Practise A2 genetic technology by explaining gene-transfer choices, PCR and gel results, and how fluorescent probes or sequence databases reveal genetic information.
The potato plant, Solanum tuberosum, is an important food crop. Crop yield is reduced if the leaves of the plant are eaten by the larvae (immature stages) of the Colorado beetle, Leptinotarsa decemlineata.
Crop scientists used recombinant DNA technology to create two genetically modified (GM) varieties of potato plant. These plants produce proteins that are poisonous to insects.
- GM potato variety A contains two new genes, S N and Bt.
- GM potato variety B contains two new genes, SN and OCII.
The new varieties were tested by having a constant number of Colorado beetle larvae introduced to the plants at time 0 hours. The number of larvae that were alive after 24,48 and 72 hours was recorded. The percentage of the larvae that had died in each time interval was calculated. This was repeated for potato plants that had not been genetically modified (non-GM).
Table 4.1 shows the percentage of Colorado beetle larvae that had died on the GM potato plant varieties and on non-GM potato plants.
Table 4.1
Suggest what is meant by recombinant DNA technology.
Adenosine deaminase (ADA) deficiency is an immune system disorder caused by a recessive autosomal mutation.
Severe combined immunodeficiency caused by a lack of ADA is called ADA-SCID.
Genetic engineering is used to make a recombinant human protein to treat people with ADA-SCID.
Outline the principles of genetic engineering.
In 2016 gene therapy to cure ADA-SCID was approved in Europe. The gene therapy involves three main steps.
- Blood (haematopoietic) stem cells are taken from the bone marrow of the person with ADA-SCID.
- The functional gene and its promoter are inserted into the blood stem cells.
- A single infusion (injection) of the gene-corrected cells is given to the patient.
Explain why a promoter has to be transferred as well as the desired gene.
The gene therapy technique used to cure ADA-SCID is not suitable for treating the genetic disease called Huntington's disease. A newer technique called gene editing could potentially be used instead to cure Huntington's disease.
Explain why gene editing is more suitable as a potential cure for Huntington's disease.
Factor VIII can be made as a recombinant human protein.
The gene that codes for human factor VIII can be synthesised from messenger RNA purified from human liver cells.
Name the enzyme that uses messenger RNA as a template to produce complementary DNA.
Outline two sources, other than messenger RNA, from which genes can be obtained for genetic engineering.
The gene that codes for human factor VIII can be transferred into mammalian cells in tissue culture.
Explain why a promoter also needs to be transferred into the mammalian cells so that human factor VIII can be synthesised.
Adenosine deaminase (ADA) deficiency is an immune system disorder caused by a recessive autosomal mutation.
Severe combined immunodeficiency caused by a lack of ADA is called ADA-SCID.
Genetic engineering is used to make a recombinant human protein to treat people with ADA-SCID.
Outline the principles of genetic engineering.
Factor VIII can be made as a recombinant human protein.
The gene that codes for human factor VIII can be synthesised from messenger RNA purified from human liver cells.
Outline two sources, other than messenger RNA, from which genes can be obtained for genetic engineering.
Traditional techniques for genetically modifying organisms use three enzymes:
- restriction endonuclease
- reverse transcriptase
- DNA ligase.
For example, these enzymes have been used to produce genetically modified (transgenic) pigs containing the GFP gene coding for green fluorescent protein, originally sourced from jellyfish.
Outline how these three enzymes could be used in genetically engineering a transgenic pig containing the GFP gene.
A new technique that aims to cause a deletion in a gene uses an enzyme called Cas9 nuclease. It is injected into zygotes along with an RNA sequence (the guide RNA) that is complementary to a target gene. The Cas9 nuclease causes a deletion in the target gene in the zygotes, preventing the expression of that gene.
The toxicity and efficiency of the new technique was tested on four groups of pig zygotes. These pig zygotes were produced by IVF using:
- ova from a female non-transgenic pig.
- sperm from a male transgenic pig whose somatic (body) cells contained one copy of the GFP gene per cell.
The pig zygotes in three groups were injected with different concentrations of Cas9 nuclease and guide RNA targeted at the GFP gene.
The fourth group of pig zygotes (control group) was not injected with Cas9 nuclease and guide RNA.
Factor VIII can be made as a recombinant human protein.
The gene that codes for human factor VIII can be transferred into mammalian cells in tissue culture.
Explain why a promoter also needs to be transferred into the mammalian cells so that human factor VIII can be synthesised.
Traditional techniques for genetically modifying organisms use three enzymes:
- restriction endonuclease
- reverse transcriptase
- DNA ligase.
For example, these enzymes have been used to produce genetically modified (transgenic) pigs containing the GFP gene coding for green fluorescent protein, originally sourced from jellyfish.
Explain why the GFP gene was chosen for testing the new technique.
Traditional techniques for genetically modifying organisms use three enzymes:
- restriction endonuclease
- reverse transcriptase
- DNA ligase.
For example, these enzymes have been used to produce genetically modified (transgenic) pigs containing the GFP gene coding for green fluorescent protein, originally sourced from jellyfish.
Some of the zygotes in each group survived and after six days each had developed into a group of cells called a blastocyst.
The blastocysts were counted using a light microscope. A filter was then added to the microscope, so that only blastocysts expressing the green fluorescent protein showed up. These were counted and the results are summarised in Table 5.1.
Table 5.1
State the best concentration of Cas9 nuclease and guide RNA to use to cause a deletion in the GFP gene and give reasons for your choice.
Myosotis is a genus of small flowering plants. Many different Myosotis species grow on the islands of New Zealand, which are an important site of Myosotis evolution. Lowland Myosotis species grow at low altitude while alpine Myosotis species grow at high altitude at the tops of mountains.
Scientists wanted to obtain molecular data to determine the evolutionary relationships of New Zealand's Myosotis species. They extracted DNA from individuals of Myosotis species collected from three different islands in New Zealand.
To carry out a polymerase chain reaction before DNA sequencing, the DNA samples were mixed with primers, deoxynucleotides and Taq polymerase and put through 35 cycles of treatment. Each treatment cycle involved one minute at , followed by one minute at and then four minutes at .
Describe what happened to the DNA at each temperature.
Traditional techniques for genetically modifying organisms use three enzymes:
- restriction endonuclease
- reverse transcriptase
- DNA ligase.
For example, these enzymes have been used to produce genetically modified (transgenic) pigs containing the GFP gene coding for green fluorescent protein, originally sourced from jellyfish.
Fig. 5.1 shows the results from a second trial of the new technique, analysed by electrophoresis.
- Lanes 1-4 show DNA from four pigs born after Cas9 nuclease was used to cause a deletion in a target gene coding for a cell surface protein.
- Lane 5 shows DNA from their surrogate mother.
- Lane 6 shows DNA from another normal pig for comparison.
The size of the DNA fragments is given in kilobase pairs (kbp) as shown in Fig. 5.1. 1 kbp is 1000 base pairs of DNA.
The target gene measures 6 kbp and codes for a cell surface protein that is essential for the disease virus PRRSV to infect cells in the pig's body.
Fig. 5.1
Explain what Fig. 5.1 indicates about the success of the new technique in causing a deletion in a gene in pigs so that they show resistance to PRRSV.
Researchers wanted to know if changes in gene expression were important in the inability of the AD mice to learn.
Groups of normal mice and AD mice either received training to allow them to learn how to swim a water maze, or they received no training. The mice in the four groups then had mRNA extracted from the memory-forming areas of their brains.
Reverse transcription of the mRNA of individuals in each group was carried out and the resulting cDNA was labelled with fluorescent nucleotides. This was then used for DNA microarray analysis using slides containing DNA sequences from 33696 mouse genes.
Explain the principles of this type of DNA microarray analysis.
Table 5.1 summarises the microarray analysis of differences in gene expression for:
- an untrained AD mouse compared to an untrained normal mouse
- a trained AD mouse compared to a trained normal mouse.
Table 5.1
State what the results in Table 5.1 show about the effect of training and learning on gene expression in brain cells.
Information about amino acid and nucleotide sequences is stored in computer databases.
Outline the advantages of using databases of nucleotide sequences to investigate evolutionary relationships between species.