Uncategorized — 27 October 2015

The science community has long employed cloning techniques to help replicate and analyse specific genes and DNA sequences in focus. Over the past few decades molecular biologists have developed procedures to simplify and standardize such cloning processes to allow vast arrays of artificial DNA structures to be more easily assembled with prolific use in the biotechnology field.

Here we look at five different cloning methods that are used today. Keep in mind, there are variations to the protocols for each method and this article only describes the general notion for each method. At the end of this article, you can find the recommended protocols for each method.

Restriction Enzyme Ligation

Long considered the traditional cloning method, restriction ligation permits a DNA fragment of interest to be inserted into a vector to be amplified.

 The general protocol:

  1. Choose Restriction sites

    First one searches for the presence of unique restriction sites in a source DNA that can be targeted with restriction enzymes to isolate the fragment of interest. PCR may be chosen as the method of insert isolation if no appropriate restriction sites can be found.

  1. Restriction enzyme digestion

    The source DNA plasmid and vector plasmid can be digested with a single restriction enzyme for non-directional cloning, or with 2 different restriction enzymes for directional cloning. Using two different enzymes that create blunt ends will also create a non-directional cloning strategy, and for any non-directional strategies, additional screening may be required to determine the desired placement of the insert in the vector.

  1. End modification

    Just before the ligation step, in order to avoid the possibility of self-ligation, the vector is dephosphorylated; the 5’ phosphate group, which the ligase needs for phosphodiester bond formation, is removed. Depending on how the insert and vector are prepared, other ‘end’ treatment may be required i.e. blunting, A-tailing and phosphorylation.

  1. Ligation

    Next a Ligase is selected to covalently join the vector and insert. Finally the recombinant DNA is transformed into E.coli and cultured on agar plates with an antibiotic. The growth of colonies therefore demonstrates antibiotic resistance and the presence of the newly acquired fragment.

The design phase:

The design and planning phase is usually the first and most important step of any cloning experiment.

  • Once the sequence or gene of interest has been decided, an appropriate vector can be chosen for insertion; here’s a list of available vectors.
  • The restriction enzymes that are required to cut the vector are now considered.
  • At this point the insert can be designed to ensure its accurate and complete insertion. This will also guide any further end modifications that might be required.

A good software platform like Genome Compiler can help design an entire cloning experiment. For Restriction Ligation cloning, Genome Compiler allows you to simulate the insertion of a fragment with compatible restriction sites for ligation into a destination vector. Primers can also be designed to PCR the insert into the vector.   You can select destination vectors that are stored in the extensive online database or you can import new ones.

Software like Genome Compiler will save time by eliminating errors from your design; the auto generation of your cloned product allows you to verify your final product before ordering and can be created in a new separate file for you to continue working on. Once finished you can easily order your insert or primers directly through the site.

What are the advantages of using this method?

  • Many different plasmids can be used and directional cloning is easily achieved
  • Cheap and flexible: there are hundreds of available enzymes, each with a specific target sequence and predictable resulting end, which are also relatively cheap
  • The process can be very efficient with purification steps to help remove the presence of any non-recombinant DNA

What are the disadvantages of using this method?

  • Sequence limitations depending on location of restriction enzyme cutting site
  • Not all restriction enzymes work equally well in all commercially available buffers

Suggested Protocols for Restriction Ligation:

Protocol 1
Protocol 2
Protocol 3
Protocol 4

Gibson Assembly

The Gibson Assembly method has been around since 2009 and is the process whereby many DNA fragments are added to a construct all within a single test-tube reaction.

The general protocol:

Using a single tube, there are three different enzymatic activities that occur:

  1. An exonuclease cuts the DNA fragments of interest leaving single-stranded 3’ overhangs to facilitate the annealing of fragments that share complementarity at one end, also known as the overlap region.
  2. A polymerase fills the gaps with the new fragments.
  3. A DNA ligase seals any nicks in the newly assembled DNA.

The DNA in focus is incubated in the Gibson buffer containing the 3-enzyme mix for up to 1 hour at 50°C, depending on the number of fragments being assembled. The end result is a double-stranded fully sealed DNA molecule.

Gibson Assembly does not rely on the presence of restriction sites within a particular sequence to be synthesized or cloned. Therefore, the user has complete control over what is assembled. Furthermore, the inclusion of unwanted additional sequence, which is often used to facilitate the cloning of multiple DNA sequences, can be avoided. Lastly, a greater number of DNA fragments can be joined in a single reaction with greater efficiency than conventional methods.

Multiple overlapping DNA fragments that are hundreds of kilobases long have been constructed in this single isothermal reaction, which are used extensively by biologists.

The design phase:

When designing a plasmid using Gibson Assembly, one must first consider the primers required. Primers must have a 5’ end that is identical to an adjacent DNA segment and a 3’ end that anneals to the target sequence. The DNA segments are then generated using PCR. The lengths of the overlapping regions required depend on how many inserts are being added. The PCR product is purified on a gel, ensuring correct size and quantity. The segments are then ready for the Gibson Assembly reaction.

Again the software program Genome Compiler is able to simulate the automatic assembly of multiple fragments into a destination vector by guiding you through the design process:

  • First, for backbone design you are directed to choose how to linearize it, either by restriction enzyme or by primer design, with options to optimise it with Primer3.
  • Single or multiple inserts can then easily be selected from their extensive database or uploaded from your own projects, and you can choose to either excise the fragments by PCR or to synthesise them externally with or without the overlapping regions incorporated.
  • The final cloned plasmid is shown with the fragments inserted and the auto-generated oligo’s designed to construct it.
  • At any point in time the process can be checked and re-run, with changed parameters, making this cloning process less error prone and more cost effective to perform.
  • Again, the final plasmid can be created as a separated file to continue working on.

What are the advantages of using this method?

  • Flexible sequence design: multiple parts assembled in a defined order at the same time in parallel
  • Parts used within assembly do not have to be free of specific sequences i.e. restriction sites
  • Fully assembled in just under 2 hours
  • DNA clones produced without any scarring
  • No PCR clean-up step required
  • Higher transformation rate for longer fragments
  • DNA is ready to be used immediately for transformation, as a template for PCR or RCA
  • Easily adapted for multiple DNA manipulations (including site-directed mutagenesis, insertions and deletions)
  • Ability to ‘stitch’ together several oligonucleotides to introduce promoters, terminators, and other short sequences into the assembly

What are the disadvantages of using this method?

  • Can require big overlaps in primers, which can come at a high cost
  • Sharp decrease in success rate when assembling more than 5 fragments at a time
  • Hairpins in sequence region can significantly reduce the efficiency of two homologous ends annealing

Suggested Protocols for Gibson Assembly:

 

Protocol 1
Protocol 2 
Protocol 3

Golden Gate Assembly

Golden Gate Assembly uses Type IIS restriction enzymes, which cut DNA outside of the recognition sites for both the DNA inserts and the cloning vectors. This ensures no scarring of the DNA sequences because the overhang sequence is not dictated by the restriction enzyme. The specific sequences of the overhangs also allow multiple fragments to be assembled at the same time and also in an ordered manner. And because the restriction site is completely removed from the ligated DNA, digestion and ligation can happen in one reaction, at the same time.

The design phase:

The DNA sequence to be cloned is produced by PCR with two Type IIS recognition sites; one with an overhang at its 5’ end, and another at the 3’ end. The recognition sites are separated by at least one base pair from the sequence overhang and if they are not palindromic they will act directionally.

The vector and the DNA fragment are mixed at the same time with the Type IIS restriction enzyme and ligase to ensure the cut linearized destination vector will irreversibly ligate with the cut PCR product. This occurs because the ligation product no longer contains any recognition sequences, thus all reactions will tend towards the desired assembly product.

Standard parts like promoters, translated regions and coding sequences have been described by various groups with a specific set of overhangs. This information has been shared generously amongst the science community to allow sequences to be freely exchanged to be used in cloning.

What are the advantages of using this method?

  • Considered a ‘one pot wonder’ with 1 restriction enzyme, 1 ligase
  • Many parts can be assembled at the one time
  • Scarless assembly
  • Very efficient process: a repeat cycle ensures no plasmids are missed
  • Re-ligation is prevented: cleaving outside of restriction enzymes sites removes them from the product
  • Particularly good for constructing combinatorial libraries where every fragment is flanked by the same two overhang sequences

What are the disadvantages of using this method?

  • Less sequence-independence than other cloning methods: restriction sites cannot be in internal locations, can be difficult to find compatible overhangs.
  • Designing with the right overhang sequences can be tedious, laborious and error-prone.

Suggested Protocols for Golden Gate assembly:

Protocol 1
Protocol 2
Protocol 3

Gateway Cloning

The Gateway cloning method was commercially established in the late 1990’s with the primary benefit that one single recombination reaction moves a piece of DNA from one plasmid into another. This simplifies the process and reduces the time compared to restriction enzyme based cloning. The proprietary set of recombination sequences and enzymes used are named Gateway Entry clones. The research community has access to the archives of all the Gateway Entry clones, which contain many of the established reading frames from human and rodent, as well as chemically synthesized clones all available for gene function analysis.

The general protocol:

The fragment of DNA being prepared for cloning must already be surrounded by specific recombination sites (also known as Gateway recombination sites; ATT sequences). Therefore the DNA fragment must first be cloned into a ‘donor plasmid’, the resulting product is called an Entry clone), a process that is still most often achieved by restriction enzyme cloning.

Once the 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 (or acquire one that already has your gene in it) and then using bacterial recombination easily transfer it into a series of plasmids.

What are the advantages of using this method?

  • Accessible libraries: quick transfer of genes into plasmids for analysis of function
  • Useful for transfer of thousands of DNA fragments into one type of plasmid or for one DNA fragment into many different types of plasmids
  • Very fast: can be done in 1.5 hours
  • Very accurate (greater than 90%)
  • Totally universal for all types of DNA fragments to be cloned i.e. PCR fragments, complimentary DNA, genomic DNA

What are the disadvantages of using this method?

  • Initial setup is timely
  • Specific plasmids need to be used
  • Clonase enzymes are expensive

 Suggested Protocols for Gateway Cloning:

Protocol 1
Protocol 2
Protocol 3

TOPO (TA) Cloning

One of the basic theories of cloning is to minimize the number of steps in the recombination process in order to reduce errors throughout the process i.e. restriction enzymes used for cutting followed by ligases used for sticking. TOPO cloning uses a single enzyme, Topoisomerase (TI) to both unwind and ligate DNA in the hope of achieving this goal of fewer errors. TI is used in the natural process of replication; when DNA is opening/unwinding it creates pressure further upstream, so to relieve this stress and prevent breakage, TI binds to DNA, cleaves and unwinds it, then re-joins the nick just created.

The general protocol:

DNA inserts must be prepared by PCR using taq polymerase, which leaves a single adenosine (A) overhang on the 3’ end of the products. The TOPO vectors that are available are already cut and have a 3’ end fused to TI. When the insert and the vector are combined there is effective hybridization between the 3’ overhang of the PCR product and the 5’ T overhang of the TOPO backbone. This reaction is completed after 5 minutes at room temperature without any restriction enzyme or ligase. The TI is cleaved out of the scene leaving a sealed DNA fragment of interest inserted into the vector.

What are the advantages of using this method?

  • Restriction sites do not need to be designed into primer: no restriction enzymes or ligases required
  • Fast: whereas restriction enzyme ligation is anywhere from 4 hours to overnight, this process takes a maximum of 1 hour
  • Efficiency rate of 95%
  • Less errors: one enzyme modifying the whole system
  • Accelerate high-throughput cloning

What are the disadvantages of using this method?

  • Preparation can be time consuming: initial preparation of DNA inserts with PCR requires specific primers
  • Careful consideration of kit selection
  • Very few plasmid backbones are available TOPO ready, and it is not feasible to create a TOPO vector yourself
  • The efficiency can vary depending on the polymerase used, and the single A overhangs degrade over time, further reducing ligation efficiency

 Suggested Protocols for TOPO Cloning:

Protocol 1
Protocol 2
Protocol 3

What’s next?

Here we have looked at 5 different cloning methods but many others exist with their own features and functions that have been carefully designed to benefit the research community. The cloning process continues to make it possible to study the structure and functioning of genes and other important DNA sequences from even the most complex genomes.

The application of the principles of cloning and modern engineering in the field of synthetic biology has allowed the standardization of DNA parts and their assembly process, advancing the research of microbiologists in leaps and bounds. And further simplifying the process are software programs like Genome Compiler that are helping biologists imagine and create DNA constructs by effortlessly simulating some of the cloning methods described in this article.

Together, this scope and breadth for cloning methods along with the newest software and DNA synthesis technology is allowing researchers to design and synthesize DNA in cheaper and more sustainable ways, allowing for even more in-depth studies of genes.

 

References

Restriction Ligation https://www.neb.com/applications/cloning-and-synthetic-biology/traditional-cloning
Gibson Assembly https://www.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/gibson-assembly
https://www.neb.com/products/e2611-gibson-assembly-master-mix
https://www.addgene.org/plasmid-protocols/gibson-assembly/
https://www.neb.com/faqs/1/01/01/what-are-the-advantages-of-this-method-compared-to-traditional-cloning-methods
Golden Gate Assembly https://www.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/golden-gate-assembly
https://www.addgene.org/plasmid-reference/cloning-choice/
Gateway Cloning https://www.addgene.org/multisite-gateway-kit/
http://www.thermofisher.com/il/en/home/life-science/cloning/gateway-cloning.html
http://www.snapgene.com/resources/gateway_cloning/
https://www.embl.de/pepcore/pepcore_services/cloning/cloning_methods/recombination/gateway/
https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/Cloning/pdfs/CO36609%20TOPO%20Cloning%20Brochure.pdf
Topo Cloning http://www.thermofisher.com/il/en/home/life-science/cloning/topo.html

 

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Dr. Lara Winter

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