Recombinant DNA technology
Recombinant DNA, which can also be known as chimeric DNA, is formed from the genetic material of two different species, that are formed in a laboratory using special techniques and then inserted into the genome of a specific organism, where the effect and expression of the DNA can then be analysed. This is possible due to the similar chemical and physical structure of DNA in different organisms, the same nucleotides are used so DNA from plants, bacteria and humans can be combined. Recombinant DNA technology plays a crucial role in medicine, allowing analysis and production of specific protein on a large scale. For recombinant DNA to be formed, two types of DNA are required, Vector DNA and Insert DNA.
Vector DNA is used to transport the insert DNA into the cell. The vector needs to contain a unique restriction site for the insertion of the new DNA, an effective origin of replication and if possible, a gene that will allow easy selection of the cells, which have successfully taken up the vector. This could be a gene that codes for antibiotic resistance that is disrupted when the insert is added, or gene that codes for fluorescent colour to appear under UV. Examples of vectors include Bacteriophages (viruses which infect bacteria) and Cosmids (genetically engineered hybrids), but the most commonly used vector is a bacterial plasmid, which can replicate independently of the bacterial chromosome. E. coli is a commonly used bacteria which acts as a genetic factory to produce a large amount of recombinant DNA.
Insert DNA is the gene that we want to be expressed in the host cell, this could be a gene coding for insulin production or for another useful protein which is inserted into the vector. This can be obtained randomly or specifically. Random isolation involves non-specific fragments of genomic DNA, isolated either by double stranded DNA, which are analysed using the genomic library, or by isolating mRNA and using reverse transcriptase to form cDNA. Specific isolation is different in that it involves specific fragments of genomic DNA or cDNA which are them amplified by the PCR technique.
The increased understanding of recombinant DNA was first sparked in the 1970s, after this was proposed by Peter Lobban. 1971 Paul Berg carried out his gene splicing experiment. Then in 1972 Herbert W. Boyer carried out another groundbreaking experiment, where ribosomal DNA was inserted into bacteria, the effect of this was that this DNA was replicated naturally 1972 and 1973 the first publications on recombinant DNA were released. Insulin was the first drug to be formed from recombinant DNA, which was carried out by Genenentech
- The first step in forming recombinant DNA is acquiring the insert of the DNA that you want to insert into your host cell, this can be done randomly or specifically as mentioned before. The enzyme required for this step is Restriction endonuclease, which cleaves DNA in a staggered fashion at specific recognition sites, this allows you to remove the specific piece of DNA required from a double strand of DNA, as this enzyme can cut through the sugar phosphate backbone.
- Once you have your insert and you have enough copies to add to multiple host cells, then you need to insert this into your vector. This is first done by cleaving the plasmid (the vector in this example) using restriction endonuclease, to produce specific ends that are complementary to the ends of the insert DNA. Then using the enzyme ligase the insert is inserted into the plasmid, annealing the two sections of genetic material to form recombinant DNA.
- Then we need to transport the recombinant DNA into the host bacterial cell. This is done by adding the plasmid and mixing, heat shock and CaCl is used to break open the cell wall so that the plasmid can move in. To repair the cell wall the cell is incubates at 37C for 30 minutes. This is the most difficult phase and not all plasmids will be successfully taken up, which is why we will need to select the successful hosts.
- The transformed cells will be able to grow on a selective medium, for example we could grow the bacteria on a agar dish that is coated with a specific antibiotic, the insertion of the new DNA will have disrupted the gene that codes for antibiotic resistance, so this means that the transformed cells will not be able to grow on the antibiotic, allowing us to positively identify those that were successful.
Now that we have our recombinant DNA we have a choice of what to do with it. We can either induce expression of a protein in the host, this can either be done directly, so producing the protein for purification, or the cloned gene could be engineered to produce a modified version of the protein. One of the most important uses for recombinant DNA is the formation of insulin, up until the 1980s animal sources where used to form insulin for diabetic injections, but now recombinant insulin can be formed as with animal sources it may be difficult to purify and there is a risk of possible contamination with pathogens. The gene coding for the production of human insulin is obtained and inserted into a bacterial host cell, where it can be produced on a large scale to provide enough for patients to inject.