Biotechnology Cloning

Methods for Cloning

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Molecular cloning method

It is the collection of experimental procedures required to isolate and expand a specific fragment of DNA into a host organism in order to create a large number of identical copies. On top of allowing the study of a single DNA sequence of interest, molecular cloning is a powerful technique that permits the generation of complex combinations of DNA fragments for the most disparate downstream applications. 

Restriction Enzyme Ligation

Restriction ligation cloning allows the insertion of a DNA fragment of interest into a vector through a “cut and paste” procedure. This inexpensive, flexible method can be broken down into a basic, two-step process.

It is important to note that restriction ligation has limitations, particularly when choosing restriction enzyme cutting site(s). Additionally, it is essential to choose buffers wisely, as not all restriction enzymes work equally well in all buffers. 

Gateway Cloning

This cloning method was commercially established in the late 1990’s and has the primary advantage 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 ligation cloning. To perform Gateway cloning, we must first prepare our DNA fragment of interest by surrounding it with specific recombination sites (also known as Gateway recombination sites; ATT sequences).  Therefore, we must first clone our DNA fragment into a “donor plasmid”.  This can be done through traditional cloning methods or by using TOPO cloning or the Gateway BP Clonase reaction.  The resulting plasmid is called an Entry Clone.

INFUSION CLONING

Infusion cloning is a simple one step cloning technique where the gene of interest is annealed into a vector based on complementary flanking sequences. In this method, the gene of interest was amplified by PCR and gene sequences of 15 nt length were amplified on both sides of the gene complementary to that of linearised vectors.

LIGATION INDEPENDENT CLONING

Ligation independent cloning is a cost effective simple cloning technique. The gene was amplified with specific gene sequences of 12 nt length complementary to modified LIC vectors. These vectors have to be linearized by PCR or by restriction digestion. Both enzyme and vector were then incubated with T4 DNA polymerase. 

Gibson Assembly

The Gibson Assembly method is the process where many DNA fragments are added to a construct all within a single test-tube reaction, producing clones without any scarring. You can assemble multiple parts at the same time to have flexible sequence design, and the ability to introduce promoters, terminators, and other short sequences into the assembly. Assembly is completed in under 2 hours.

Golden Gate Assembly

Golden Gate Assembly uses two Type IIS Restriction Enzymes, which cut DNA outside of the actual recognition site for the enzyme. The recognition sites are separated by at least one base pair from the sequence overhang, ensuring no scarring of the DNA sequence because the overhang sequence is not dictated by the restriction enzyme. If the recognition sequence is not palindromic, we can assemble multiple fragments at the same time and in an ordered manner. 

TOPO (TA) Cloning

TOPO cloning uses a single enzyme, Topoisomerase I (TI) to both unwind and ligate DNA. TI is used in the natural process of replication; the opening/unwinding of DNA 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.

BI- or MULTI-CISTRONIC CLONING

Cloning two or more genes can be possible by using this technique. Here the genes to be expressed are separated by a specific sequence. Currently two strategies are used for expressing two or more genes.

  • Internal ribosome entry site (IRES) elements 
  • 2A peptides 

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