The requirements for successful gene cloning to consider are the general steps; the fundamental supplies like your target DNA, vectors, related reagents; and downstream processes.
Basic supplies to clone a fragment of target DNA:
- Target DNA
- Host cells and vectors for gene cloning
- Cloning enzymes
- Host cells and vectors for expressing the cloned DNA
- Growth media for host cell culture
Beyond the basic requirements listed, PCR, electrophoresis, and cell culture resources are also necessary.
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The following steps give you a general idea of the scope of the molecular gene cloning process. Each protocol you encounter will have specific steps and key techniques to optimize the process, but this section lets you look at the typical workflow for molecular gene cloning.
- Isolation and preparation of the source DNA that you want to clone.
- Preparation of the cloning vector.
- Combining the vector and DNA fragments suitably so they form the recombinant DNA molecule.
- Introducing this recombinant DNA (vector + insert) into the host recipient.
- Selecting the host cells that have the correct recombinant DNA introduced into them.
- Ensuring the insert is expressing itself to serve the purposes it was cloned (mass-production of a foreign protein etc.). Often, transgene expression is carried out using a strain or cell line different from the one that was used to clone it.
The first step of gene cloning involves identifying and preparing the desired DNA fragment referred to as the fragment of interest (FoI).
So, to begin, you will first need to isolate the cDNA or genomic DNA. Both are considered to be your source DNA.
Once isolated, the target DNA sequence in your source cDNA or genomic DNA needs to be amplified through PCR before it can be inserted into a vector.
As with any PCR, you need to confirm your results using DNA gel electrophoresis to be certain you have the fragment you intend.
The quality and integrity of the isolated and prepared target DNA are essential for successful gene cloning experiments.
A cloning vector carries the cloned DNA fragment into the desired host organism. But it not only functions as a vehicle delivering your target DNA fragment, it also ensures efficient replication, expression, and maintenance of the cloned DNA fragment within the host organism.
Examples of gene cloning vectors include plasmids, cosmids, and phages.
There are four crucial elements that cloning vectors must have for successful gene cloning: an origin of replication, a selectable marker, a multiple cloning site, and a promoter.
- Origin of replication (ori)
- Selectable marker
- Multiple cloning site (MCS)
Something very important to note is that within gene cloning, there are two types of vectors needed, the cloning vector and the expression vector.
The cloning vector is responsible for cloning your target gene of interest.
The expression vector enables the cloned gene to be expressed.
Expression vectors are DNA molecules used in gene cloning to facilitate the expression of a specific gene or genes in a host organism.
By cloning a gene of interest into an expression vector, researchers can introduce the vector into a suitable host organism, such as bacteria or mammalian cells.
The host cells then replicate the vector and transcribe and translate the cloned gene, leading to the production of the desired protein.
Your cloning and expression vectors may be different, or they might be the same depending on your experimental needs.
Within any gene cloning setup, there are certain enzymes that are necessary to carry out the procedure. Most importantly are your restriction enzymes that cut DNA at specific points, and DNA ligase that join DNA fragments together.
To clone your target DNA fragment into the vector, both the fragment and the vector might need to be cut and then stitched back together.
This is where an enzyme class called restriction endonucleases, also known as restriction enzymes, are useful. Restriction enzymes cut DNA at very specific cut sites.
Within a vector’s multiple cloning site (MCS) are multiple restriction sites where restriction endonucleases cut.
To ensure compatibility between the digested vector and the insert, choosing the most appropriate restriction enzymes is crucial in a cloning reaction.
Restriction enzymes are specifically selected to generate compatible sticky ends between the digested vector and insert.
Figure 1. Shows how restriction enzymes help generate sticky ends between digested vector and the insert
For a much deeper explanation about how restriction enzymes work and their role in molecular cloning, take a look at the restriction enzyme section of this article.
Once you have your digested DNA fragment and the vector with their sticky ends, you will have to join the DNA fragment with the plasmid backbone through covalent bonding. This is facilitated by an enzyme called DNA ligase.
Figure 2. Schematic representation of a DNA ligation reaction during cloning.
The desired host organism is a critical component in gene cloning experiments because it serves as the recipient for the introduction and propagation of the cloned DNA and its expression.
Most of the time, people doing gene cloning will use E. coli strains. Other bacteria, yeast, mammalian or plant cells also may be used.
Horizontal gene transfer methods are used to introduce the recombinant vector construct into the recipient host. There are several methods for doing this:
Bacterial host organisms like Escherichia coli (E. coli) are commonly used due to their ease of manipulation and rapid growth.
Yeast and mammalian cells offer advantages in studying eukaryotic gene expression and protein function.
Plant cells are utilized for cloning plant genes and investigating plant molecular biology.
Growth media, also known as culture media, are solid or liquid mixtures that provide the necessary nutrients, vitamins, and minerals for cell growth and proliferation during gene cloning experiments.
They serve as an environment for the host organisms to propagate, to replicate, maintain and express the recombinant DNA.
The culture media composition can vary depending on the host organisms and gene cloning experimental requirements.
Typically, culture media contains a carbon source such as glucose, a nitrogen source such as amino acids or ammonium salts, salts and other essential nutrients.
Liquid culture media are typically used for growing bacterial or yeast cells while solid media, such as agar plates are used to isolate and select individual transformed colonies.
Growth media are also extensively used when you want to culture the host cells for expressing the recombinant DNA you’ve cloned in those cells.
Choosing your growth media for your host cell culture depends on the specific host organism being used. Different organisms have different nutritional requirements.
For example, if the expression cell line is mammalian, you would need to use the appropriate cell culture methods, which are different from bacterial cell culture.
Commonly used growth media for host cell culture:
- LB (lysogeny broth/ Lauria broth): commonly used for bacterial cell culture.
- TB (Terrific broth): used for bacterial cell culture.
- YPD (Yeast extract Peptone Dextrose): used for growth of yeast cells.
- Minimal media: used for selective bacterial growth or for yeast strains with specific nutrient requirements.
- DMEM (Dulbecco’s Modified Eagle’s Medium): commonly used for mammalian cell culture.
- RPMI 1640: another widely used medium for mammalian cell culture.
In addition, antibiotics or selective agents may be added to media to select cells carrying the recombinant DNA. This type of media is known as selective media.
Selective media enables host organisms that contain the desired recombinant target DNA to grow and be identified.
Components within selective media allow transformed or transfected cells to grow while not being inhibited by non-transformed cell growth.
Using selective media enhances the efficiency and accuracy of gene cloning experiments because it enables researchers to select cells that carry the desired genetic material.
Downstream applications of gene cloning depend on specific research goals like studying a gene’s function or mass-producing a gene or protein. Based on your goals, after cloning, the next processes can include cell culture, PCR, sequencing and protein expression.
Even from a basic science perspective, gene cloning is immensely important. Scientists clone genes to study their functions and how they interact with other genes or proteins.
After gene cloning, several downstream procedures are typically performed to isolate, purify, and analyze the cloned gene or its protein product. These processes may vary depending on the specific application and goals of the cloning project. Some downstream steps are:
- Isolating the recombinant plasmid containing the cloned gene of interest
- Isolated plasmid DNA is often subjected to restriction enzyme digestion/PCR to confirm the presence of the cloned transgene.
- Verification by DNA sequencing such as Sanger sequencing or Next-generation sequencing. This is done next to ensure the accuracy and identify any errors in the sequence or mutation.
- If the goal of gene cloning is to express the gene and produce its protein product, then expression analysis is done. This involves introducing the recombinant plasmid into an appropriate expression host such as bacteria, yeast, insect cells or mammalian cells to express the cloned gene. The expression of the cloned gene is assessed through techniques like western blot, ELISA, etc.
- Next, protein purification is necessary to obtain a pure and active protein product. Various protein purification techniques like affinity chromatography, ion exchange chromatography, size exclusion chromatography etc. are used.
- After purification, the cloned protein can be characterized to determine its biochemical properties such as isoelectric point, enzymatic properties, interactions with other biomolecules by using techniques such as mass spectrometry, circular dichroism spectroscopy, protein interaction assays etc.