How does gene cloning work?

1) Firstly, we need a sufficient amount of DNA to clone a gene. For this, a bit of DNA is retrieved from a cell and amplified by a PCR. Common ways in extracting DNA is:

Sonicating: compressed sound is administered to the cell, so that it agitates it and causes lysis; the DNA is then put in a alcoholic solution to make it easier to retrieve.

   



Osmosis: application of using diffusion to retrieve DNA.

 
A PCR (Polymerase Chain Reaction) is a commonly used technique in laboratories that can make multiple copies of a specific strip of DNA through thermally stable polymerase enzymes (enzymes used in the assembling of DNA; in this case Taq polymerase) and DNA primers (strands of nucleic acids required for the synthesis of DNA). In PCR, the sample bit of DNA is first heated in high temperatures so that the DNA separates into 2 pieces of single-stranded DNA. These strands are then used as templates to build duplicate strands by an enzyme called Taq polymerase. Then the duplicate strands are used to build another set of strands and so on. The complete PCR process is performed in a machine called a Thermocycler.


Watch Video on how PCR works







Diagram of how PCR works






A much complex diagram of the function of PCR; to all the interested ones out there!









2) Secondly, a vector is obtained, which is a piece of DNA that duplicates itself (replicates) in a bacterium. These are usually circular pieces of DNA called plasmids (separate in form), found mainly in bacteria, which are self-replicating (capable to make copies of the chromosomal DNA found in bacterium) and function under stable conditions. Using a vector, a gene can be copied many times (replicated) in a single bacteria; the maximum is millions of bacteria in one litre flask. 





Diagram of a Bacterium; here you can find the plasmid and the bacterial chromosome.






Plasmids: How plasmids replicate chromosomal DNA found in Bacteria.



3) Next, restriction digestion is applied in order to cut the preferred gene out of the amplified DNA product. A restriction enzyme (in particular, a “sticky-ends” restriction enzyme) is used so that the bits of amplified DNA that are all over the place, will stick to each other, making the job much easier.





Application of the Restriction Enzyme: DNA fragments stick to each other because of their 'sticky' ends, as shown in this diagram.



The gene is then cut out of the DNA fragments through a technique called gel electrophoresis. 


• Gel electrophoresis requires the use of a piece of gel, which is usually made of a seaweed extract called agarose. This gel will have a lot of resistance to the DNA pieces because of its consistency, making it useful. There will be wells (holes) in one end of the gel so that the DNA bits can be inserted into it. An electric current is applied so that the DNA pieces move to the other end [DNA fragments are negatively charged because of its phosphate groups, causing it to move towards the positive end of the gel (one without wells)]. However because of the resistance described prior, only smaller pieces of DNA will make it through, since larger fragments will move slowly. This way the smaller pieces of DNA can be separated, which will contain the preferred gene(s).  

1. Here you can see the piece of gel (agarose); there are wells (holes) in the left end of the gel piece for loading DNA fragments.

















2. When the DNA fragments containing the gene is inserted into the gel, an electric current is run through the gel (as you can see in the diagram the current is heading right).












3. Due to the gel's resistance to the DNA fragments, the DNA pieces are separated according to their size. 



4. Voila, the process is complete! Smaller DNA bits are near the right end of the gel (down) and bigger DNA bits are near the left end/middle (up).

4) After the gene is extracted, the same method is used to extract the preferred vector.





5) The vector and gene are both then mixed and ligase enzyme is added. This enzyme connects the two pieces (or ends) together. In other words, it is similar to glue. 



Sample diagram showing how ligase enzyme 'connecting' works; DNA ligase is applied to the ends of the two DNA fragments (the gene and vector) so that they stick. 



Before and After Ligation




6) The DNA (now connected with the vector) is inserted back into a bacteria cell; one way of doing this involves making the bacteria's cell membrane permeable so that a liquid containing the DNA piece can pass through it, thus adding the DNA to the cell. The bacteria cell is later left to recover and grow. 



Gene Cloning: Reproducing Bacteria cells (a)






Gene Cloning: Reproducing Bacteria cells (b)










 

7) Finally you have your desired project, cloned genes. However, you must screen for any mistakes that happened, (e.g. if some of the vectors don't have the gene, any mutation, etc.). Most of the time, bacteria cells that have incorporated the desired gene appear in a different colour (white) than bacteria cells that have not; this is since because an cell that has a DNA target cloned into its vector has another gene disrupted, which in return causes the cell to turn white. Using this method, the white cells can be picked and grown, and you have your cloned genes. 



Sample diagram of Gene Cloning and the resulting white colonies of 'cells.'




Sample Diagrams of Gene Cloning:  











Click here for a animation of Gene Cloning (Recommended) 


Click here to watch a video on Gene Cloning (Recommended)