Obtaining the Gene
Obtaining the Gene
Genetic engineering aims to remove a desired gene and transfer it to another organism where it can be expressed. This means that the required protein can be synthesised within the new organism.
This process is extremely important and can result in the production of life-saving drugs, such as insulin, or plants that are resistant to certain diseases or insect pests.
How this is done:
- Firstly, it is necessary to obtain the desired gene.
- The gene is then cloned to get many copies of the required length of DNA.
- A copy of the gene is inserted into the host DNA, using sets of enzymes.
- The cells containing the host DNA can then make the desired protein in large quantities.
There are various methods that are used...
If the amino acid sequence of the desired protein is known, the DNA code can be worked out and the DNA made in the lab by stringing together the correct order of nucleotides.
Note:Many proteins are extremely large, therefore this would be a tedious process.
- Isolate the messenger RNA (mRNA) for the desired gene and make a single copy of the complementary DNA using the enzyme reverse transcriptase and another copy is made by adding DNA polymerase so that a doubled stranded length of DNA is made. (The original reverse transcription enzymes were first discovered in retro viruses.)
- Isolate the gene from the entire genome. To do this the DNA must first be cut into fragments and the one containing the desired gene must be identified. The enzymes used to cut the DNA are called restriction enzymes or restriction endonucleases.
The fragments need to be put into an organism that will express the protein.
There are several ways of getting DNA fragments into host cells:
- Electroporation - temporary holes are made in the membrane using electrical pulses.
- Microprojectiles - tiny beads of tungsten or gold coated with DNA fired by a gun into the cell.
- Using viruses as vectors.
- DNA in liposomes - fuse with the cell membrane.
Note: These methods only introduce DNA into the cell; they do not incorporate it into the host DNA.
The main requirement of the organism is that large quantities of a protein can be produced as cheaply and easily as possible, it needs to be an organism that:
- Grows fast.
- Is easily manipulated.
- Has a simple chromosome (prokaryotic cells do not have a nuclear envelope).
- Contains naturally occurring vectors (see later).
A good option therefore is to use yeasts or bacteria.
There are many restriction enzymes (R.E), each one of which will cut at a specific DNA sequence.
The names of the restriction enzymes indicate from where the enzyme was isolated. (For example, EcoR1 comes from E.coli.)
The target sites are palindromic (reads the same both ways, like "Hannah" or "Madam") and may cut at the same place in both strands creating blunt ends as in Hpa11 or at different places in the 2 strands leaving so-called sticky ends as in EcoR1.