Edit DNA Ends

How do I modify the ends of a linear DNA sequence?

This lesson describes how to:

  • Polish (trim or backfill) or partially polish (backfill) double stranded DNA overhangs
  • Add or remove 5' phosphates
  • Covalently close a double stranded DNA end
  • Add or remove a  3' terminal nucleotide

Open or Create a DNA sequence of Interest

In this example we create a sticky ended linear DNA fragment by selecting the KpnI site on pEGFP-1, then shift-click on the NotI site to select the KpnI-NotI restriction fragment.

Click menu File → New File from Selection to generate a new linear sequence file representing the KpnI-NotI restriction fragment.

The 5' phosphorylated nucleotide overhangs (sticky ends) generated by simulated digestion with KpnI and NotI are depicted in Map and Sequence views.

"Polish" the DNA Ends

Click Edit → Edit DNA Ends... to open the "Edit DNA Ends" dialog.

The two ends of the DNA fragment are displayed.

You can click within the top or bottom strands of either end and manually edit to alter the nucleotide sequences.

Use the Undo and Redo buttons at the top of the Edit DNA ends window if you need to correct mistakes in editing.

For 3' overhangs (in the case of KpnI), click Remove to simulate "polishing" and removal of the 3' overhang by exonuclease activity (for example, using T4 DNA polymerase or Klenow fragment).

To backfill 5' overhangs (in the case of NotI), select Fill in 5' overhang, then click Fill in to simulate backfilling by a DNA polymerase in the presence of a dNTP mixture.

Click dNTPs, and choose specific nucleotides if you want to partially backfill with a subset of dNTPs, then click Fill In.

To trim 5' overhangs, (in the case of NotI), select Remove 5' overhang, then click Remove to simulate enzymatic removal of the 5' overhang (for example, using Mung bean nuclease).

Add or Remove 5' Phosphate Groups

Choose Unmodified to simulate removal of a 5' phosphate by a phosphatase enzyme (for example, using Bovine Intestinal Alkaline Phosphatase).

Choose 5' phosphorylated to simulate enzymatic addition of a 5' phosphate by a kinase (for example, using T4  Polynucleotide Kinase).

Covalently Close a Blunt End

For blunt ends, you can choose to simulate covalent closure of the forward and reverse DNA strands.

Add or Remove 3' Terminal Nucleotides at a Blunt End

Simulate the non-templated addition of 3' terminal nucleotides by TAQ polymerase to a blunt ended fragment.

Click Add to add a 3' Terminal nucleotide, click Remove to remove a '3 Terminal nucleotide.

The default addition is an "A" nucleotide as "A" is the nucleotide preferentially added by Taq polymerase in the presence of a mixture of all dNTPs. Use the dropdown to choose to simulate addition of other single nucleotides, mixtures of nucleotides (R - A or G, Y - C or T), or non standard nucleotides (U - uracil).

See the User guide lesson Simulate TA TOPO® cloning and the video TA and GC cloning to learn how 3' terminal nucleotides are used to facilitate TA- and GC- cloning procedures.