Gene cloning is one of the most important steps in recombinant DNA Technology. Now-a- days researchers are using different cloning techniques depending on purpose, time, cost, ease of use and availability of resources. A few of the techniques are briefly explained below.
1. Using restriction enzymes:
This is the traditional cloning technique where a gene of interest is inserted in to a vector by a cut and paste method. Restriction digestion of both gene of interest and vector was performed by using restriction enzymes that cut the gene at a specific sequence. Several enzymes like EcoR I, BamH I, Nco I, Nde I, Xho I etc.,are used. Digestion with some of these enzymes result in a sticky end and some in blunt ends. After the digested fragments are cleaned up, the digested gene and vector are ligated to form a recombinant plasmid using DNA ligase enzyme. There are so many standardized protocols for this technique, but in some cases optimization of buffer is required and time of restriction digestion also plays a major role. When using two restriction enzymes (double digestion) buffer used should be compatible for both the enzymes. If there is no compatible buffer, gene and vector should be first digested with one enzyme followed by the other.
2. TA cloning or TA/TOPO cloning:
TA cloning is an easy, rapid cloning technique that consumes less time compared to traditional cloning method. Here a single enzyme is used for both digestion and ligation called Topoisomerase I that digest DNA at specific site 5’-(C/T)CCTT-3’ and ligates at 3’ phosphate group of thymidine base. This can be performed within 5 to 10 minutes.
Polymerase enzyme used is also very important in this technique, like Taq DNA polymerase. Taq enzyme add poly A tail to the PCR product which is complementary to the thymidine residues and thus can easily be ligated.
3. GATEWAY CLONING
Gateway cloning is a molecular cloning technique developed in the 1990s and gained wide importance in life science research. In this method, specific primers are used to insert specific gene sequences on both sides of the desired gene by PCR. Gateway cloning is a two step process, where two enzyme mixes, BP clonase and LR clonase, are used. The gene of interest flanked with specific sites attB is first cloned into an entry vector with specific attP sites flanking on both sides by using BP Clonase resulting in attL sites. Later this entry clone is used to transfer the gene into expression vector flanked by attR site using LR Clonase. This technique can be used to clone genes into multiple vectors ranging from bacterial or insect or mammalian expression systems. It can also be used to clone multiple genes into a specific vector like CMV promoter.
4. 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 was amplified on both sides of the gene complementary to that of linearised vector. Then with the help of infusion enzyme the gene can easily be cloned into the vector. In this procedure any vector of any size can be used to clone the gene of interest.
5. LIGATION INDEPENDENT CLONING
Ligation independent cloning is 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. This polymerase has 3’ to 5’ exonuclease activity resulting in overhangs that are complementary to both gene and vector. Later dCTP was added to get back its polymerase activity. The resulting clone will have nicks which are later repaired by E.coli cells.
6. 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 - In eukaryotes translation occurs only from 5’end hence only one translation end. But this IRES elements have the capability to start translation irrespective of 5’end. So, if this IRES elements are incorporated between two genes both can easily be expressed. But this method has few disadvantages like their large size (500bp), often the second gene express less compared to the first
- 2A peptides - Small peptides of 20 aa length are used by researchers to overcome some of the problems faced by using IRES. 2A peptide sequences usually start with GSG residues and end with PGP, where the cleavage of the second protein occurs between G and P and the second one will start with proline residue. This technique is successfully used to clone more than two genes in a single multi-cistronic
7. GIBSON ASSEMBLY
Gibson assembly is a cloning technique where multiple DNA fragments can be joined in a single isothermal reaction (constant temperature of 50 ℃). In this method, three enzymes are used. An exonuclease, that cleaves the 5’ end of DNA fragments allowing them to anneal with the other DNA fragment. A polymerase to fill the gaps after the two genes anneal. Finally, a Ligase to join the two fragments removing the nicks. In a single tube up to 5 DNA fragments can be assembled using this technique. But to join large number of fragments (up to 15 fragments), the reaction can be performed in two tubes. In the first step exonuclease and annealing steps are done and in the next step polymerase and ligase enzymes will complete the assembly.
8. GOLDEN GATE CLONING
Golden gate cloning is a seamless cloning method in which multiple DNA fragments are joined together without any nicks. Two enzymes are used in this cloning method, Type II restriction enzymes and DNA ligase enzyme. Type II restriction enzymes like Bsa I, BsmB I, Bbs I. These enzymes will cut the DNA fragment outside the recognition sequence resulting in non palindromic over hangs. The reaction is carried out at 37 ℃ and 16 ℃ for restriction digestion and ligation respectively. This is an irreversible method of gene assembly, once the gene is inserted in the vector it can never be cut with the same restriction enzyme. This method is carried out in two steps, first single gene construct is made with a promoter, ORF and terminator. In the second step, several single gene constructs are joined to get multigene constructs. For the second step, modular cloning system (MoClo) and Golden braid 2.0 can be used.
References:
- Thieme F, Engler C, Kandzia R, Marillonnet S. Quick and Clean Cloning: A Ligation- Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes. Agarwal S, ed. PLoS ONE. 2011;6(6):e20556. doi:10.1371/ journal.pone.0020556.
