Genetic Engineering
- Genetically modified organisms are organisms that have had their DNA altered through recombinant DNA technology.
- Recombinant DNA technology involves the transfer of fragments of DNA from one organism, or species, to another.
- Transgenic organisms can successfully express a gene from any organism, as the genetic code and mechanism of protein production (transcription and translation) are universal.
- DNA fragments are created by:
- Using restriction endonucleases to cut at recognition sites near the desired gene
- Converting the mRNA of the desired gene to cDNA, using reverse transcriptase. Double stranded DNA is then synthesised using DNA polymerase
- Synthesising the gene using a gene machine. The gene sequence is determined by the primary protein structure
- The isolated gene is then modified by the addition of a promoter and a terminator region.
- A vector is used to transfer the isolated gene into a host cell. This is mainly a plasmid.
- Restriction endonucleases are used to cut plasmids open, creating sticky ends. The same endonuclease isolates the gene, so the sticky ends of the desired gene and the plasmid are complementary. DNA ligase joins them together
- To reintroduce the desired DNA into bacterial cells, the recombinant plasmid must pass through the cell surface membrane of a bacterial cell (transformation).
- Transformation involved mixing the bacteria and plasmids in a medium containing Ca2+ ions, which increased membrane permeability. Changes in temperature also make the bacterial cell surface more permeable.
- The transformed host cells can be cultured as an in vivo method to amplify DNA fragments.
Marker Genes
- Transformed bacteria can be detected using marker genes
- The plasmid contains 2 marker genes
- The first marker gene is used to identify which bacteria have successfully taken up a plasmid. It is a antibiotic resistance gene, so transformed bacteria are identified by growing on a medium containing the antibiotic
- The second marker distinguishes between bacteria that have taken up an empty or recombinant plasmid. When a recombinant plasmid is formed, the desired gene is inserted in the middle of the second marker gene making it non-functional. Therefore, bacterial cells that express the second marker gene do not contain the recombinant plasmid
- The second marker gene has easily identifiable phenotypes such as:
- Producing a fluorescent protein
- Providing resistance to a different antibiotic
- Producing an enzyme whose action can be identified
Polymerase Chain Reaction (PCR)
- PCR is a method of amplifying DNA by artificial replication in vitro.
- It requires: DNA sample of around 10,000 base pairs, nucleotides, Taq polymerase (stable at high temperatures), primers complementary to 3’ of DNA sample and a thermocycler to carry out the automated process.
The Use of Genetically Modified Organisms (GMOs)
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- The risk of GM bacteria can be reduced by modifying the bacteria so that they are unable to produce an essential nutrient or amino acid and cannot survive outside the lab.
Gene Therapy
- Gene therapy is the mechanism by which genetic diseases are treated or cured by masking the effect of a faulty allele through the insertion of a functional allele.
- Firstly, a healthy allele from healthy cell tissue is isolated. The allele is inserted into the cells using vectors.
- If the mutated allele is recessive, a dominant allele is inserted. If the mutated allele is dominant, DNA is inserted into the middle of the allele to silence it.
- Somatic therapy involves altering the alleles in body cells. The altered allele is not passed onto the offspring
- Germ-line therapy altering the alleles in the sex cells. The altered alleles are passed onto offspring
- Germ-line therapy has ethical concerns such as the potential of designer babies or the potential impact gene insertion could have on other genes.