Molecular cloning is a primary procedure in contemporary biosciences. This involves introducing a specific piece of DNA (most often a gene) into a cell where it does not generally belong. Furthermore, this process ensures that this foreign DNA fragment replicates (copies itself) and expresses itself (through transcription) in the new host.
A basic schematic representation of the molecular cloning process is depicted below in Figure 1.
Figure 1. Schematic overview of molecular cloning
In this article, we provide a brief discussion on the molecular cloning steps using the most common cloning method, the restriction enzymatic digestion of plasmid and fragment of interest, as a prototype example.
This is followed by a small section comparing some specialized cloning techniques.
6 major steps of molecular cloning
- Isolation and preparation of the insert.
- Preparation of the vector.
- Combining the vector and insert DNA fragments suitably so they form the ‘recombinant DNA’ molecule.
- Introducing this recombinant DNA (the vector, which now has the 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.).
DNA insert preparation for molecular cloning
The first step in molecular cloning is to identify and prepare your DNA fragment of interest (FoI).
Often, the FoI is sought from a source of DNA which may be scarce or contaminated.
For example, consider the situation of cloning a gene from a fossilized organism. Therefore, the first step involves amplifying the target DNA sequence several times using PCR.
Primers can be appropriately designed such that the amplified insert fragment contains specialized sequences such as restriction enzyme sites or homologous recombination sequences at both ends (Figure 1). Fidelity of the fragment of interest to be cloned is generally checked by sequencing prior to cloning into the vector.
Alternatively, the fragment of interest can be purchased from companies that synthesize nucleic acid fragments based on ordered sequences. In this approach, specialized upstream and downstream overhangs are ordered as part of the synthetic DNA sequence.
A “vector” transfers the DNA fragment of interest (FoI) into the host cell just as an insect acts as a vector transmitting diseases.
To understand the structural elements of a vector, and how they are useful in molecular cloning, let us look at a plasmid as a prototype example.
Plasmids are one of the most commonly used vectors in molecular cloning. A plasmid has the following basic features:
- Circular piece of double-stranded bacterial DNA.
- Is replicated independent of the bacterial genomic/ chromosomal DNA.
- The genes encoded on a plasmid are expressed (transcribed and translated) separately from those on the bacterial core genome. Two cells of the same bacterial strain, one harboring the plasmid DNA and the other without it, are markedly different phenotypically owing to the genes on the plasmid.
A plasmid is a very useful tool during molecular cloning because your fragment of insert gets packaged into the plasmid. When the plasmid is replicated, the fragment is also replicated, and the inserted DNA within the plasmid gets transcribed and translated along with the other plasmid genes.
- The fragment of insert can be packaged into it.
- When the plasmid replicates in the host cell, the cloned fragment also gets replicated.
- The insert DNA cloned in the plasmid gets transcribed and translated along with the other genes of the plasmid, using the host cell’s expression machinery.
Figure 2: Schematic diagram of a plasmid
As shown in figure 2, here are the important structural elements of a plasmid:
1. Origin of replication (ori): The origin of replication, or ori, is the site where replication of the plasmid starts. This region also contains sequences that regulate this process. Control of plasmid replication can be such that a plasmid has either a high or low copy number. The ori of a plasmid is different from that of the host cell’s genomic DNA; however, the host cell’s replication machinery (DNA polymerase and other enzymes, nucleotides etc.) is used.
2. Selectable marker: The plasmid backbone has at least one gene that distinguishes cells having the plasmid from cells that do not have the plasmid. Most commonly, plasmids have genes that confer resistance to antibiotics. Thus, bacteria with the plasmid will grow on a plate with the antibiotic selection pressure, while the ones without the plasmid will die out. Since such genes serve as a ‘marker’ for positive selection, they are termed ‘selectable markers’. How they are critical in molecular cloning is described later.
3. Multiple Cloning Site (MCS): This is the site on a plasmid where the transgene can be cloned. This region has multiple restriction enzyme cleavage sequences, which enables cloning the insert fragment at a defined location on the plasmid and in a specific orientation. For details please see the section below on “Restriction enzyme digest of plasmid and fragment of interest.”
4. Promoter: In this region, the RNA Polymerase enzyme binds and drives expression of the genes downstream. Please note from figure 2, the MCS, where the transgene is cloned, is downstream of the promoter; hence transcription of the cloned fragment is ensured. The promoter can be constitutively active. Alternatively, a conditional promoter drives transcription in response to selective stimuli. Therefore, expression of the transgene can be engineered to be constitutive or highly regulated by choosing a suitable plasmid vector.
A vector provides three main advantages. First, vectors have selectable markers, enabling proper cell selection. Second, vector structure allows precise insertion of the gene of interest. And third, vectors have the necessary machinery needed for cloning.
Detailed list of advantages:
- The vector has certain genes within it, like those coding for proteins, that confer resistance to certain antibiotics. Such genes are called “selectable markers” because they enable selection of recipient cells that have the vector, possibly along with the insert DNA fragment introduced into them over recipient cells that do not have the vector.
- Suitable genetic architecture, for example, multiple cloning sites (MCS) flanked by characteristic restriction endonuclease cleavage sites (discussed in detail later in the text)
- The vector has suitable genetic machinery; for example, proper nucleic acid sequences to drive both replication and transcription of the insert, a necessary requisite of successful cloning, inside the host cell.that enables chemically suturing the FoI at a precise location within the vector molecule.
The primary vectors used in molecular cloning are plasmids, cosmids, viral vectors and artificial chromosomes. Each has important features that determine which is the best for use.
- Double-stranded circular DNA found in bacteria.
- MCS allows integration of the fragment of interest (usually not more than 2-3kb in size) at a precise location.
- Replicates and expresses its genes independent of the bacterial genome using the host cell machinery.
- Transferred into host cells via bacterial conjugation or transformation.
- Many copies per cell.
- Type of plasmid with the “cos” site from the Lambda phage.
- Can be maintained in mammalian host cells.
- Fragments of interest as large as 45kb can be cloned.
- Transferred into host cells via transduction.
- Genetically modified viruses that carry the fragment of interest.
- Used to integrate the fragment of interest within the host cell genome.
Artificial Chromosome (AC)
- Used for cloning very large sized fragments. For example, a bacterial artificial chromosome (AC) can integrate (up to 350 kb). And a yeast AC can integrate (up to 10,00 kb).
- 1 copy per cell.
- Has typical features of a chromosome: origin of replication, centromere, telomere.
Restriction enzyme digest of the plasmid and fragment of interest
The multiple cloning site (MCS) of the plasmid vector has multiple restriction endonuclease (RE) cleavage sites.
For a specific cloning reaction, the REs are appropriately chosen to produce compatible “sticky ends” between the RE-digested vector and the insert. Typically, the same REs are used to digest both the insert and vector DNA molecules.
Figure 3A schematically depicts the mechanism of RE action, and how that is exploited in a cloning reaction is described in Figure 3B.
Figure 3. Restriction enzyme (RE)-mediated production of complementarity between vector and insert DNA
Now that we have restriction enzyme-digested inserts and plasmid molecules, an enzyme called DNA Ligase is used to covalently stitch (ligate) the fragment of interest (FoI) into the plasmid backbone.
DNA ligase catalyzes phosphodiester linkages between adjacent nucleotides of the plasmid and the fragment of interest (FoI), in this case. Further, digestion of the FoI and plasmid by the same restriction enzymes (REs) produces single-stranded overhangs which are complementary to each other and tend to readily “stick” (base pair) with each other. This allows the insert to be chemically sutured to the digested plasmid at both ends resulting in a re-circularized plasmid, but this time harboring the FoI.
At this point, the recombinant plasmid molecule has been constructed. Figure 4A schematically describes a ligation reaction. Figure 4B describes how this is used in molecular cloning.
Figure 4. Schematic representation of a DNA ligation reaction during cloning
Bacterial cells are treated appropriately, which make them “competent,” or able to take up the recombinant plasmid prepared in the previous step.
To learn more about competent cells, their importance, how they work and how they’re made, we have two really useful articles with more detail.
Once these “competent cells” indeed take up the plasmid construct, they are phenotypically “transformed” owing to the genes encoded by the plasmid, most notably the antibiotic resistance selectable marker.
Hence this procedure is called “bacterial transformation.” After this step when the host bacterial cells are plated onto solid media containing the appropriate antibiotic (selection pressure), only those that indeed have taken up the plasmid survive as colonies and are thus selected over the non-transformed cells.
While colonies obtained in the last step have the plasmid, it still needs to be determined whether the plasmid is indeed the recombinant construct harboring the fragment of interest (FoI) or if it is merely the blank vector that has self-ligated (and thus, recircularized) at the ligation step.
Many sophisticated short-cuts, such as the blue-white screening method, are now available to visually screen correct recombinant clones versus those with the blank plasmid.
Two traditional methods for screening include the “colony PCR method,” and the “diagnostic restriction enzyme (RE)-digest method.”
- Colonies (from the transformation plate) are directly used as sources of template DNA (the corresponding plasmids) in the PCR reaction
- Primers for this PCR reaction are designed such that they bind to specific sequences on the plasmid just upstream and downstream of where the insert is supposed to be cloned.
- Thus, when the products of the PCR reaction are analyzed using DNA agarose gel electrophoresis, the recombinant colonies give a higher base pair band versus those with just the blank plasmid; the insert fragment accounts for this higher PCR fragment on the gel.
- Primers can be optimized for the various variations of colony PCR.
Diagnostic restriction enzyme (RE)-digest method to screen transformed colonies
In the diagnostic RE-digest method, the recombinant construct gives a different band pattern on the agarose gel than the blank plasmid.
This second method involves two extra steps: plasmid isolation and RE-digest.
Most commonly, molecular cloning is performed to overexpress a transgene (gene of interest from an organism), or to study the phenotype conferred by it.
So it is critical to ensure that the FoI is indeed getting expressed in the target host cell. For this, Northern blot or quantitative Real-time PCR is employed to monitor transcript expression of the FoI. If the FoI is a protein coding gene, Western blot may be utilized to detect expression of the final transgene product.
As evident from Figure 1 and described in detail using the restriction endonuclease (RE) digestion mediated cloning as an example (Figures 3-4), the basic underlying principle in molecular cloning involves:
- creating single-stranded complementary sequences between the vector and the insert DNA molecules,
- which then are annealed and recombined.
The various cloning techniques basically vary in the machinery they use at either of these two steps.
As shown in table 1, instead of RE-mediated single-strand overhang production, other techniques may use specialized exonucleases for this purpose of generating overhangs.
Similarly, instead of ligation, bacterial cellular machinery (see LIC in table 1) may be harnessed to seal the nicks between nucleotides of adjacent fragments that are getting stitched together in the cloning reaction. A brief comparison between common techniques, including their methodology, advantages and disadvantages is listed in table 1.
Table 1 Comparison of common cloning techniques.
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Written By | Pallabi Roy, PhD.