Cloning DNA Using A Restriction Enzyme Stimulation

Zahra Ahmed
9 min readJan 29, 2021

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- rest in peace Dolly the sheep -

Honestly, this is crazy. I mean come on, what did Dolly, the world’s most famous sheep, have to do to get so much recognition? She literally looks like she eats all day, takes a few walks then naps. Well, I can assure you, she did all of those alright.

Other than the fact that she’s the first mammal to be cloned from an adult cell, there’s absolutely no reason as to why she’s different from teenagers going through a global pandemic (surprisingly spot-on 😳)

Dolly deserves a bit more appreciation — so here’s a bit about her. Dolly was born in the lab, so not from the usual reproductive process involving egg and sperm, but from DNA taken from an adult sheep’s mammary gland (aka the gland that produces milk to feed offspring).

Her birth proved to the scientific community that specialized cells could actually be used to create an exact copy, whether it be a plant or animal cells.

So far, we’ve covered a) why a sheep is going down history b) why youth are no different than sheep.

But, let’s take a step back and forget about the sheep fiasco. The fact that we as humans have advanced this far, to not only read and identify genomic sequences but actually clone DNA is mad rad.

Cloning has always been so techy and futuristic in the movies — I mean come on, Jurassic World set some high standards about cloning dinosaurs!

But in reality, cloning genes is now so efficient that it’s actually a standard laboratory technique. However, I can assure you, just because it’s happening in labs, doesn’t mean we’re going to open a dino theme park any time soon.

Welcome to the world of molecular cloning (and not Jurassic park)

What is molecular cloning? Let’s start by breaking up the phrase:

  • “Molecular” means something that has to do with or is made up of molecules.
  • “Cloning”, is the action of producing genetically identical copies.

Together, molecular cloning is making large amounts of identical copies of molecules. So, DNA cloning is making identical copies of genetic material, aka DNA molecules.

To oversimplify, the process of cloning is like sending out mass cold emails. Here’s a quick run-through ↙

  1. DNA is first cut from its original source then pasted into a host — people copy generic cold messages from other platforms, and start sending them out to different professionals.
  2. The host then replicates itself in hopes of copying the new DNA — people continue sending out mass cold emails in hopes of connecting with different professionals.
  3. The hosts that successfully replicated the new DNA get to live — if the cold email was effective, professionals get back.
  4. The hosts that were not successful to replicate the new DNA die off — if the cold email was not effective, then it goes to junk or trash.

Not the best example of cold emailing, but both works 😳 In the laboratory, there’s a standard workflow approach towards cloning. Let’s get at it, and do some cloning.

Step 1: DNA Isolation

The DNA that is intended to clone is first “cut” from the organism source using restriction enzymes.

Restriction enzymes recognize the DNA, and bind to specific sequences, known as restriction sites. Restriction enzymes cut DNA by making two incisions (one through each strand of the double helix).

This incision produces either creates sticky ends or blunt ends.

Diagram of sticky end and blunt end cutting

Sticky ends are like “staggered ends” on a DNA molecule. There are unpaired nucleotides; they form short, single-stranded overhangs. During the process of ligation, sticky ends find each other faster because of their attraction for each other (peep the next step).

On the other hand, blunt ends are a cut straight down — the bases on the ends are flat. They are less likely to find each other, so the ligation process requires more DNA.

Important terminology: the DNA “cut” from the organism is referred to as the DNA insert.

Step 2: Ligation

Once we’ve isolated the DNA from the organism, it needs to go through a ligation process. The ligation process is where we insert the fragment of DNA into a plasmid, so it can replicate.

Big words, let’s break down, starting with plasmids! ↙️

Purpose of a plasmid?

  • A plasmid is a type of vector — they act like vehicles. Plasmids take the desired DNA fragment into a host cell to duplicate.

What does a plasmid look like & where can it be found?

  • Plasmids are small circular DNA molecules that replicate independently in bacteria. They are commonly found in bacteria, some archaea and eukaryotes like plants and yeast.

Main parts of a plasmid

  • Promoter — The promoter region controls RNA polymerase binding and transcription factors
  • Origin of Replication (ORI) — It is the particular sequence in the plasmid where replication is initiated.
  • Multipe cloning site — The place where DNA binds with restriction enzymes.
  • Antibiotic resistance gene — A gene that gives plasmids the ability to be unaffected by antibiotics.

That’s a bit about plasmids, now let’s actually get into ligation.

Plasmids are split with restriction enzymes. The “opening” allows the insertion of the DNA, which then can be spliced together with the plasmid.

An enzyme called DNA ligase permanently joins the DNA fragments when the ends come together. Ligase is an enzyme that can attach double-strand breaks in DNA.

spliced DNA fragments → superglued together

The end result of a) cutting a gene from its original source b) pasting it in a different organism produces a recombinant DNA molecule.

Definition of recombinant DNA → molecules formed in a lab using genetic recombination.

Step 3: Transformation

The plasmid (that now contains recombinant DNA) enters into bacterial host cells through small pores created in the cell membranes.

During this process, the host cells copy the plasmid’s DNA along with their own DNA. In other words, they’re creating multiple copies of the inserted DNA → the cells are being cultured.

Step 4: Selection

Many of the cultured cells may not contain a plasmid with the DNA insert, because the transformation process is not always successful. Things just sometimes don’t work out.

So after transformation, bacteria is grown on a nutrient-rich food plate, and this is where the antibiotic resistance gene in the plamid comes into play.

Cultures are grown in the presence of an antibiotic, which means that only bacteria transformed with the plasmid containing the resistance gene will be able to grow.

And that's it. That’s how you clone DNA. The process may not seem as high-tech, and crazy scientist stuff, but hey it's the first step towards cloning unicorns. 🦄

[Branching off to a different topic for a bit] This is how I feel about emerging science technologies, like gene editing, synthetic biology, bio-engineering, you name it.

Fascinated = Terrified

Why? Because we need to draw the line somewhere.

What if in the near future ( see, it’s not all about the future anymore baby 😎) when we get to understand genomic sequence structures more in depth, a biohacker decides to create a genetically engineered virus that eradicates humans who like, I don’t know, oranges?

Sounds crazy, but it’s so critical to look into, discuss, and implement the laws and ethical aspects of these technologies. I know for a fact that Jennifer Doudna, her team and the rest of the scientific world is having a very similar discussion about CRISPR technology.

Funnily enough, it’s going to be our generation (gen z) establishing these regulations when current pioneer technologies become emerging. It’s wild to think of what power we’re going to hold 😱

Different Methods of Cloning

As cloning is such an important step in different scientific technologies, scientists are implementing the use of various techniques that depend on external factors like time, costs, and even resources.

A couple of techniques include:

  • Restriction enzyme based cloning
  • Gibson assembly
  • Golden gate cloning

Let’s go deeper into restriction enzyme-based cloning. ↙️

Restriction Enzyme Method

Now, why would I want to explore the restriction enzyme method— the name is literally self-explanatory! Restriction enzymes are used to digest and ligate the DNA insert with the plasmid — done.

However, it’s always important to go back to the roots and learn about the pioneers. Wait, is this becoming a history lesson? Well kind of, but not really.

The fact is, without restrictions enzymes, biotechnology startups and leading industries would not have been where they are today. Restriction enzymes were the first application used for cutting DNA into fragments, identifying genes, and also recombining DNA molecules from different genomes.

It was literally the first step, and obviously, I wanted to try it out myself.

How editing a plasmid crashed my computer…

Yeah, my laptop died while working on this stimulation… because I forgot to charge it. The plasmid had nothing to do with it 😂

the benchling platform

I ran the stimulation on Benchling! It’s an amazing software where you can work on DNA sequence editing, designing and other cool stuff! Benchling article collections are very helpful too — it’s a must check out!

So since I decided to take a restriction enzyme-based approach, I performed the digest and ligate assembly on the platform.

The goal was to insert a JfyP gene (a fictional gene) into a pET-31b plasmid.

step 1: selected the backbone of the pET-31b plasmid

Here’s how I did it:

First I identified the backbone of the plasmid to isolate the multiple cloning site — this is where I’m going to insert the JfyP gene.

It’s located between the orange and purple triangles on the top of the plasmid. They represent ClaI and AlwNI (the restriction sites).

step 2: selected the sequence of the JfyP gene

On the JfyP gene, I selected the sequences between the ClaI and AlwNI restriction sites (the bases in the grey box).

side note → Benchling has awesome interactive visual components! As a 15-year-old, the specific feature I’m going to mention blew my mind! Apart from actually seeing the base pairings, I can also visualize the ligation process.

In the Preview below, the bolded letters represent the base sequences of the plasmid. The grey letters are the end bases of the JfyP gene. From the diagram, we can notice the incision of the plasmid created sticky ends.

-preview

We can also see how the JfyP gene sequencing on the right side of the Preview matches up with the plasmid genes in step 3. The grey “C” and bolded “G” pairs up with the starting bases in the JfyP ClaI restriction zone!! The bases literally go hand in hand! 🤯

end result

After inserting the fake gene into the plasmid, this is how the end result looks like. The JfyP gene is now in the place of the multiple cloning site.

Cool stimulation, now what?

There are many different laboratory practises that use cloning like:

  • Reproductive cloning — the process of making genetically identical copies of an organism.
  • Therapeutic cloning — the process of making multiple copies of a cell to treat a disease.

It also has wide range of applications, for example:

  • Genetic analysis — With cloning, we can have access to a greater amount of DNA, thus making analyzing genetic material in detail possible.
  • Gene therapy — DNA sequences can be replicated to produce proteins directly into cells where defective genes are present.

On more of a personal note, I want to learn more about the fields in genomics, molecular biology, anything biotechnology-related really. I find hands-on stimulations the best way to learn new concepts while also having fun. If anyone has any project ideas or recommendations, please do let me know :)

I’m a 15-year-old student researching biotech applications and learning more about applying engineering principles to life forms! If you enjoyed my article and would like to connect, here’s my LinkedIn and monthly newsletter.

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