Have you ever heard the phrase “measure twice, cut once?” In woodworking, this means making sure you’re cutting the wood the correct length before you actually cut it, since you can’t really uncut wood.
What does that have to do with DNA plasmids? I’m so glad you asked! In my early days as a graduate student, my advisor altered this phrase to suggest that I check twice and order once when ordering new DNA oligonucleotides to clone new DNA plasmids. Her advice is just as valid for the DNA plasmids themselves – always check and make sure they are what you think they are.
To check that your plasmid has your gene of interest you could:
- Cut the gene of interest out of your plasmid and verify its size
- Sequence the gene of interest
- Sequence the entire plasmid
DNA plasmids are tools that enable detailed biological studies at the gene and molecular levels. Yet it is critically important to make sure that you know exactly what is in your plasmid. Working with the wrong plasmid can easily set your research back years even if it just has one little, tiny error.
In this article we’ll provide some background on the options for verifying your plasmids and weigh the pros and cons of each choice.
In this article:
Cut the Gene of Interest Out of the Plasmid
Sequence the Gene of Interest (Sanger Sequencing)
Sequence the Entire Plasmid (Nanopore Sequencing)
Deciding Between These Verification Techniques
Historical perspective
Not too long ago when you requested a plasmid from another lab or a colleague, you would get an accompanying diagram something like the one in Figure 1. So, assuming the diagram was correct, you had some information about the plasmid you were working with. For example, you would know:
- The gene being expressed
- The antibiotic resistance being used for expression
- Maybe the promoter or restriction enzyme sites used to clone the gene of interest into the plasmid
Figure 1.
Example of a hand drawn plasmid map – this is useful because it tells you the
gene of interest that is being expressed (Gene X) and the resistance (AmpR),
but it is lacking a lot of other important information about the plasmid.
Don’t get me wrong, that’s all really useful information. But to draw an analogy, that would be like trying to drive across the United States with just a single sign at the beginning that says “that way.” You might need a little more information to make it to the other side of the country.
What I’m trying to say is that even with a neat diagram like Figure 1, there’s still so much information you wouldn’t know about this plasmid:
- Which gene isoform is being used?
- Are there any mutations in the gene of interest?
- Are there any mutations in the plasmid backbone, especially mutations that would render the plasmid nonfunctional?
- Gene of interest
- Selection marker
- Replication origin
- Promoter
- Multiple Cloning Site
Key regions of the plasmid where a mutation might render it nonfunctional or dysfunctional include the:
By the way, if you’re looking for a quick refresher on what any of these key components of a plasmid are – check out this article.To verify any of these key features, you would need to check the plasmid and see what’s really in there. Let’s go through a few options for checking your plasmid of interest.
Cut the Gene of Interest Out of the Plasmid
So, one low resolution way to check and make sure your collaborator shared the right plasmid with you is to use restriction enzymes to cut your gene out of the plasmid backbone.
This would tell you if the gene in the plasmid is roughly the same size as you’d expect, and whether the correct enzymes were used if the plasmid was assembled with traditional restriction enzyme-based cloning (Figure 2). But it wouldn’t provide you with much more granular information than that.
Figure 2. Cutting
a gene of interest out of a plasmid. Left, restriction enzymes (scissors) cut
the plasmid on each side of the gene of interest (Gene X). Right, agarose gel
of DNA plasmid without (-) and with (+) restriction enzyme treatment. Restriction
enzymes cut the gene of interest out of the plasmid resulting in a smaller DNA
band that runs faster on an agarose gel.
You might be thinking “why do I need to cut the gene out of the plasmid, why can’t I just run uncut versions of the plasmid without the gene versus with the gene?”
In theory, this should work just fine, but in practice the results are often difficult to decipher. For most genes, the plasmid is much larger than the inserted gene, so telling if there is a difference and by how much is challenging. Furthermore, plasmids often supercoil which would make this approach intractable.
Figure 3. Example of an electrophoresis gel that ran a plasmid with the gene insert as well as a plasmid without the gene insert. Because the gene insert is so small, relative to the plasmid, it is difficult to determine the size of the gene this way.
Also, since cutting the gene out of your plasmid will destroy the thing you were trying to create in the first place, it’s best to do this on only a portion of your plasmid. Then, for any plasmids that look like they have your gene, use the uncut aliquot that you set aside for all downstream purposes.
Sequence the Gene of Interest (Sanger Sequencing)
If you wanted more information, you could send the plasmid for sequencing. Traditionally, that means Sanger sequencing – a technique named after its inventor Frederick Sanger (Sanger et al, 1977). With this traditional technique, you could determine the DNA sequence of your gene of interest, and maybe a little bit of the surrounding area.
The typical read length using Sanger sequencing is around 500-800 base pairs long (Crossley et al, 2020). Since this typical read length is roughly an order of magnitude shorter than the length of most plasmids, you need to dictate which part of the plasmid you want sequenced.
To do this you design short oligonucleotide primers that are about 15-30 nucleotides in length and are complementary to the region of the plasmid that you want to sequence. In the sequencing reaction the primer will bind to the complementary region of the plasmid and provide a docking site for polymerase to start making a copy of your gene of interest for sequencing.
Usually when using this method, the most critical part of the plasmid to sequence is the gene of interest. If your gene is very small, you might only need a single primer. But you will get complete coverage of most short and medium length genes by using two primers at the beginning and end, respectively, of your gene of interest (Figure 4).
Figure 4. When
using Sanger sequencing, you design an oligonucleotide primer to each end of
the gene (purple and orange arrows). This enables the sequencing of each side
of the gene (light purple and light orange boxes). If the gene is particularly
large, additional primers may be needed (Figure 5).
If you’re sequencing a really long gene, you would need to
use additional primers within the gene to obtain the full sequence (Figure 5).
Figure 5. For long genes such as Gene Y, additional primers (green arrow) may be needed to sequence the entire length of the gene.
As you can tell, this approach provides you with a lot more information than just cutting your gene of interest out of a plasmid. You would now know the sequence of the entire (or nearly the entire) gene of interest, and likely enough of the surrounding region to confirm the flanking restriction enzyme sites and maybe even the promoter.
Remember though, although this approach provides you with the sequence of your gene of interest – you are still in the dark regarding the sequence of the rest of the plasmid, including important features such as the origin of replication and the antibiotic resistance gene.
Sequence the Entire Plasmid (Nanopore Sequencing)
One limitation to Sanger sequencing is that it only works for small to intermediate lengths of DNA. However, your plasmid is at least several thousand nucleotides long, so you would have to tile a lot of primers together to sequence the entire thing!
Luckily, nowadays there is a much easier and more cost-effective way to sequence entire plasmids – nanopore sequencing. This technique is called nanopore because a bacterial pore protein pulls DNA, or RNA through a synthetic membrane (Figure 6).
Figure 6. In nanopore sequencing, there is a nanopore protein (blue) that threads DNA or RNA (gray) across a synthetic membrane bilayer (pink and yellow). There is an electric current across the membrane and different nucleotides perturb that current to different magnitudes allowing decoding of the nucleic acid sequence.
There is an electrical current going through the pore and the synthetic membrane, and this current is perturbed to different extents depending on which type of nucleotide is currently in the pore. While there is a bit of intense math that goes into this process, the essential point is that the changes in electrical current are used to decode in DNA or RNA sequence (Marx, 2023).
In theory, there is no limit to how long of a sequence nanopore technology can process. In practice, the average read length is around 10,000 to 20,000 base pairs, and the maximum read length is millions of base pairs (Wang et al, 2021). So, for most purposes, including sequencing plasmids, it is not the technology that limits the read length but rather the DNA or RNA sample.
This all means that this technique will allow you to sequence your entire plasmid, instead of just the gene of interest (Figure 7). Therefore, you will know the sequences of all important regions in the plasmid, and if the plasmid is not working for some reason, you can use those sequences to help identify what the problem might be.
Figure 7.
Nanopore sequences your entire plasmid (purple box) giving you sequence-level
details about the gene of interest (Gene X), antibiotic resistance (AmpR), and all
other important features. Compare this figure to Figures 4 and 5 for the
regions sequenced by Sanger sequencing.
Deciding Between These Verification Techniques
So which technique should you use to verify your plasmid? Well, it depends on the information that you want.
If you just mixed up your tubes that you previously sequenced and want to make sure you know which one is which – cut your gene of interest out and verify you have the right tube. Other than that, I would say you always want to sequence verify your plasmid before using it. If you want to cut the gene out first as a preliminary step in selecting promising clones before sending them for sequencing, go for it. But, you will get that same information (restriction enzyme sites, gene length) from sequencing too.
Ok, now before we decide between sequencing techniques let me give a little historical context. I’m a little bit old. In grad school, I had to walk up hill in the snow both ways (this is a joke). But we didn’t have nanopore sequencing, so I had to sequence all of my plasmids with Sanger sequencing. However, if I were cloning nowadays, I would skip Sanger sequencing and use nanopore every time.
With nanopore, you sequence the entire plasmid with the same next-day turnaround, and it’s roughly the same price as if you Sanger sequenced your gene of interest from both 5’ and 3’ directions (Figure 4). If you’re working with a big gene then you definitely want to use nanopore sequencing instead of tiling a bunch of primers together – you’ll save money, time, and mental effort.
Now there are a few particular sequences that nanopore sequencing struggles with for technical reasons (see Wang et al, 2021 for further details). If that particular sequence is very important in your plasmid for some reason, then go ahead and use Sanger sequencing to confirm this region is correct. Otherwise, in most cases you should just use nanopore to get the entire sequence and correct this known error in silico (in your computer) after sequencing your plasmid.
So, there you have it – 3 easy ways to verify your DNA plasmid. Please, please, please - make sure to do the responsible thing and always sequence your plasmids before you use them!
