Molecular Cloning Techniques
Molecular cloning or the creation of recombinant DNA is an essential process used in scientific research and discovery. With molecular cloning scientists can amplify and manipulate genes of interest and then insert them into plasmids for replication and protein expression. So how do scientists recombine DNA?
There are many methods that have been utilized over the years to move around pieces of DNA. Oftentimes several approaches will work for any specific cloning project; however, it is likely that for any given project there is an ideal approach. This may be due to speed, cost, availability of starting materials or just personal preference. Check out our blog on choosing the right cloning method for your research project.
The following guide will highlight several of the most popular cloning methods used to create recombinant DNA.
Restriction Enzyme Cloning
Restriction enzyme (endonuclease) based molecular cloning is the "classic" cloning method, and for many reasons, remains one of the most popular today. Restriction enzymes, which are naturally produced by certain bacteria and archaea, cleave double stranded DNA (dsDNA) at specific sequence sites in the DNA. In restriction cloning, scientists utilize specific restriction enzymes to cut dsDNA of interest into fragments containing precise 5' or 3' single-strand overhangs (sticky ends), or no overhang (blunt ends). Two pieces of DNA that have complementary overhangs, or which are both blunt-ended, can then be fused together during a ligation reaction with T4 DNA ligase.
Restriction enzyme cloning benefits from the hundreds of available enzymes, many of which are relatively cheap. They also cut specific target sequences, that range from 4 to 13 base pairs, and produce predictable resulting ends in the DNA fragments. Given its prevalence, the vast majority of plasmids used for DNA cloning and expression contain several popular restriction enzyme sites. You can easily move (subclone) any piece of DNA that already has restriction sites on either side of it into any plasmid that has the same sites in the same orientation within its multiple cloning site. Due to their short length, it is also easy to add restriction sites to any piece of DNA during PCR amplification, allowing for it to then be digested and ligated into your desired plasmid. It is important to note that restriction enzyme target sites can be repeated throughout a specific DNA sequence, which can make it difficult at times to identify compatible restriction enzymes, that cut your insert or backbone at only the desired location for your cloning project. Restriction enzyme cloning also leaves behind a short scar in the DNA sequence and can be time consuming compared to other cloning methods.
Gateway® Recombination Cloning
Gateway® cloning is a recombination based cloning method. The benefit of Gateway® is that moving a piece of DNA from one plasmid into another is done via a single recombination reaction, drastically simplifying the process and reducing the amount of time required for cloning.
To utilize this approach, the fragment of DNA that you would like to clone into a plasmid must already be surrounded by specific recombination sites (in this regard, not so dissimilar from restriction enzyme cloning). To do this your DNA fragment must first be amplified with specific Gateway attB1 and attB2 sites attached to the 5’ and 3’ ends of DNA sequence. This fragment can then be cloned into a gateway donor plasmid which contains compatible attP sites via a proprietary BP clonase (creating an entry clone). The entry clone now has recombined attL sites flanking your DNA fragment of interest.
Now that your DNA fragment has been cloned into a donor plasmid, it can be rapidly shuttled into any compatible Gateway® Destination vector, which contain attR sites via LR clonase enzymes. Thousands of Gateway® destination plasmids have been made with different promoters, tags and fluorescent proteins. Thus, you can clone your gene of interest one time into a donor plasmid (or acquire one that already has your gene into it) and then using bacterial recombination easily move it into a series of plasmids that allow you to do many different molecular biology techniques (such as fusing it with different tags, putting it under a variety of promoters and into backbones with different selection cassettes). Although gateway cloning is a simple and efficient cloning method, gateway vectors and recombination enzymes can be quite expensive. In addition it is quite difficult to switch to another cloning method, such as restriction cloning, once your project has been completed via gateway cloning.
Toposiomerase based cloning, often called TOPO® cloning or TA cloning, is a method that relies on the hybridization of the complementary base pairs adenine (A) and thymine (T). TOPO® cloning utilizes the Taq polymerase which naturally leaves a single adenosine (A) overhang on the 3' end of PCR products. The complementary thymine (T) comes from a pre-cut, linear, cloning ready TOPO® vectors that has a DNA topoisomerase I fused to the 3’ end. The topoisomerase acts as a ligase that joins the A and T compatible ends together. TOPO® cloning thus does not need restriction enzymes or an exogenous ligase providing an incredibly quick and easy way to clone a fresh PCR product into a plasmid.
The major disadvantage of TOPO® cloning is that very few plasmid backbones are available TOPO® ready, and it is not feasible to create a TOPO® vector yourself. Additionally, the efficiency can vary depending on the polymerase used, and the single A overhangs degrade over time, further reducing ligation efficiency. TOPO® ready Gateway® Entry plasmids are also available, allowing for rapid cloning of PCR products into donor plasmids without the need for restriction enzyme cloning.
For more information and tips check out our Plasmid 101 blog post on TOPO cloning.
Gibson Assembly (Isothermal Assembly Reaction)
Isothermal cloning, more commonly known as Gibson assembly (protocol), takes advantage of the properties of 3 common molecular biology enzymes: 5' exonuclease, polymerase and ligase. 5' exonuclease digests the 5' end of dsDNA fragments to generate 3' single-stranded overhangs. DNA polymerases synthesize DNA molecules using the 4 nucleotides and lastly DNA ligase fuses DNA strands together.
In Gibson assembly DNA fragments with 20-40 base pair homology at their ends can be easily ligated together in one isothermal reaction. First the 5’ exonuclease chews back the 5’ ends of your DNA fragments generating long overhangs that anneal to each other due to their homology. DNA polymerase then closes the gap created by the 5’ exonuclease and finally ligase seals the nicks in the DNA to create one piece of double stranded DNA.
A major benefit of Gibson cloning is that it allows for the simple assembly of multiple fragments of DNA in the chosen orientation, and without the need for any unwanted sequence at the junctions (such as a restriction enzyme or Gateway recombination sites). Any double stranded DNA fragments can be used, so if properly designed, any insert fragment (PCR product or synthesized oligo) with appropriate overhangs can be efficiently ligated into any plasmid backbone independent of how it was originally designed to be cloned into. A drawback of this system is that Gibson assembly works best when combining DNA fragments over 200 base pairs. Anything shorter than 200 base pairs has the potential to be completely chewed back by the 5' exonuclease.
Type IIS Assembly (Golden Gate & MoClo)
Type IIS systems, such as Golden Gate and Modular Cloning (MoClo), take advantage of the unique properties of type IIS restriction endonucleases. These endonucleases cut dsDNA at a specified distance away from the recognition sequence. Cutting distal to their recognition site allows for the creation of custom overhangs, which is not possible with traditional restriction enzyme cloning. Scientists have utilized this approach to create compatible custom overhangs that can then be efficiently assembled together.
The advantage of type IIS systems are two-fold. First, the entire cloning step (digestion and ligation) can be carried out in one reaction with a single type IIS restriction enzyme, since the resulting overhangs will be distinct and preserve the directionality of the cloning reaction. Second, the restriction site is encoded on both the insert and plasmid in such a way that all recognition sequences are removed from the final product, with no undesired sequence ("scar") retained.
A disadvantage of type IIS assembly cloning systems is that like restriction enzyme sites, type IIS sites can be found throughout DNA sequences. Thus it is important to confirm that there are no additional sites present within the fragments you want to assemble before you get started. Check out Addgene's website for easy to use MoClo and Golden Gate cloning kits for your next cloning project.
Ligation Independent Cloning
Ligation Independent Cloning (LIC) relies on the 3'-5' exonuclease activity of T4 DNA polymerase. An exonuclease is an enzyme which removes nucleotides from the end of a DNA strand. In LIC, the T4 DNA polymerase’s exonuclease activity creates “chewed-back” overhangs of 10-12 base pairs on the 5' end of both the vector and insert. These overhangs can easily anneal creating a circular product with four nicks that are repaired by the bacteria after transformation. LIC does not require site-specific recombination or a ligation step, making it an easy, cheap and rapid cloning method.
So how does this work exactly? LIC depends on the addition of only one free dNTP to the reaction. In the presence of a single free dNTP, T4 polymerase will continue to function as an exonuclease until a base is exposed on the single strand overhang which is complementary to the free nucleotide. Given this opportunity, T4 will resume its polymerase activity, add back the free base, and become stuck at this point (with no other free bases to add). Complementary overhangs are built into the PCR primers for the insert, based on the destination vector sequence and choice of restriction site. Because of the relatively long stretches of base pairing in the annealed product, ligation is rendered unnecessary. The product may be transformed directly into E. coli, where the nicks will be repaired by the normal replication process. It is important to note that LIC has difficulty assembling DNA fragments with repetitive sequences and DNA that ends in sequences that form complex secondary structues. You can view an example of a LIC protocol on our website.
Yeast-mediated Cloning and Oligonucleotide Stitching
Yeast-mediated cloning is very similar in principle to Gibson cloning, but instead of an in vitro reaction with purified enzymes, it takes advantage of the powerful recombination abilities of yeast. Similar to Gibson, this method can efficiently fuse two (or more) fragments of dsDNA that have 30 or more bases of overlapping homology. One major advantage is that much larger final products can be generated (up to 100kb) compared to other cloning methods that utilize bacteria where it becomes progressively more difficult to clone plasmids larger than 10kb. Another advantage is the ability to perform oligonucleotide stitching, in which pieces of DNA that share no end homology can still be fused together in a seamless manner. To accomplish this, you just need to introduce into the yeast the two (or more) fragments of DNA that you would like fused along with custom ordered DNA oligos of 60-80 base pairs in length, with 30-40 base pairs of homology to the ends of the two fragments that you would like to fuse. A major disadvantage is that you need to be set up to grow, transform and purify DNA from yeast.