recombinant DNA technology
Referred to as Recombinant DNA technology, as the processes involve combining DNA, from different organisms or from different sources, in a single organism.
The following steps are necessary;
- The required gene is obtained.
- a copy of the gene is placed-packaged and stablised-in a vector.
- The vector carried the gene to the recipient cell
- The recipient expresses the gene through protein synthesis.
Obtaining the gene to be engineered-The mRNA produced from transcription of the gene can be obtained from cells where that gene is expressed. E.g. the mRNA for insulin is obtained from beta cells in islets of Langerhans in the pancreas. The mRNA can be used as a template to make a copy of the gene. The gene can be synthesised using an automated polynucleotide sequencer. A DNA probe can be used to locate the gene on DNA fragents and the gene can be cut from a DNA fragment using restriction enzymes.
Placing the gene in a vector-The gene can be sealed into a bacterial plasmid using the enzyme DNA ligase. This is by far the most common vector method used in genetic engineering. Genes may also be sealed into virus genomes or yeast cell chromosomes. Vectors often have to contain regulatory sequences of DNA. These ensure that the inserted gene is transcribed in the host cell.
Getting the gene into the recipient cell
The gene, once packaged in a vector, can form quite a large molecule that does not easilt cross the membrane to enter the recipient cell.
- Electroporation-a high-voltage pulse is applied to disrupt the membrane
- Microinjection-DNA is injected using a very fine micropipette into the host cell nucleus.
- Viral transfer-the vector is a virus, this method uses the virus' mechanism for infecting cells by inserting DNA directly.
- Ti plasmids used as vectors can be inserted into the soil bacterium Agrobacterium tumefaciens. Plants can be infected with the bacterium, which inserts the plasmid DNA into the plant's genome.
- Liposome-DNA is wrapped in lipid molecules. These are fat-soluble and can cross the lipid membrane by diffusion.
Restriction and ligase enzymes
Recombinant DNA techniques often involve the cutting and sticking together of DNA strands. E.g. a useful gene may need to be cut out of the chromosome on which it has been found, the sealed into a plasmid vector.
Enzymes known as restriction enzymes-restriction endonucleases-are used to cut through DNA at specific points. These enzymes were first extracted from bacterial cells, where they perform a natural defence function against inection by viruses. There are now more than 50 different commonly used restriction enzymes.
A particular restiction enzyme will cut DNA wherever a specific base sequence occurs and only where that sequence occurs. This sequence is called the restriction site, and is usually less than 1- base pairs long. In most of the restiction enzymes in use, the enzyme catalyses a hydrolysis reaction which breaks the phosphate-sugar backbones of exposed bases known as a sticky end.
When separate fragments of DNA need to be stuck together, an ezyme known as DNA ligase is used to catalyse a condensation reaction which koins the phosphate-sugar backbones of the DNA double helix together. This enzyme is the same as that used in natural DNA replication to seal DNA nucleotides together to form new DNA strands
In order to join together DNA fragments from different sources both need to have originally been cut with the same restriction enzyme. This means that the sticky ends are complementary and allows the bases to pair up and hydrogen bond together. DNA ligase can then seal the backbone.
Where DNA fragments from different organisms are joined in this way, the resulting DNA is caled recombinant DNA.