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Redbook Choosing a Molecular Cloning Technique


Restriction Enzyme | Gateway | TOPO | Gibson | Type IIS | Ligation Independent Cloning | Oligo Stitching

Summary

When it comes to moving pieces of DNA around, many methods have been used over the years. 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. The following guide compares several of the most popular cloning methods to help you make the decision of which is best for your specific cloning project.


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. In a restriction digest , enzymes are utilized to cut double stranded DNA 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 . Restriction enzyme cloning benefits from the hundreds of available enzymes, each with a specific target sequence and predictable resulting end, which are also relatively cheap. 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 ( protocol here ).


Gateway® Recombination Cloning

Gateway® cloning is a recombination based cloning method. The primary benefit of Gateway is that the reaction that moves a piece of DNA from one plasmid into another is done via a single recombination reaction, drastically simplifying the process and reducing the time compared to restriction enzyme based cloning. To utilize this approach, the fragment of DNA that you would like to clone must already be surrounded by specific recombination sites (in this regard, not so dissimilar from restriction enzyme cloning). This requires that you first clone your DNA fragment into a Donor plasmid (creating an Entry clone ), a process that is still most often achieved by restriction enzyme cloning. Once your DNA fragment has been cloned into an Donor plasmid, it can then be rapidly shuttled into any compatible Gateway® Destination vector. Thus, you can clone your gene of interest one time by restriction enzyme cloning 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). Click here to view the Santalucia lab Multisite Gateway C3 cloning kit.

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TOPO® Cloning

TOPO® cloning , often called TA cloning, is a method that takes advantage of the fact that taq polymerase leaves a single adenosine (A) overhang on the 3' end of PCR products. TOPO® vectors are sold cloning ready, which means that they are already cut and their 3' end is fused to DNA topoisomerase I . This results in efficient hybridization between the 3' A overhang of the PCR product and the 5' T overhang of the TOPO® backbone, providing an incredibly quick and easy way to clone a fresh PCR product into a plasmid. The major disadvantage 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. Often TOPO® cloning is used as an initial step in the cloning of PCR products, allowing you to sequence them to make sure that they are mutation free before subcloning into another vector using restriction enzyme digestion (which is why many TOPO® plasmids add commonly used restriction enzyme sites on either side of the insert). 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.

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Isothermal Assembly Reaction (Gibson Assembly)

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 double stranded DNA fragments to generate 3' single-stranded overhangs. If this is done in a reaction mixture that included two (or more) DNA fragments that have 20-40bp of homology at their ends, the resulting complementary "sticky ends" (much like with restriction enzymes, but with greater length of complementarity) will find each other and anneal. At this point the polymerase fills in any remaining regions of single-stranded DNA and then the ligase fuses the nicks, resulting in a single DNA fragment. A major benefit of this method 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 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.

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Type IIS Assembly (Golden Gate/MoClo)

Type IIS systems, such as Golden Gate , Green Gate , and MoClo , take advantage of the unique properties of type IIS restriction endonucleases, which cut dsDNA at a specified distance from the recognition sequence. This feature allows for the creation of custom overhangs, which is not possible with traditional restriction enzyme cloning. The advantages of this system are two-fold. First, the entire cloning step (digest and ligation) can be carried out in one-pot with a single 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.

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Ligation Independent Cloning

Ligation Independent Cloning employs the 3'-5' exonuclease activity of T4 DNA polymerase in order to create 5' overhangs on both the vector and insert. 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. View an example LIC protocol here .

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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 60-80bp in length, with 30-40bp 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.

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