- Introduction to genetic engineering
- Intro to biotechnology
- DNA cloning and recombinant DNA
- Overview: DNA cloning
- Polymerase chain reaction (PCR)
- Polymerase chain reaction (PCR)
- Gel electrophoresis
- Gel electrophoresis
- DNA sequencing
- DNA sequencing
- Applications of DNA technologies
Overview: DNA cloning
Definition, purpose, and basic steps of DNA cloning.
- DNA cloning is a molecular biology technique that makes many identical copies of a piece of DNA, such as a gene.
- In a typical cloning experiment, a target gene is inserted into a circular piece of DNA called a plasmid.
- The plasmid is introduced into bacteria via a process called transformation, and bacteria carrying the plasmid are selected using antibiotics.
- Bacteria with the correct plasmid are used to make more plasmid DNA or, in some cases, induced to express the gene and make protein.
When you hear the word “cloning,” you may think of the cloning of whole organisms, such as Dolly the sheep. However, all it means to clone something is to make a genetically exact copy of it. In a molecular biology lab, what’s most often cloned is a gene or other small piece of DNA.
If your friend the molecular biologist says that her “cloning” isn’t working, she's almost certainly talking about copying bits of DNA, not making the next Dolly!
Overview of DNA cloning
DNA cloning is the process of making multiple, identical copies of a particular piece of DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of interest (perhaps a gene for a medically important human protein) is first inserted into a circular piece of DNA called a plasmid. The insertion is done using enzymes that “cut and paste” DNA, and it produces a molecule of recombinant DNA, or DNA assembled out of fragments from multiple sources.
Diagram showing the construction of a recombinant DNA molecule. A circular piece of plasmid DNA has overhangs on its ends that match those of a gene fragment. The plasmid and gene fragment are joined together to produce a gene-containing plasmid. This gene-containing plasmid is an example of recombinant DNA, or a DNA molecule assembled from DNA from multiple sources.
Next, the recombinant plasmid is introduced into bacteria. Bacteria carrying the plasmid are selected and grown up. As they reproduce, they replicate the plasmid and pass it on to their offspring, making copies of the DNA it contains.
What is the point of making many copies of a DNA sequence in a plasmid? In some cases, we need lots of DNA copies to conduct experiments or build new plasmids. In other cases, the piece of DNA encodes a useful protein, and the bacteria are used as “factories” to make the protein. For instance, the human insulin gene is expressed in E. coli bacteria to make insulin used by diabetics.
Steps of DNA cloning
DNA cloning is used for many purposes. As an example, let's see how DNA cloning can be used to synthesize a protein (such as human insulin) in bacteria. The basic steps are:
- Cut open the plasmid and "paste" in the gene. This process relies on restriction enzymes (which cut DNA) and DNA ligase (which joins DNA).
- Insert the plasmid into bacteria. Use antibiotic selection to identify the bacteria that took up the plasmid.
- Grow up lots of plasmid-carrying bacteria and use them as "factories" to make the protein. Harvest the protein from the bacteria and purify it.
Let's take a closer look at each step.
1. Cutting and pasting DNA
How can pieces of DNA from different sources be joined together? A common method uses two types of enzymes: restriction enzymes and DNA ligase.
A restriction enzyme is a DNA-cutting enzyme that recognizes a specific target sequence and cuts DNA into two pieces at or near that site. Many restriction enzymes produce cut ends with short, single-stranded overhangs. If two molecules have matching overhangs, they can base-pair and stick together. However, they won't combine to form an unbroken DNA molecule until they are joined by DNA ligase, which seals gaps in the DNA backbone.
Our goal in cloning is to insert a target gene (e.g., for human insulin) into a plasmid. Using a carefully chosen restriction enzyme, we digest:
- The plasmid, which has a single cut site
- The target gene fragment, which has a cut site near each end
Then, we combine the fragments with DNA ligase, which links them to make a recombinant plasmid containing the gene.
Diagram depicting restriction digestion and ligation in a simplified schematic.
We start with a circular bacterial plasmid and a target gene. On the two ends of the target gene are restriction sites, or DNA sequences recognized by a particular restriction enzyme. In the plasmid, there is also a restriction site recognized by that same enzyme, right after a promoter that will drive expression in bacteria.
Both the plasmid and the target gene are (separately) digested with the restriction enzyme. The fragments are purified and combined. They have matching "sticky ends," or single-stranded DNA overhangs, so they can stick together.
The enzyme DNA ligase joins the fragments with matching ends together to form a single, unbroken molecule of DNA. This produces a recombinant plasmid that contains the target gene.
2. Bacterial transformation and selection
Plasmids and other DNA can be introduced into bacteria, such as the harmless E. coli used in labs, in a process called transformation. During transformation, specially prepared bacterial cells are given a shock (such as high temperature) that encourages them to take up foreign DNA.
The DNA produced by ligation (which may be a mix of desired plasmids, side-product plasmids, and linear DNA pieces) is added to bacteria. The bacteria are given a heat shock, which makes them more apt to take up DNA by transformation. However, only a tiny minority of the bacteria will successfully take up a plasmid.
A plasmid typically contains an antibiotic resistance gene, which allows bacteria to survive in the presence of a specific antibiotic. Thus, bacteria that took up the plasmid can be selected on nutrient plates containing the antibiotic. Bacteria without a plasmid will die, while bacteria carrying a plasmid can live and reproduce. Each surviving bacterium will give rise to a small, dot-like group, or colony, of identical bacteria that all carry the same plasmid.
Left panel: Diagram of plasmid, showing that it contains an antibiotic resistance gene.
Right panel: all the bacteria from the transformation are placed on an antibiotic plate. Bacteria without a plasmid will die due to the antibiotic. Each bacterium with a plasmid makes a colony, or a group of clonal bacteria that all contain the same plasmid. A typical colony looks like a small, whitish dot the size of a pinhead.
Not all colonies will necessarily contain the right plasmid. That’s because, during a ligation, DNA fragments don’t always get “pasted” in exactly the way we intend. Instead, we must collect DNA from several colonies and see whether each one contain the right plasmid. Methods like restriction enzyme digestion and PCR are commonly used to check the plasmids.
3. Protein production
Once we have found a bacterial colony with the right plasmid, we can grow a large culture of plasmid-bearing bacteria. Then, we give the bacteria a chemical signal that instructs them to make the target protein.
The bacteria serve as miniature “factories," churning out large amounts of protein. For instance, if our plasmid contained the human insulin gene, the bacteria would start transcribing the gene and translating the mRNA to produce many molecules of human insulin protein.
A selected colony is grown up in a large culture (e.g., a 1-liter flask). The bacteria in the large culture are induced to express the gene contained in the plasmid, causing the gene to be transcribed into mRNA, and the mRNA to be translated into protein. The protein encoded by the gene accumulates inside of the bacteria.
Once the protein has been produced, the bacterial cells can be split open to release it. There are many other proteins and macromolecules floating around in bacteria besides the target protein (e.g., insulin). Because of this, the target protein must be purified, or separated from the other contents of the cells by biochemical techniques. The purified protein can be used for experiments or, in the case of insulin, administered to patients.
Uses of DNA cloning
DNA molecules built through cloning techniques are used for many purposes in molecular biology. A short list of examples includes:
- Biopharmaceuticals. DNA cloning can be used to make human proteins with biomedical applications, such as the insulin mentioned above. Other examples of recombinant proteins include human growth hormone, which is given to patients who are unable to synthesize the hormone, and tissue plasminogen activator (tPA), which is used to treat strokes and prevent blood clots. Recombinant proteins like these are often made in bacteria.
- Gene therapy. In some genetic disorders, patients lack the functional form of a particular gene. Gene therapy attempts to provide a normal copy of the gene to the cells of a patient’s body. For example, DNA cloning was used to build plasmids containing a normal version of the gene that's nonfunctional in cystic fibrosis. When the plasmids were delivered to the lungs of cystic fibrosis patients, lung function deteriorated less quickly.
- Gene analysis. In basic research labs, biologists often use DNA cloning to build artificial, recombinant versions of genes that help them understand how normal genes in an organism function.
These are just a few examples of how DNA cloning is used in biology today. DNA cloning is a very common technique that is used in a huge variety of molecular biology applications.
Explore outside of Khan Academy
Do you want to learn more about restriction enzymes? Check out this scrollable interactive and this simulation from LabXchange.
Do you want to learn more about the role of DNA ligase in gene cloning? Check out this scrollable interactive and this simulation from LabXchange.
Do you want to learn more about bacterial transformation? Check out this simulation from LabXchange.
Do you want to learn more about the selection of transformed bacteria? Check out this simulation from LabXchange.
LabXchange is a free online science education platform created at Harvard’s Faculty of Arts and Sciences and supported by the Amgen Foundation.
Want to join the conversation?
- Why does the DNA need to be in the form of a plasmid when pasted in a bacteria and not just the string of DNA cut by the restriction enzyme?(10 votes)
- There are several reasons:
1) Linear DNA is unstable because there are enzymes present within all organisms (including bacteria) that degrade linear DNA molecules.
2) Vectors contain a sequence (known as the origin of replication) that causes the DNA to be replicated within the bacteria — this is necessary to maintain at least one copy of the new DNA per bacterium as the cells divide.
3) Vectors usually contain at least one sequence that allows selection for the vector (e.g. antibiotic resistance) — this is discussed in this article.
4) Vectors can also be used to do different things with the DNA. A common example of this would be an expression vector — this causes the DNA to be transcribed and translated and would allow you to examine the protein encoded in the cloned DNA.
Does that help?(25 votes)
- Are identical twins also clones ?(4 votes)
- The short answer is no. While they share a lot of DNA, there are also mutations that naturally occur within your cells, so no two people can share exactly the same DNA.(20 votes)
- When the bacteria taking up the plasmid. How can we be sure that the bacteria used aren’t having any plasmid in it? To make sure that when we grow it on agar the bacteria got the recombinant DNA. What if. The original bacteria we use to take up the recombinant plasmids are already having its own plasmid. So how can we differentiate them then?(5 votes)
- Good question :-)
It could be difficult to know if you were just using a random bacteria isolated from nature — especially since there are likely to be many thousands of different plasmids (1730 were present in a sequence database as of 2009). We could sequence all the DNA inside the bacteria, but that is still a lot of work ...
However that doesn't matter as much as you might think.
For example, assume we are using a plasmid that contains a marker (selectable gene) encoding resistance to ampicillin. All we need to know is that the bacteria were are transforming are not already resistant to ampicillin. This is easy to test — we just try growing the bacteria in the presence of ampicillin, if they don't then we can use our plasmid.
In practice microbiologists have domesticated strains of bacteria (a favorite is Escherichia coli — often abbreviated to E. coli) that have been studied for decades. In almost all cases you would be using one of these well characterized strains and so would not need to worry about whether there were unknown plasmids.(11 votes)
- Do the bacteria ever make mistakes in the replication process? If not, why? If they do, how can we still call Dolly the sheep a clone if the original sheep is actually slightly different?(5 votes)
- This is DNA cloning, not the actual cloning of organisms. For more information on cloning, visit this webpage: https://en.m.wikipedia.org/wiki/Cloning. Hope you find it useful! :)(5 votes)
- Why bacterias without plasmids will die?What are the functions of plasmid?(3 votes)
- Plasmids are usually present in bacteria, and plasmids can replicate its own DNA independently of the bacteria, which is why it is often used in DNA cloning. Plasmids usually have an antibiotic resistant gene, so the bacteria won't die in the antibiotic. For more information, visit this webpage: https://en.m.wikipedia.org/wiki/Plasmid. I hope this answers your question! :)(7 votes)
- How is the cutting and joining of DNA monitored?(4 votes)
- To assess whether a "digest" (restriction enzyme cutting reaction) is complete, we usually run a small sample of the digest on an agarose gel§ with a "ladder" sample containing fragments of known sizes and a small sample of uncut DNA.
This comparison allows us to see whether we got fragments of the expected sizes and how much uncut plasmid still remains.
We generally don't directly check whether a ligation has worked — ligation is very reliable and it is usually easier to just transform the DNA into a new host bacteria.
We then purify plasmid DNA and use restriction digests, PCR †, or sequencing to test whether we got the desired outcome.
§For details see:
†For details see:
- How can we clone a gene that is unknown sequence ?(4 votes)
- You can use partial digestion method and expression in bacterial vectors and finally antibiotics to select your cloned vectors.
Look at this:
particularly this paper has protocol:
https://www.researchgate.net/publication/10637344_Partial_characterization_of_a_transposon_containing_the_tetA_determinant_in_a_clinical_isolate_of_Acinetobacter_baumannii using tetA resistance gene as a marker on a plasmid (resistance to Tetracycline).(2 votes)
- Could you have a vector other than the bacterial plasmid
for instance a bacteriophage(3 votes)
- Yes, though every case I know of involves a phage based plasmid (known as a "phagemid") that is manipulated as a bacterial vector before being converted into a bacteriophage.
I haven't personally used phagemids and suspect they are no longer commonly used, but you can learn more about this technology here:
Does that help?(3 votes)
- In "Steps of DNA cloning" step 2, how can a plasmid be transformed into bacteria? Shouldn't it be "Insert the plasmid into bacteria"?(2 votes)
- Transformed with CaCl and heat - called heat shock.
Ca makes bacterial membrane porous and susceptible to the plasmid.(2 votes)
- After you finish cloning the gene into the plasmid, how could you check to see if your gene is actually inside the plasmid? Could restriction enzymes be used to determine this?(2 votes)
- Many scientist send their plasmid samples to a laboratory for sequencing, so they do not have to do it by themselves.(2 votes)