This practice session will give you a review of the basic functions of the ApE plasmid editor and how to visualize linear and circular DNA sequences. ApE is a free, simple to use DNA sequence editor written by Wayne Davis at the Department of Biology, University of Utah. ApE is quick and intuitive and has all the tools and functions you need to design your cloning strategies without getting in the way.

If you have already installed ApE, skip to the next section “Enter a sequence in ApE”.

1. Go to: www.biology.utah.edu/jorgensen/wayned/ape or Google “ape plasmid” to find the download page.
2. On the top of the page are symbols for OSX or Windows.
3. Click on the symbol for your operating system:

The file for Windows is called “ApE_win_current.zip”. If you unzip the file, you will have a file called “ApE.exe”. You can use ApE.exe directly without installation. You should see something like the window below when open.

You can enter sequence directly into ApE by placing the cursor in the text window and typing using the letters “a”, “g”, “c”, “t” or “n”. These letters represent the four bases of DNA (Fig 4) and “n” represents an unknown base. These five letters are the only ones permitted by ApE.

Enter the sequence “tttttt” (six “t”s) in the ApE window like in

ApE assumes that DNA is double stranded. This means that the DNA molecule we entered is really a double stranded molecule with both a sense (watson) and an antisense (crick) strand:

5'-tttttt-3' (watson) |||||| 3'-aaaaaa-5' (crick)

We only see the top strand (“tttttt”) and DNA sequence is always presumed to be in the 5' to 3' direction.

We can change the point of view of out molecule by clicking the button on the toolbar OR choose Edit>Reverse-Complement from the menu. Now you should see the sequence shown in Fig 8.

If you click the button again or choose Edit>Reverse-Complement, you will see “tttttt” again. It is very important to understand that these are two equally valid representations of the same DNA molecule. The sequence does not change in any way after this operation. We only choose which way we choose to see the double stranded DNA molecule.

Question 1: What is the sequence of the DNA molecule in Fig 9? What is the reverse-complement?

Question 2: Can you think of any type of DNA or RNA molecule that does not have a complementary sequence?

DNA can be either linear (like a human chromosome) or circular (like most plasmids). This structure can not be determined from the primary sequence. For this reason, we have to tell ApE whether the DNA is linear or circular.

If our molecule (tttttt) was circular, For our molecule this means that there is a bond between the first and last thymines and between the first and last adenines:

Enter “aaaaaa” into the ApE window. Click on the “linear” button to toggle the sequence to “circular” (Fig 11).

Click the “Graphic map” icon on the toolbar OR choose “Enzymes>Graphic map” from the menu. You should see a new window appearing showing a circular molecule of 6 bp (Fig 12).

Fig 12*

Now close the window showing the circular molecule and display the molecule as if it was linear. You should now see a figure like in Fig 13.

Fig 13: a linear sequence in ApE

It is important to select the proper format, especially for making a graphic map, but also for simulation of restriction analysis.

Change origin for a circular DNA molecule The origin of a circular DNA molecule is located at 12 O'clock in the image of the molecule. For example the circular plasmid “plasmid1” below.

>plasmid1 aattcg

Plasmid1 represents the circular molecule in Fig 14 where the origin is indicated in blue.

origin

![ref1]

The circular plasmid pUC19 is a popular cloning vector for E. coli. The sequence for this plasmid is available in the same folder as this document in a file called:

“plasmid_pUC19_in_raw_sequence_format.txt”.

Raw means that the file only contains the nucleotides of the sequence. The sequence is 2686 bp long. Open the sequence in ape by copy-paste or by using the file/open function in ApE. The sequence should look like Fig 19. The sequence is also available at Genbank with accession number L09137. The sequence should have 2686 bp.

Fig 19*

Question 4: What are the last five characters of the pUC19 sequence?

Question 5: Change the origin of the pUC19 sequence as indicated by the symbol ▲ below:

“tcgcgcgtttc...”. Now the sequence should start with “tttcggtgat...”. What are the last seven characters of the new sequence?

The new sequence represents the same circular molecule as before.

ApE has of course many more features than was covered in this tutorial. If you are interested, there are some good tutorials on-line as documents or videos. There are links to these tutorials at the end of this document.

Question 6:

This is an individual exercise for each student. The input data can be found in The TP02 Google spreadsheet where you can find your name in the leftmost column.

One column called Sequence1 contains a DNA sequence that represents a double stranded circular DNA molecule. Change the origin of this molecule so that it starts with the sequence indicated in the “New origin” column. Use the same method as in the previous question (5).

Put your answer in the "Answer" column. Please answer with a raw DNA sequence as indicated for the first example student "Max Maximus". If your name is ***not\ in the list, please inform your instructor.

There are many tutorials on YouTube regarding the installation and use of ApE. See also this blog post about cloning a cDNA in the pCR Blunt II TOPO vector.

ApE is a popular editor which is free, stable and fast. It also seems to work well on all operating systems. There are alternatives that might be more user-friendly and have more features that you can use if you want. Note that all TP classes will assume that you have ApE. There is a compilation of alternatives on researchgate.

Some popular alternatives are:

• Serial Cloner (Free, offline) (Win, Mac, Linux)
• Benchling (Free, online only)
• SnapGene (Free trial period, offline) (Win, Mac)
• GenomeCompiler (Free, online)

© Björn Johansson 2024