Producing DNA Fragments
- Development of technology that allows genes to be manipulated, altered and transferred from organism to organism, even transform DNA itself, has become very advanced in recent years.
- It has enabled scientists to have better understanding of organisms and it has also been very beneficial for medical application.
- A number of human diseases result from the individual being unable to produce various metabolic chemicals themselves; many of these chemicals are proteins, for example insulin.
- This is caused by a specific portion of DNA - the product of a gene.
- Treatment of such deficiencies previously involved extracting the chemical from a human or animal donor and introducing it into the patient; but rejection by the immune system and risk of infection may occur.
- The cost is also very expensive.
- There are advantages in producing large quantities of 'pure' proteins from other sources; as a result, techniquies have been developed to isolate genes, clone them and transfer them into a microorganism.
- The microorganisms are then grown to provide a 'factory' for the continous production of the desired protein.
- The DNA of two different organisms that has been combined in this way is called recombinant DNA and the organism is then known as a genetically modified organism.
- The process of making a protein using the DNA technology of gene transfer and cloing involves a number of stages:
- Isolation of the DNA fragments that have the gene for the desired protein.
- Insertion of the DNA fragment into a vector.
- Transformation - the transfer of DNA into suitable host cells.
- Idenfication of the host cells that have successfuly taken up the gene by the use of gene markers.
- Growth/cloning of the population of host cells.
- Before a gene can be transplanted, it must be identified and isolated from the rest of the DNA.
- To do this, two method which involv enzymes are used: reverse transcriptase and restriction endonuclease.
Using Reverse Transcriptase
- This is done by the use of retroviruses, best known as HIV.
- Retroviruses genetic information is in the form of RNA. They are used, because they are able to synthesise DNA from RNA using an enzyme called reverse transcriptase.
- The enzyme is called reverse transcriptase, because it catalysese the production of DNA from RNA, which is the reverse to usual transcription of RNA from DNA.
- The process of using reverse transcriptase to isolate a gene is as follows:
- A cell that readily produces the desired protein is selected, for example, the beta cells of the islets of the Langerhans from the human pancrease which produce insulin.
- These cells have large quantities of mRNA which code for insulin, which is then extracted from the cells.
- Reverse transcriptase is then used to make a single strand DNA from RNA; this DNA is known as complementary DNA (cDNA), because it is make up of the nucleotides which are complementary to the mRNA.
- To make the other strand of DNA, the enzyme DNA polymerase is used to build up the complementary nucleotides on the…