“DNA cloning/ gene cloning or molecular cloning is a technique used to make identical or similar copies of a DNA or gene.”
Gene cloning is a traditional method or technique used in molecular genetics to make DNA copies. The DNA is a polynucleotide chain, a tiny and colorless entity with the prime function of encoding various proteins.
Its size isn’t adequate to see by the naked eye or under a microscope, furthermore, a copy of a specific gene isn’t sufficient to conduct an experiment. So we need a huge amount of DNA. We need millions of copies of DNA or a specific gene, although DNA can’t replicate outside our body. Replication is an enzyme-governed catalytic process for the synthesis of DNA, in vivo.
The original concept of gene cloning was restricted to only ‘clone’ the DNA, gene or any polynucleotide chain, however, later on, it became an important tool for recombinant DNA technology.
Gene cloning was a popular and fascinating thing in the ‘80s but was later on replaced by the automated polymerase chain reaction technique. PCR is a fast, reproducible, automated and cost-effective technique. It can generate way more copies of DNA within hours than gene cloning.
When we heard about ‘cloning’, dolly sheep came into our imagination, the first artificially cloned Dolly sheep was born in 1996. Although the history of cloning is far too old. The concept of cloning DNA just came to light during the 70s after the discovery of DNA in 1953. Cloning DNA has many groundbreaking and tremendous applications at that time. And are actually real!
As we said, the life span of gene cloning was too short and replaced by other techniques. Still, in recent times such techniques have pivotal importance in molecular genetics and scientists are still using them.
So what exactly is gene cloning? How it is used and what are the applications and limitations? Let us find out.
What is gene cloning?
A clone can be anything identical, here it is DNA or gene. Making copies or clones of DNA is known as gene cloning.
Technically, the gene cloning technique combines restriction digestion, ligation, synthesis by artificial means and generates copies of a specific gene or DNA. It has significantly important applications in genetic engineering, recombinant DNA technology and gene therapy.
The story of DNA began with the discovery of the chemical structure of the DNA by J. Watson and F Crick in 1953. Their finding revealed the molecular structure of the DNA by postulating that the DNA has three key ingredients: Sugar, Phosphate and Nitrogenous bases. DNA is present in almost all life forms, thus, considered as a ‘molecule of life’ for every organism on earth.
Timely various techniques were evolved to study the DNA, nonetheless, the exact history of who first cloned the DNA was unknown! In 1903, Webber J first time used the term ‘Clone’. In 1973, the first animal gene was cloned by Morrow and Cohen et al., from the collaborative work between Stanford university and UCFS.
A biochemist from the Stanford university Berg P was the first person who manufactured the recombinant DNA in 1972. Note that recombinant DNA is artificially prepared DNA of two different origins.
The eukaryotic DNA fragment of Clawed frog Xenopus was isolated and cloned into E Coli. in 1986, Royer- Pokora et al., first time cloned the gene for the inherited human disorder chronic granulomatous disease.
Gene cloning is referred to as a process of copying DNA or gene through recombinant DNA, transformation and gene isolation in order to get copies of a gene for various downstream applications.
Gene cloning is the process of synthesizing DNA artificially using a vector.
Steps and procedure:
A simple looking yet complicated technique gene cloning comprises steps like gene isolation, plasmid insertion and transformation into a suitable bacterial host. The use of the antibiotic resistance gene along with the GOI is a selectable marker to isolate the gene of interest at the end.
DNA extraction: Isolation of DNA from the selected organism.
Isolating DNA or gene of interest: Isolating a fragment of DNA using restriction digestion or chemical lysis.
Constructing a vector: Insertion of DNA into a suitable vector/ vehicle to transfer it into the host.
Introduction into the host cell: insertion of recombinant DNA into the host bacteria by chemical, physical or enzymatic technique.
Selecting transformed cells: Selection of transformed cells through selectable marker antibiotic resistance gene.
Isolating cloned genes: Isolation and purification of gene copies from the host.
Step 1: DNA extraction:
DNA extraction is the first step in gene cloning, in fact, in any genetic experiment. Genomic DNA from the organism whose gene we wish to clone is isolated and purified. There are so many methods of DNA isolation available. Several common techniques are Enzymatic, chemical or physical methods. Here is a list of techniques:
- Proteinase K DNA extraction method
- Phenol- chloroform DNA extraction method
- CsCl density gradient DNA extraction method
- CTAB DNA extraction method
- Spin-column DNA extraction method
- Magnetic bead-based DNA extraction method
If you wish to learn more about each technique, you can read this article: Different types of DNA extraction methods, or if you want more in-depth knowledge you can buy my ebook: DNA extraction to PCR.
Step 2: Isolating gene of interest:
Now we have the entire genomic DNA of an organism. It contains many genes, DNA sequences and junk DNA, we need a specific gene or DNA. There are plenty of techniques available to cut out GOI but the popular and effective way is through restriction digestion.
Restriction digestion is an enzymatic process governed by a restriction enzyme that cleaves DNA at a specific location and allows prospective researchers to isolate a GOI. The fragment is isolated by gel electrophoresis and purified.
Other chemical and physical methods are also available to cleave the DNA of interest, however, are not so effective as restriction digestion.
Step 3: selecting a vector:
Vector transfers our gene in the host genome, vector selection is an important step in the process of gene cloning.
Vector is a kind of vehicle that transfers the GOI to the target location. The vector carries the GOI to the host cell, replicates and makes copies of DNA there. Plasmid DNA, BAC, PAC and YAC are the popular vector systems used in gene cloning.
To use a vector or plasmid in the gene cloning or DNA cloning experiments, it must have several characteristics,
- Self-replication: A vector can replicate using its own replication machinery.
- Marker site: The vector must have a site to insert the selectable marker gene, for example, the antibiotic resistance gene.
- Presence of recognition site: It should have a decent recognition site for the restriction enzyme to cleave.
- High gene carrying capacity: It must have a heavy gene carrying capacity.
- It can’t interfere with the host DNA replication.
- It should have a decently fast replication speed.
Vectors with such characteristics are selected for the gene cloning experiment.
Note that the suitable vector selection also depends on the size of DNA which we wish to clone. Once the process is done. The plasmid (a type of vector) is cleaved by restriction digestion, ligated with the GOI and transferred to the host. The artificially synthesized vector now has two separate DNA viz, its own DNA and the GOI.
See the above image.
Related article: Plasmid DNA- Structure, Function, Isolation And Applications.
Step 4: introducing into a host cell:
We need multiple copies of DNA therefore we need a host organism whose replication or multiplication rate is faster, like the bacteria and doubles within minutes.
A suitable and non-harmful bacterial system is chosen to insert the recombinant plasmid DNA for gene cloning. The gene of interest replicates there and forms protein sometimes if needed. The artificial insulin protein was first manufactured like this!
Techniques like physical, chemical or artificial insertion are employed to penetrate DNA in the host cell. Electroporation, microinjection and chemical insertion are common techniques used routinely. After successful insertion, it starts replicating and generates copies of DNA.
If you are smart enough, you may have a question, how can we distinguish transformed vs non-transformed cells? The answer is in the next step.
Step: 5: selecting transformed cells:
DNA is tiny and colorless! We can’t identify DNA by microscopic analysis but there are ways to do so. The use of a selectable marker, the word we used many times in this article is the best solution for this purpose.
Along with our GOI, a selectable marker as an antibiotic resistance gene is incorporated in the plasmid DNA. Under a selective media, with an antibiotic, non-transformed cells can’t grow as they don’t have an antibiotic resistance gene.
But fortunately, our plasmid-carrying bacteria (with an antibiotic resistance gene) can grow. We can pick colonies for further processing. To grow transformed bacteria faster the culture should have all the necessary nutrients and amino acids, and be processed under ideal cell culture conditions for 72 hours.
Step 6: Isolating cloned gene:
Once we get a successful culture, we have a huge amount of transformed bacteria cells with our gene of interest. Next, we have to isolate it from the host DNA. Using the plasmid DNA extraction protocol, the plasmid DNA (having the GOI) can be isolated.
The isolated DNA is further purified and used for the downstream process. Restriction enzymes (we have used previously) are allowed to digest the GOI to re-isolate our product. The final product is collected to construct a DNA library.
If you wish to learn more on DNA libraries, you can read these two articles:
- The Process of cDNA Synthesis and cDNA Library Preparation.
- Genomic DNA Library- Preparation and Applications
Gene cloning has tremendous applications in diverse fields. To produce artificial protein the gene of interest is allowed to replicate with the host genome and manufacture artificial protein. For example, human insulin, human growth factors, etc.
Bacteria are rapidly growing organisms that make their own proteins from a few genes. When we insert a gene of interest in the bacterial genome they transcribe the foreign gene as theirs, make the mRNA and form a protein product. Such mRNA can directly be used for gene expression and transcriptomics studies.
Advantages and applications:
The main advantage of gene cloning is that it gives multiple copies of a gene or DNA we wish to study.
The process is not harmful to the researcher.
It helps in gene expression studies as well.
Few applications of gene cloning are here;
It was used widely in gene and DNA analysis. Simply by gene cloning the presence or absence of a gene can be determined.
Also by constructing the recombinant plasmid, the function of a gene can be determined. Gene knockout, gene knockdown, and transgene construction are also some other applications.
However, the traditional gene cloning technique- to make copies of DNA is replaced by a robust, rapid and cost-effective PCR technique. Using the Taq DNA polymerase, PCR can amplify or synthesize genes in rapid time.
The structure or sequence of a gene can also be determined by cloning. A gene of interest is isolated and sequenced to know the sequence variations within.
Construction of genetically modified organisms:
The technique of gene cloning is widely employed in the construction of genetically modified organisms using the recombinant DNA technique.
A transgene or recombinant DNA can be inserted in bacteria, plants, mice and other organisms to alter the traits, phenotype and study the function of a gene or disease model.
One of the fascinating uses of gene cloning is in gene therapy to cure genetic disorders. A faulty gene or DNA sequence can be repaired or replaced by the healthy one. If you want to learn more about gene therapy, you can read this article: What is gene therapy and how does it work?
Mutational studies and identification of mutations:
Traditionally the gene cloning technique was used in the identification of mutations and other mutagenesis studies, however, it is now replaced by the polymerase chain reaction technique, as we said it is rapid and cost-effective. It is also used in site-directed mutagenesis studies as well.
Production of recombinant or artificial protein products is one of the most groundbreaking applications of gene cloning. We already have discussed how it happens. Proteins like insulin and growth factors were traditionally synthesized using the present technique.
Limitations of gene cloning:
DNA cloning/ gene cloning or molecular cloning was one of the groundbreaking discoveries in the 80s, but have some limitations and drawbacks.
The present technique is time-consuming. It takes at least 3 to 4 days to get results. Steps like culturing and restriction digestion take days to complete.
The chance of contamination makes it more questionable.
The present technique is also costlier and tedious to perform.
The accuracy and the yield of the experiment are also too low.
The objective of gene cloning is to make copies of DNA, but the technique is not so handy. The limitations of gene cloning were overcome after the discovery of the Polymerase chain reaction in 1983. The polymerase chain reaction is a rapid, cost-effective, accurate and high-yield technique.
The number of DNA copies obtained after the experiment is far more than the conventional gene cloning technique. The PCR saves time and money and provides ease in the experiment, this makes it a powerful “tool” in genetics and genomics.
Zhou Guoling, Yang Guangsheng, Fu Tingdong. Gene cloning techniques Hua Zhong Nong ye da Xue Xue Bao. Journal Huazhong (Central China) Agricultural University. 2001 ;20(6):584-592.