TP03
Molecular cloning often involve insertion of a gene or other DNA fragment into a genetic vector (usually a plasmid). This new combination is called a recombinant DNA molecule.
Cloning is necessary in order to study the function of a gene, since the cloning change the context of the gene from its original environment.
Cloning is also almost always necessary in order to express the gene in a different organism, since controlling sequences such as promoters and terminators needed are different between organisms.
It is necessary to plan a cloning experiment before doing the actual experiments. This planning should contain all the manipulations needed to reach the final construction. This exercise will show you how to make simple sequence manipulation on your computer and how to assemble the sequence of recombinant DNA molecules following a cloning strategy.
The main purpose of in-silico cloning is to predict the final sequence outcome of a cloning strategy or recombinant DNA construction project on the computer before performing the actual experiments in order to errors in the cloning strategy.
The final sequence is needed in order to confirm a successful cloning.
The cloning procedures in this document are done by restriction enzyme digestion followed by ligation.
You will practice cloning using a restriction enzyme with:
- 5' overhang cut
- 3' overhang cut
- blunt cut
- outside cut (type IIS enzyme)
The DNA editor ApE is used for these exercises, but any other editor could be used.
Restriction enzymes cleave DNA so that the phosphate remains on the 5' side of the cut (see below).
Restriction enzymes are classified depending on the structure of the cut they make in double stranded DNA. If the cut in the upper (Watson) strand comes before the cut in the lower (Crick) strand, the resulting ends have 5' overhangs (see HindIII below). If the cut in the Watson strand comes after the cut in the Crick strand the resulting ends have 3' overhangs (see PstI below). If the cuts are in the same position, the cut is blunt (see EcoRV below).
vs 3' overhangs & blunt ends
Open a new empty window in ApE or similar DNA editor. Copy the DNA sequence below and paste it into the ApE window.
ggtaccATGAAATAAggtacc
This sequence represents a short gene consisting of a start codon (ATG), one amino acid alanine codon (AAA) and a stop codon (TAA). The ggtacc are recognition sites for a restriction enzyme called Acc65I. The sequence above represents the double stranded linear DNA molecule shown below.
ggtaccATGAAATAAggtacc
|||||||||||||||||||||
ggtaccTACTTTATTggtacc
Now we need to find out how the enzyme Acc65II cuts the DNA; we need to know which bonds are going to be broken by the enzyme.
To do this go to Enzymes>Enzyme Selector in ApE and look at the Acc65II enzyme. Just put the cursor above the enzyme name as shown below:
The ^ symbol above can be understood as a cut in each strand (see below).
-G^G-T-A-C-C-
| | | | | |
-C-C-A-T-G^G-
This information is also available from the on-line restriction enzyme database (see below).
Many companies sell this enzyme.
Question 1: Does Acc65I produce blunt cut or sticky ends?
Question 2: Which one of the following enzymes produce a blunt cut:
- BamHI
- EcoRI
- SmaI
When we digest our molecule with Acc65I, we obtain three molecules of double stranded DNA (see below). The fragment we want to clone is the middle part.
In order to find the Acc65I, site in the sequence, go to Enzymes>Enzyme Selector and click “Highlight” at the bottom of the page.
The resulting Acc65I highlighting in the main sequence window can be seen below.
Note how ApE indicate the cut sites in each strand.
Open the pUC19 sequence in another ApE window, but keep the first sequence open as well. The pUC19 sequence can be found here.
This file should be present in the same folder as this document. Highlight the Acc65I site in pUC19 as well (see below).
Copy the sequence between the cuts in the Watson strand (see above and below) and paste the sequence into the pUC19, exactly where the Acc65I cuts in pUC19.
You have now simulated a cloning experiment with a restriction enzyme (and DNA ligase) that resulted in a recombinant plasmid 🙂.
We call this plasmid pUC19_Acc65I_insert.
Question 3:
The partial checksum for pUC19_Acc65I_insert is cdseguid=rVzaVq and the size is 2701 bp. What is the complete checksum of the new plasmid? How many Acc65I sites can you find in the new plasmid?
Since the sticky ends on each side of the molecule are the same, there is another way to combine the two molecules (see below.
The insert fragment in the second example above has been cloned inverted. This will happen for 50% of the recombinant molecules in a real experiment. Simulate the cloning of the molecule in reverse in pUC19 using a fresh copy of the pUC19 plasmid sequence.
You can obtain the second sequence above by using the reverse complement button: 
on the insert or plasmid sequence. We call the resulting plasmid pUC19_Acc65I_insert_b.
Question 4: The pUC19_Acc65I_insert_b has 2701 bp and the partial seguid is cdseguid=rVzaVq. What is the complete seguid and how many Acc65I sites can you find in the recombinant plasmid?
Some other enzymes such as KpnI or SacI cuts the DNA in a staggered manner so 3' sticky ends are produced. As you can see below, KpnI recognizes the same sequence as Acc65I, but the cut produces 3' overhangs.
Copy the sequence below into a new ApE window.
ggtaccATGCCCTAAggtacc
Reopen a fresh copy of the pUC19 sequence file. Recreate the same cloning experiment as in the Acc65I example, but with KpnI.
As before, we copy the insert between the cuts in the Watson strand and paste where KpnI cuts in the pUC19.
Create the sequence of the recombinant molecule in ApE as described above. The KpnI fragment can also enter in two directions as in the example with Acc65I. Simulate the cloning of the KpnI fragment in reverse as well.
We call the resulting plasmids pUC19_KpnI_insert and pUC19_KpnI_insert_b.
Question 5: Fill in the missing information in the table below:
| Sequences | Size (bp) | Partial seguid | Complete seguid | Number of KpnI sites? |
|---|---|---|---|---|
pUC19_KpnI_insert |
2701 | cdseguid=I37jqk |
? | ? |
pUC19_KpnI_insert_b |
" | cdseguid=oNRmtX |
? | ? |
Some restriction enzymes produce compatible ends, like BglII and BamHI below. These enzymes are isocaudomers. These can ligate to each other as shown in the example below:
Note that the hybrid site is not recognized by either enzyme. This means we can not cut it with either BglII or BamHI.
Question 6:
The sequence below has recognition sites for BglII. Simulate the cloning of the sequence below in both orientations using pUC19:
agatctATGAAATAAagatct
The resulting sequences are called pUC19_BglII_BamHI_insert and pUC19_BglII_BamHI_insert_b. Fill in the missing information below:
| Sequences | Size (bp) | Partial seguid | Complete seguid | Number of BamHI sites? | Number of BglII sites? |
|---|---|---|---|---|---|
pUC19_BglII_BamHI_insert |
2701 | cdseguid=tPVJ7W |
? | ? | ? |
pUC19_BglII_BamHI_insert_b |
" | cdseguid=jN9osk |
? | ? | ? |
Not all restriction enzymes cut inside the recognition sequence. The restriction enzyme BsaI cuts for example on the side, one and four nucleotides away from the recognition sequence (see below). More details about this enzyme can be found at rebase. This enzyme is used for the Golden Gate cloning.
As you can see above, the BsaI site is not symmetric and the recognition site is not a palindrome as in the previous examples.
Reopen the pUC19 sequence file in a new window. Enter the sequence below in a new ApE window:
GGTCTCAgatcATGAAATAAgatcAGAGACC
This molecule is cut by BsaI in the figure below:
The overhang sequence is NOT defined by the recognition sequence, the actual overhan sequence depends on the sequence next to the site. The sequence in this example was designed to be cloned in a restriction site produced by BamHI for example.
Simulate the cloning of this fragment in the pUC19 vector. Create versions with both directions of the insert..
Question 7: Fill in the missing information below:
| Sequences | Size (bp) | Partial seguid | Complete seguid | Number of BamHI sites? |
|---|---|---|---|---|
| pUC19_BsaI_insert | 2699 | cdseguid=BUFuz1 |
? | ? |
| pUC19_BsaI_insert_b | " | cdseguid=k-jmfm |
? | ? |
Question 8: Give an example of a cloning strategy error that could affect these experiments.
Question 9:
Your task is to simulate the cloning of the 1.5 kb BglII – EcoRV fragment from the vector pUG6 into the BamHI – SmaI sites of pUC19. Get pUG6 here . It is also available from GenBank under the accession number AF298793.
This is a directional cloning since the enzymes are not all compatible, see details for how they cut using the same technique as in Question 3. This also means that there is only one result and not two as in the previous examples.
The size of the recombinant vector should be 4184 bp and the partial checksum is cdseguid=THGA3G. What is the complete checksum?
Question 10:
This is an individual exercise for each student. The input data can be found in a Google spreadsheet where you can find your name in the leftmost column.
One column called sequenceZ contains a DNA sequence that represents a double stranded linear DNA molecule.
The columns re1 and re2 contain two restriction enzymes that cut the sequenceZ. The restriction enzymes also cut the plasmid pUCmu. The sequence of the pUCmu can be found here.
Your task is to simulate the cloning of sequenceZ in pUCmu after digestion of both with the two enzymes The resulting sequence where sequenceZ is cloned into pUCmu is called pUCmu_Z. Put your sequence in the indicated cell.
Please answer with raw DNA sequences as indicated for the first example student "Max Maximus”. There is a 3 min YouTube video (very bad sound) here that shows how to solve this question here.
© Björn Johansson 2013-2025