Molecular cloning is a basic technique in molecular biology and biotechnology laboratories. It is a useful tool to study a gene, modify the gene, reintroduce the modified gene into the natural host or another host, or to produce protein. You can perform molecular cloning by incorporating a piece of DNA into a plasmid (a recombinant DNA) to make more identical recombinant DNA in a living host. Things to consider before you clone are the availability of your cloning materials, the goal of your research (such as to sequence a DNA fragment or to produce a protein), and the difficulty level to perform your protocol.
MATERIALS FOR CLONING
To begin with, you have to prepare some important materials for cloning:
The source for a DNA fragment or “DNA insert” can be genomic DNA, complementary DNA, plasmid DNA, PCR product, or synthetic DNA. The DNA insert must contain particular sequences at the end of the fragments compatible with the prepared vector. You can add these particular sequences onto your DNA insert by PCR.
A vector is a DNA molecule which can carry a DNA insert to generate a recombinant DNA and replicate in a particular host.
Some examples of vectors are:
- Cosmid: a large DNA vector containing λ phage DNA sequence. It can carry large DNA fragment up to 45 kilobases into the host.
- Artificial Chromosomes: a large DNA vector which can perform the functions of a chromosome.
- Plasmid: a small extrachromosomal circular DNA which can replicate in a cell, commonly used in cloning.
Elements in a plasmid vector:
- Origin of Replication (ORI): a required sequence or element on the plasmid for its replication inside the host.
- Selectable Marker: a required element for cloning to select a host, which carries the DNA construct. The only host cell growing in the growth medium containing a particular selective agent has the DNA construct with a selectable marker inside the cell. For an example, selectable markers in the DNA construct often contain antibiotic resistance genes. A transformed host has the antibiotic resistance gene on the DNA construct, therefore it can grow in the medium with that particular antibiotic. On the other hand, the host cell without the DNA construct can’t survive in the selective medium.
- Multiple Cloning Site (MCS): an element on the plasmid fragment which contains restriction enzyme sites to allow DNA insertion. Compatible restriction enzymes cut on the MCS of plasmid and a DNA insert during preparation step of cloning.
- Promoter Region: a region which drives the protein expression of the cloned DNA.
- Protein Tag: a particular sequence which produces a protein with specific function, and it is usually attached to the recombinant protein. An example of a protein tag is luciferase or GFP, to monitor or quantify the protein.
- Poly-adenylation signal: an element containing poly-A which is important to produce a protein.
After a DNA fragment is incorporated into the plasmid vector, the next cloning step is to perform a transformation step. In this transformation step, the recombinant DNA is introduced into the competent cell by chemical reaction or electroporation. Competent cells are cells which are temporarily permeable to extracellular DNA.The host organisms which are commonly used in the laboratories are Escherichia coli and Saccharomyces cerevisiae.
Selective medium is a growth medium containing a selective agent to grow the transformed host. When you choose antibiotic selection for cloning, your growth medium must contain antibiotics. The most common antibiotics used for selection are Ampicillin, Kanamycin, and Chloramphenicol.
There are several different approaches to clone and you will need to find the right approach for your research. Below are some examples of popular cloning methods to generate a recombinant DNA construct:
Restriction Enzyme Based Cloning
Restriction enzymes are enzymes which cut DNA near at a specific short nucleotide sequence called a restriction site. The restriction enzyme based cloning method depends on the activity of restriction enzymes to ‘cut’ both a vector and a DNA insert and the method also depends on a DNA ligase to ‘paste’ the DNA fragment into the vector. This method is useful when have one DNA insert to incorporate into the plasmid.
By using PCR, you can add restriction enzyme sites on your DNA insert to accommodate this method. Your DNA insert must not contain an internal restriction site similar to the restriction site on your plasmid. Your restriction enzyme can cut your DNA insert at this internal restriction site and produce unwanted smaller pieces of DNA fragments.
You can choose to use one restriction enzyme or two enzymes to cut your DNA fragment and vector. When using two enzymes, both enzymes must be compatible or work well in the same restriction enzyme buffer.
Restriction Enzyme Based Cloning. 1. Short sequences containing restriction sites are added into the 5’ ends of primers for DNA amplification by PCR. 2. Both the vector and DNA fragment are digested with restriction enzymes to create cohesive ends. 3. The vector and DNA fragment are ligated. 4. The recombinant DNA enters the host cell during transformation.
PCR cloning relies on a process called ligation, which is a method of inserting a DNA fragment into a vector using DNA ligase. The reason ligation is important for this step is because it is responsible for inserting the PCR product into a ‘T-tailed’ plasmid.
PCR amplified inserts contain an adenine residue at the 3’ end of the DNA fragments (‘A-tailed’ ends). A ‘T-tailed’ plasmid vector has a single 3’ deoxythymidine (T) at each end of the arms of a linearized plasmid. Therefore, these PCR products can be ligated into ‘T-tailed’ vectors by using DNA ligase, and this step is followed by transformation.
You can choose this method when your restriction enzymes are not compatible or you find an internal restriction enzyme site in your DNA insert.
One disadvantage of this method is you will need a specific ‘T-tailed’ vector to perform PCR cloning. But ‘T-tailed’ vectors may not have supportive elements for your protein research, such as promoter region or protein tag.
PCR Cloning. 1. PCR Product with A-tailed ends is combined with T-tailed vector. 2. During ligation, PCR product is inserted into the vector.
Ligation Independent Cloning (LIC)
Ligation independent cloning (LIC) is performed by generating short sequences at the end of a DNA insert that match to the short sequences of a plasmid vector. Enzymes with 3’ to 5’ exonuclease activity chew 3’ ends and generate cohesive ends between the DNA fragment and the linearized vector. The two materials are then combined for annealing step. During transformation, the host organism repairs the nicks on the recombinant DNA.
The advantage of this method is it won’t create any new restriction sites or unwanted sequences in the final DNA construct.
LIC Cloning. 1. Short sequences which matches with sequences on the plasmid are added into the 5’ ends of primers for DNA amplification by PCR. 2. Plasmid is linearized by using restriction enzyme. 3. Both DNA insert and vector are treated with 3’ to 5’ exonuclease to create cohesive overhangs. 4. Both DNA and vector are annealed. 5. After transformation, the host cell repairs the nicks on the recombinant DNA.
Seamless Cloning (SC)
The seamless cloning (SC) technique (similar to LIC) depends on matching short sequences at the ends of a DNA fragment to the short sequences on a plasmid vector. SC method requires an enzyme with 5’ to 3’ exonuclease activity to create 3’ overhangs, a DNA polymerase to fill in gaps, and a DNA ligase to seal the nicks.
The advantage of LIC and SC over the restriction enzyme based cloning is it allows insertion of more than one DNA fragment into a vector. In addition, when you find an internal restriction enzyme site on your DNA fragment, you can use LIC or SC as an optional cloning method.
Seamless Cloning. 1. Short sequences are added into the 5’ ends of primers for DNA amplification by PCR. 2. Vector is digested by a restriction enzyme. 3. Both DNA fragment and vector are treated with an enzyme with 5’ to 3’ exonuclease activity to create cohesive overhangs. 4. During ligation, the DNA fragment is inserted into the vector.
This method requires site-specific DNA recombinase enzymes, which exchange and recombine DNA pieces with particular recombination sites.
The first step in this method is to insert a DNA fragment into an entry vector generating an entry clone. Another way to create an entry clone is by swapping and recombining a donor vector into an entry clone.
After creating an entry clone, the next step is to swap and recombine the entry clone into a destination clone. The benefit of this approach is it can be used to place more than five elements into a single vector. It is commonly used to identify protein-binding interactions or to optimize protein expression, purification and solubility. To perform this method, you will need a particular plasmid which has recombination sites.
Recombinational Cloning. 1. DNA fragment is inserted into an entry vector to create an entry clone. 2. Entry clone and destination vector are combined by a recombinase enzyme to create a destination clone.
In this article, we briefly explained about five molecular cloning methods which are commonly used by researchers. Molecular cloning has been used for many different purposes in a variety of research fields. In agriculture, cloning can shorten the time required to insert and develop a new beneficial trait in crops, such as a drought tolerant trait or a pest resistant trait. On the other hand, molecular cloning can be used to produce therapeutic proteins, such as a clot dissolving protein and interferon, or to synthesize other useful proteins, such as insulin, growth hormones, and monoclonal antibodies. Overall, the application of molecular cloning continues to improve and create more remarkable developments in the fields of agriculture, pharmaceutical industry, and biomedical research.
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