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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.

There are many methods that allow you to move around pieces of DNA. Oftentimes several approaches will work for any specific cloning project; however, for any given project there is usually 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.

This guide highlights several of the most popular cloning methods for the creation of recombinant DNA.

Restriction Enzyme Cloning

Restriction enzyme (endonuclease) molecular cloning is the "classic" cloning method, and 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 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, which range from four to 13 base pairs, and produce predictable cleaved 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, usually within the multiple cloning site (MCS). 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 MCS. 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. This 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.

Read more in our Restriction Cloning blog post.

The top half of the figure features a donor plasmid containing an insert and an antibiotic resistance gene. Two primers flank the insert, one containing an EcoRI site and one containing a NotI site. Plasmid is PCR amplified to incorporate these sites and digested with the restriction enzymes to leave overhangs (sticky ends). The bottom part of the figure shows the plasmid backbone (recipient) with a promoter, selection marker, and antibiotic resistance gene, along with a multiple cloning site with XhoI, EcoRI, HindIII, and NotI sites. Backbone is digested with EcoRI and NotI to create overhangs. The digested backbone is ligated with the digested insert from above, to create a new plasmid containing the insert.
Figure 1: Restriction enzyme cloning of your gene of interest (insert) into a plasmid backbone. Created with BioRender.com.

Gateway Recombination Cloning

Gateway cloning is a recombination-based cloning method. During Gateway, 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 — similar to 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 the 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 you have made an entry vector containing your DNA of interest (or obtained one of the thousands of entry vectors deposited with Addgene), it can be rapidly shuttled into any compatible Gateway destination vector, which contains attR sites, via LR clonase enzymes. Thus, you can clone your gene of interest one time into a donor plasmid or acquire one that already has your gene in it. Then you can use bacterial recombination to easily move it into any destination plasmid that fits your experimental goal. 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.

Addgene's collection contains thousands of Gateway donor, Gateway entry, and Gateway destination vectors with different promoters, tags, selection markers, and fluorescent proteins to fit a variety of experimental conditions. Read more in our Gateway Cloning blog post.

The top of the figure shows the BP reaction. It starts with a gene flanked by attB1 and attB2 sites plus a donor vector containing the ccdB gene flanked by attP1 and attP2 sites. Using a BP clonase, the gene and donor vector recombine, making a byproduct (ccdB gene flanked by attR1 and attR2 sites) and the entry clone (a plasmid with the gene flanked by attL1 and attL2 sites). The bottom of the figure shows the LP reaction. It starts with the entry clone from above, plus a destination vector containing the ccdB gene flanked by attR1 and attR2 sites. Using an LR clonase, the entry clone and destination vector recombine, making a toxic byproduct (a plasmid with the ccdB gene flanked by attP1 and attP2 sites) and the expression clone (a plasmid with the gene flanked by attB1 and attB2 sites).
Figure 2: Summary of Gateway cloning. Created with BioRender.com.

TOPO 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 A overhang on the 3' end of PCR products. The complementary T comes from a pre-cut, linear, cloning-ready TOPO vector that has a DNA topoisomerase I covalently bound to the phosphate group on the free 3' T. 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.

Read more in our TOPO Cloning blog post.

The gene insert is treated with Taq polymerase and dATP, producing ‘A’ overhangs. A TOPO-ready backbone vector contains TOPO recognition sites (CCCTT). The TOPO enzyme is linked to the 3' phosphate on the 'T' overhang of the linearized vector. TOPO acts as a ligase to ligate the gene insert with its overhangs, and the TOPO enzyme is released.
Figure 3: Summary of TOPO cloning. TOPO = topoisomerase I. Created with BioRender.com.

Gibson Assembly (Isothermal Assembly Reaction)

Isothermal cloning, more commonly known as Gibson assembly, takes advantage of the properties of three common molecular biology enzymes: 5' exonuclease, polymerase, and ligase. 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. Second, DNA polymerase then closes the gap created by the 5’ exonuclease. Finally, ligase seals the nicks in the DNA to create one piece of dsDNA.

A major benefit of Gibson cloning is that it allows for the assembly of multiple fragments of DNA in the chosen orientation at a time, and without the need for any unwanted sequence at the junctions (such as a restriction enzyme or Gateway recombination sites). Any dsDNA 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. 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 degraded by the 5' exonuclease.

Read more in our Gibson Assembly blog post.

Insert 1, insert 2, and a linearized vector have paired homology ends: insert 1, homology A and B; insert 2, homology B and C; linearized vector, homology A and C. These components are mixed with a 5 prime exonuclease, a polymerase, and a ligase. The homology ends match up, giving a final, circularized vector containing inserts 1 and 2.
Figure 4: Summary of Gibson assembly. Created with BioRender.com.

Golden Gate and MoClo

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 advantages 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 endogenous 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.

Read more in our Golden Gate Cloning blog post.

Three plasmids are shown with different inserts. On each 3 prime and 5 prime end are unique homologous sequences along with type IIS restriction enzyme recognition sites. From left to right, homology sequences for the inserts are: insert 1, homology A and B; insert 2, homology B and C; insert 3, homology C and D. A destination vector is shown with a promoter, followed by homology A, two type IIS recognition sites, and homology D. The destination vector is combined with the three insert plasmids, plus a type IIS enzyme and a ligase. This creates a final vector, containing (from 3 prime to 5 prime) a promoter, homology A, insert 1, homology B, insert 2, homology C, insert 3, and homology D.
Figure 5: Summary of Golden Gate cloning. Created with BioRender.com.

Ligation Independent Cloning

Ligation Independent Cloning (LIC) relies on the 3' to 5' exonuclease activity of T4 DNA polymerase. 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 bacteria repair after transformation. LIC does not require site-specific recombination or a ligation step, making it an easy, cheap, and rapid cloning method.

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 that is complementary to the free nucleotide. 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 normal replication process will repair the nicks. 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 structures.

Read more in our SLIC blog post.

On the left, a gene insert is amplified using PCR to add homology ends. A T4 polymerase and dCTP nucleotide are added to chew back the homology ends, leaving overhangs on the 5 prime ends. On the right is a vector backbone containing a sequence homologous to the gene insert homology ends. The sequence contains a restriction enzyme recognition site. The backbone is linearized using a restriction enzyme. A T4 polymerase and dGTP nucleotide are added to check back the exposed ends of the backbone, leaving overhangs on the 5 prime ends. The linearized backbone and gene insert are annealed to create a final circularized plasmid.
Figure 6: Summary of LIC. Created with BioRender.com.

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. By simply transforming into yeast two (or more) fragments of dsDNA that have 30 or more bases of homologous ends, the endogenous DNA repair pathways will produce the fused product. One major advantage is that much larger final products can be generated (up to 100 kb) compared to other cloning methods that utilize bacteria, where it becomes progressively more difficult to clone plasmids larger than 10 kb.

Another advantage is the ability to perform oligonucleotide stitching, in which pieces of DNA that share no homology ends can still be fused in a seamless manner. To accomplish this, you can transform into yeast the fragments of DNA to be fused along with custom synthesized DNA oligos that span each junction. These oligos should be 60–80 bp long, with 30–40 bp of homology to each of the larger fragments. The fragments should have complementary ends with 30–40 base pairs of homology. While this approach doesn't work in other cell types, it can be a major time- and cost-saver for labs working with yeast.

The top of the figure depicts yeast-mediated plasmid cloning. Two inserts and a linearized vector with overlapping homology sequences are introduced into a yeast cell. The inserts and vector are introduced into yeast, where recombination occurs to create a complete circularized plasmid. The bottom of the figure shows yeast-mediated oligonucleotide stitching. Three inserts are shown with two small oligonucleotides, one with homology to insert 1 and 2, the other with homology to insert 2 and 3. The inserts and oligonucleotides are introduced into yeast, where recombination occurs to create a complete linear section of DNA.
Figure 7: Summary of yeast-mediated plasmid cloning and oligonucleotide stitching. Created with BioRender.com.

Additional Resources

Addgene Webpages

Addgene Protocols

Addgene Blogs

Content last reviewed: 22 October 2025