA Assembly for more details.

Option 1: Use Inverse PCR to Amplify/Linearize an Entire plasmid

Choose the Linearization Point

Open a circular plasmid sequence, switch to "Sequence view" and click within the sequence to set the point for linearization. The nucleotide to the right of the cursor will represent the first position of the linearized plasmid.

Note the coordinate of the first position (in this example position 469).

Click Edit → Select Range.

In the "Range Selection" dialog, enter the coordinate of the first nucleotide in the leftmost field.

Enter the coordinate of the first nucleotide - 1 in the right field. Confirm the predicted fragment size (in this example 2686 bp) matches the size of the complete circular plasmid.

Click Select to select the entire sequence, starting at the desired linearization point.

Skip to the section "Open the PCR Dialog".

Option 2: Use Inverse PCR to Amplify/Linearize a Portion of a Plasmid

Select the Region to Remove

In Map view, click on a feature to select it for removal, or in Sequence view, select a specific sequence region for removal.

Invert the Selection

To invert the selected region, click Edit → Invert Selection. The selected region will be amplified by Inverse PCR.

Open the PCR Dialog

To open the PCR dialog, click Actions → PCR....  

Choose the Primers

To automatically design primers, type the desired Tm and click Choose Primers.

Review the Primers

In the PCR dialog, review the primer names and phosphorylation state.

Optional: If required, add a phosphate group to at least one primer by clicking one of the 5' Phosphorylated check boxes.

At least one 5' phosphate is required if your plan to simulate recircularization by blunt-end ligation with DNA ligase.

Verify the Polymerase Selection

Verify that the Polymerase: menu shows the Creates Blunt Ends selection.

Name the Amplified Fragment

When you are ready to simulate inverse PCR, type the name of the amplified fragment, then click PCR.

View the Fragment

The linear fragment will be shown in a new window.

Click File → Save to save the new PCR product sequence to an appropriate location on your computer.

Circularize the Fragment

To circularize the fragment, click Actions → Circularize... .

Type the name of the circularized vector and then click Circularize.

View the Circularized Product

The circular product will be shown in a new window.

Click File → Save to save the new sequence to an appropriate location on your computer.

Review the History

To review the steps performed, switch to History view.