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Cloning Standards

DOULIX allows you to order either ready-to-use construct or multiple fragments to assemble your plasmid by yourself. If you choose to build it yourself, DOULIX offers you several different cloning standards. When you choose a cloning method, DOULIX will adjust accordingly and deliver you the perfect fragments to run your cloning protocol. 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.

When it comes to moving pieces of DNA around, many methods have been developed over the years; Each one with its cons and pros. The following table compares several of the most popular cloning methods according to useful parameters:

  • Multiple fragments assembling
  • One-pot Synthesis
  • Cost per reaction
  • Excise and Replace
  • Fragment size
  • Scar-less
  • Accuracy
Restriction Enzyme Cloning Golden Gate DNAMate Gibson Assembly Ligation Independent Cloning Gateway Recombination Cloning
Multi-Assembly * * * * * * * * * * * * * * * * * * * *
One-pot * * * * * * * * * * * * * * * * * * *
Cost * * * * * * * * * * * * * *
Excise/Replace * * * * * * * * * * * * * * * * *
Fragment Size * * * * * * * * * * * * * * * * * * * * * * * *
Scarless * * * * * * * * * * * * * * * * * * * * *
Accuracy >80% >80% >80% >80% - >80%
Available on Doulix coming soon yes yes yes yes yes

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 enzyme are used to cut double stranded DNA into fragments containing precise 5' or 3' single-strand overhangs (sticky ends). Two pieces of DNA that have complementary overhangs 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. This allows synthetic biologists to design constructs with multiple unique restriction sites that can be used to replace any piece of DNA region between any two restriction sites or easily move a piece of DNA into any other plasmid harboring the same two restriction sites. The downside of restriction enzyme cloning is that you can hardly assemble more than 3 fragments simultaneously, which requires multiple cloning steps to built-up an entire plasmid.

Golden Gate or Type IIS Assembly or MoClo Assembly

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. The downside of Golden Gate Assembly is that it does not allow to easily excise and replace DNA pieces once the full-length construct is assembled making post-synthesis manipulation, such as combinatorial library construction and fragment replacement, cumbersome.

goldengate

DNAmate or DNA Modular Assembling Technology

The DNAmate is a Type IIS assembling systems, as Golden Gate or MoClo, that allows the single-pot directional assembly of up to 8 DNA fragments. Each fragment has a specific prefix and suffix that harbor a type IIS recognition sequence and a unique restriction site. This design allows to combine the best of both worlds since it offers the simultaneous cloning of multiple fragments of the Golden Gate and the possibility to manipulate at will the final construct using the unique restriction sites. The downside of DNAmate is that it leaves short undesired sequences (“scars”) between two adjacent fragments. This limitation is solved by carefully designing the scars so to not interfere with construct functions.

dnamate

Gibson Assembly or Isothermal Assembly Reaction

Isothermal cloning, more commonly known as Gibson assembly, efficiently joins multiple overlapping DNA fragments in a single-tube. It takes advantage of the interplay of 3 enzymes:

  • The 5’-exonuclease creates single-stranded 3´ overhangs into DNA fragments that facilitate the annealing of fragments that share complementarity at one end (overlap region).
  • The polymerase fills in gaps within each overlap region.
  • The DNA ligase seals nicks in the assembled DNA.

The final is a double-stranded fully sealed DNA molecule that can serve as template for PCR, RCA or a variety of other molecular biology applications, including direct transformation. The downside of Gibson Assembly is that it does not allow to easily excise and replace DNA pieces once the full-length construct is assembled making post-synthesis manipulation, such as combinatorial library construction and fragment replacement, cumbersome.

gibson

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. The downside of restriction enzyme cloning is that you can hardly assemble more than 3 fragments simultaneously, which requires multiple cloning steps to built-up an entire plasmid. In addition, ligation independent cloning does not allow to easily excise and replace DNA pieces once the full-length construct is assembled making post-synthesis manipulation, such as combinatorial library construction and fragment replacement, cumbersome.

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 by restriction enzyme cloning. Once your DNA fragment has been cloned into a 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 and then easily move it into a series of plasmids using bacterial recombination. The downside of Gateway® Recombination Cloning is that you can hardly assemble more than 3 fragments simultaneously and your gene of interest is limited to Gateway compatible plasmids significantly reducing your downstream application and lab-to-lab sharing. In addition, the Gateway® Recombination Cloning does not allow to easily excise and replace DNA pieces once the full-length construct is assembled making combinatorial library construction and fragment replacement cumbersome.

gateway

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.