How do I modify the ends of a linear DNA sequence?
This lesson describes how to:
- Manually create single stranded overhangs
- 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 will use a sticky ended KpnI-XbaI restriction fragment. For more information, see Extract a Restriction Fragment from a Sequence.
Open or create a linear double stranded DNA fragment.
Switch to Sequence view.
The 5' phosphorylated nucleotide overhangs (sticky ends) generated by simulated digestion with KpnI and NotI are depicted in Map and Sequence views.
Partial restriction enzyme site left due to digestion will be bracketed, for example <KpnI>.
Click Edit → Edit DNA Ends... to open the "Edit DNA Ends" dialog.
Manually Edit Overhangs
Use the Undo and Redo buttons at the top of the Edit DNA ends window if you need to correct mistakes in editing.
Manually Delete a Single Stranded Region
In the "Edit DNA ends" dialog, for either the left or right end, select a region on one strand and type delete, or alternatively, click the Delete Single Strand to remove the region.
The edited end will be displayed.
Click OK to keep the changes.
Manually Backfill an Overhang
In the "Edit DNA ends" dialog, select the complementary region on the strand to be backfilled.
Click Make Double Stranded to backfill the single stranded region.
The end is backfilled and "polished" to create a phosphorylated blunt end.
Simulate Enzymatic "Polishing" of DNA Ends
Polish 3' Overhangs
In the "Edit DNA ends" dialog options are provided for enzymatic polishing of ends.
For 3' overhangs (in this example for KpnI), click Remove to simulate "polishing" and removal of the 3' overhang by exonuclease activity (for example, using T4 DNA polymerase or Klenow fragment).
The 3' overhang will be removed to create a blunt end.
Backfill 5' Overhangs
To backfill 5' overhangs (in the case of XbaI), select Fill in 5' overhang, then click Fill in to simulate backfilling by a DNA polymerase in the presence of a dNTP mixture.
The 3' recess will be filled to create a blunt end.
Partially Backfill 5' Overhangs
If you want to perform a partial backfill with a subset of nucleotides then click the "dNTPs" button and choose specific nucleotides if you want to uses, then click Fill In.
The end will be filled as far as possible using the specified nucleotides.
Polish 5' Overhangs
To simulate enzymatic removal of a 5' overhang (for example, using Mung bean nuclease), use the dropdown menu and choose Remove 5' overhang, then click Remove.
The 5' overhang will be removed to create a blunt end.
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.
- To add a 3' terminal nucleotide to a blunt end, use the dropdown to specify the nucleotide and click Add.
- To remove a 3' terminal nucleotide and create a blunt end, click Remove.
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.