Gene cloning promises immense upside potential in the field of microbiology. It can provide medical solutions to numerous genetic problems. While the technology is still in the nascent stages, it has somewhat sown the seeds of revolution for microbiology.
There are numerous methods to clone genes. The technology first caught the limelight when scientists successfully cloned Dolly. Dolly was a sheep that lived on for 6 years, and then she was euthanized. The reason behind this was that after 6 years, Dolly developed serious health issues, including arthritis and lung tumors.
Since then, there’s been numerous research in the field. We’ve come a long way from there. Here are 8 techniques that can be used for cloning a gene:
Using Restriction Enzymes
If this was to be explained in layman’s terms, the restriction enzyme method is the CTRL + C and CTRL + V method. This means the specific gene is cut up and then pasted into the vector. The gene is treated with enzymes to get the waste parts digested and extract the gene of interest. Basically, the vector and gene of interest is extracted out to attain a specific gene sequence.
Several enzymes are used in the process depending upon the needs of the process. EcoR, Nco I, BamH I, etc., are just a few of the many enzymes used for this process.
TA Cloning or TA/TOPO Cloning
This is an advanced method of TA cloning that’s faster and more efficient than the traditional methods. Instead of using a variety of genes for different purposes, one single enzyme is used for digestion and ligation.
With Topoisomerase I as the primary enzyme, the process gets completed in between 5 to 10 minutes.
This technique was developed in the 90s and has quickly become a process of importance in biological researches. The process takes place in two steps, and BP Clonase and LR Clonase are used as the enzyme mixes.
This technique is useable for various purposes and works well with bacterial, mammalian, or even insect DNA.
The method uses complementary flanking sequences where the gene of interest is put to the vector by annealing. The good part with this method is that it isn’t size restricted and that vector of any size can be used to conduct the procedure.
Ligation Independent Cloning
This is a relatively simple and quicker method of cloning. The vectors are linearized by either PCR or via restriction digestion. (Lynda, 2002)
This method is used when there’s a need to clone 2 or more genes. This process is in the developmental stage, with two methods currently deployed.
1. Internal ribosome entry site (IRES) elements
This is a method to detect the IRES elements that may remain unexpressed between the two genes when other methods are deployed. The method is efficient but fails to deliver results when the particle size becomes large (500Bp)
2. 2A Peptides
This method efficiently deals with the shortcomings of IRES. The method helps researchers to easily clone more than a couple of genes in a singular multi-cistronic.
This is one technique that works with multiple DNA strands to combine them into a single isothermal reaction. The practise requires a constant temperature of 50 degrees. The process can be worked up in a single PCR tube, but when the size of DNA fragments increases, it may need to be performed in a couple of tubes.
Golden Gate Cloning
This process is conducted to join DNA fragments without cutting the fragments. The enzymes are placed so strategically that they themselves work upon the DNA strands to cut the DNA sequences that are outside of the recognition of the domain of the enzyme.
The above mentioned are the methods of cloning a gene. These are used in various conditions depending upon the requirements of the processes or research. One main apparatus that is key to all the above-mentioned procedures is a PCR tube. MBP provides the best microbiology apparatus to labs and research facilities around the world.
If you need PCR tubes or other microbiology equipment, then feel free to contact us.
Lynda, M. L. (2002). High Throughput Methods for Gene Cloning and Expression. Elsevier, 1-7.