Recombinant DNA technology - AL only (3.8.4.1)
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Genetic engineering involves manipulating an organism’s genome. One of the most common ways this is carried out involves inserting desired genes into a bacterial plasmid, creating recombinant DNA.
This recombinant DNA can be inserted into an organism, creating a transgenic or genetically modified organism.
Gene insertion is possible as transcription and translation are universal biological processes.
Enzymes called restriction endonucleases can be used to cut the desired gene from an organism.
Restriction endonucleases can cut a gene in different ways, producing either blunt ends or sticky ends.
The mRNA of the desired gene for genetic engineering can also be isolated from the main strand. This method is easier when the desired gene is being isolated from an organism with a large genome.
The mRNA for the desired gene is isolated, then treated with reverse transcriptase enzymes. This produces complementary DNA (cDNA).
Genes can be manufactured in a lab:
- The desired sequence of amino acids is determined.
- The amino acid sequence is used to determine the mRNA codons and the complementary DNA triplet code.
- The sequence is checked to ensure ethical, safety, and biosecurity guidelines are followed.
- The computer designs small strands of nucleotides called oligonucleotides.
- The oligonucleotides are built up and joined together to make the gene.
- The complementary DNA strand is then assembled.
- The DNA is replicated using PCR.
The DNA with sticky ends can be inserted into a bacterial plasmid, allowing the gene to be cloned, stored or transferred to another organism.
Creating genes in a lab can be advantageous. There are no introns or exons, so no processing of mRNA is required.
The desired gene can be easily transcribed and translated by bacterial cells.
Following extraction, the desired gene is placed into a vector, which is usually a bacterial plasmid.
The plasmid is edited to contain two markers: one is a selectable marker, such as a gene conferring antibiotic resistance, and the other is a detectable marker, such as a fluorescent marker or an enzyme that changes colour under specific conditions.
The plasmid is opened at the same site as the marker genes using a restriction endonuclease. DNA ligase joins the plasmid and the desired gene together. This results in one of the marker genes failing to function.
The functional markers allow screening to determine which bacteria have taken up the desired gene.
Genes are inserted into a vector between a promoter and a terminator:
Promoter: a section of the plasmid where RNA polymerase and transcription factors join, this is the start point of transcription
Terminator: a section of the plasmid is where the RNA polymerase is released, ending transcription.
Following gene insertion, not all bacterial cells will take up the modified plasmid.
Issues can also occur in modification when DNA ligase either joins the plasmid without the desired gene or joins the gene into a non-viable plasmid.
The presence or absence of various marker genes will enable the identification and isolation of only bacteria carrying the desired recombinant plasmid.
Following the take-up of the successfully recombinant plasmid shown above, the host bacteria will exhibit antibiotic resistance but will not fluoresce. The bacteria are then allowed to multiply on an agar plate infused with antibiotics. This allows only the correct bacteria, the non-fluorescent spots, to be extracted and grown to create the cell culture.
Animals can be genetically modified to produce drugs and proteins, this is called pharming.
Mammals, such as sheep and goats, can be genetically modified so that proteins, such as proteins involved in clotting, are produced in their milk. These proteins can then be purified and used medicinally to treat conditions such as haemophilia.
Genetically modified plants can be produced using the bacteria Agrobacterium tumefaciens. This bacterium causes tumour growth in plants. Rapid mitosis in tumours can be utilised to transfer desired genes into plant cells.
Plants can be modified to be resistant to weed killers, such as glyphosate, or to produce their own insecticides.
Gene therapy involves treating genetic diseases which are caused by faulty alleles, such as haemophilia, cystic fibrosis and sickle cell disease.
These diseases may be treated by replacing the faulty allele with a functioning allele.
One of the main advantages of gene therapy is that gene therapy can cure genetic disorders which are otherwise very difficult to treat.
However, manipulating the genome could have unintended consequences. Inserting the functioning allele could cause other genetic issues depending on where in the genome the allele is inserted.