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(1) DNA Profiling DNA profiling is being used as part of an international research project to identify twenty thousand marine fish species by the year 2010. Scientists will identify the sequence of bases in a specific region of mitochondrial DNA which does not show much variation within species, but between species is very different. The DNA is sourced from muscle tissue using standard DNA extraction techniques. (a) Describe what DNA profiling is. (b) PCR and gel electrophoresis are two of the techniques used in DNA profiling. For each of these two techniques discuss BOTH, how the technique is carried out and why the technique is used. (c) Discuss the human need or demand for DNA profiling technology in relation to identifying marine fish species. (2) Genome Analysis “Sheep genomics” is a trans-Tasman partnership involving Australian and NZ research organisations. The project will ultimately lead to sequencing of the sheep genome. This will allow a virtual sheep genome map to be created, according to Australian researcher Dr Brian Dalrymple. Around the world genome analysis is currently being carried out on several species. (a) Describe what genome analysis is. (b) Two techniques used in genome analysis are the development of DNA sequencing and DNA chips. Discuss how and why BOTH DNA sequencing and DNA chips are used in genome analysis. (c) Use the above examples of genome analysis to discuss why people are spending huge amounts of time and money to carry out this application in sheep. (3) Transgenesis Chymosin is an enzyme that digests milk proteins and can be used to clot milk into curds. Traditionally Chymosin was obtained from the stomach secretions of suckling calves less than 10 days old but is now produced in an application of Transgenesis by using ‘transgenic bacteria’ – these are bacteria which have had the chymosin gene added to their DNA. (a) Describe what Transgenesis is. (b) Discuss how restriction enzymes and DNA ligase are used produce transgenic bacteria, such as the chymosin producing bacteria. (c) Discuss why producing chymosin has changed from extraction from stomach secretions of suckling claves less than 10 days old to the use of transgenic bacteria. (4) Gene Cloning Since Banting and Best discovered the hormone, insulin in 1921, diabetic patients, whose elevated sugar levels are due to impaired insulin production, have been treated with insulin derived from the pancreas glands of abattoir animals. The hormone, produced and secreted by the cells of the pancreas regulates the use and storage of food, particularly carbohydrates. Although bovine and porcine insulin are similar to human insulin, their composition is slightly different. Consequently, a number of patients' immune systems produce antibodies against it, neutralising its actions and resulting in inflammatory responses at injection sites. Added to these adverse effects of bovine and porcine insulin, were fears of long term complications ensuing from the regular injection of a foreign substance, as well as a projected decline in the production of animal derived insulin. These factors led researchers to consider synthesising Humulin by inserting the insulin gene into a suitable vector, the E. coli bacterial cell, to produce an insulin that is chemically identical to its naturally produced counterpart through Gene Cloning. (a) Describe what Gene Cloning is in relation to the example given. (b) Restriction enzymes and ligation are two key techniques used in Gene Cloning. Discuss how each technique is carried out and include an explanation as to why each technique is used to clone the lysozyme gene using a plasmid vector. (c) Discuss the human needs or demands for this process and its particular usefulness. In your discussion include potential advantages and disadvantages of this form of the production of the enzyme. (1) DNA Profiling - Answers (a) Achieved: DNA profiling is a method that uses microsatellities or STRs in DNA to identify different species or individuals OR create patterns (ladders) from which the DNA can be compared to ascertain relatedness of two or more samples or to identify specific features when compared to a known sample. (b) Achieved: describes how OR why the TWO techniques are used e.g. PCR is used to increase the amount of DNA you have. PCR can change a very small sample of DNA into a large sample very quickly. e.g. Gel electrophoresis separates DNA fragments by their size. Merit: explains how or why ONE of the two techniques are used e.g. WHY - PCR is used to amplify the DNA sample so that many tests can be taken from the original sample of tissue taken from the victim. HOW – Raising the temperature breaks the hydrogen bonds between the two strands because they are relatively weak bonds. Lowering the temperature anneals the primers to the DNA strand, raising the temperature again allows the DNA Polymerase to add nucleotides to the primers. e.g. WHY - Gel Electrophoresis is used because the technique separates the DNA fragments by their size making it possible to analyse the pattern produced by fish species. Thereby identifying differences between species and recognising the pattern produced by each species. HOW – Electric field through agar gel separates fragments according to size. DNA is negatively charged so moves towards the positive electrode. The smaller fragments are able to move faster because of their size so in a set time they move further from the starting point. The spread out pattern produced by each species is different because the length of the microsatellite is different so this gives a way of recognising each of the different fish species. Excellence: discusses how AND why ONE of the two techniques is used. (c) Achieved: describes human need or demand. e.g. The use of DNA profiling enables fish species to be accurately identified OR this allows biodiversity / conservation issues to be addressed OR evolutionary links identified OR data on fish species will also enable fish quotas / stocks to be ascertained. Merit: explains human need or demand. e.g. DNA profiling is faster and more accurate than any other method of identification so that data on fish species can enable fish quotas / stocks to be ascertained to ensure they are sustainable. Excellence: discusses human need or demand e.g. This use of DNA profiling is justified as it enables fish species to be identified faster and more accurately than any other method. This allows biodiversity and conservation issues to be addressed more accurately, as data on each fish species is more accurate. Without the use of DNA profiling different species of fish that look similar may have been misidentified and this would have made it appear that there were more of a species present than was actually the case. When the stocks of a fish species are able to be quickly and accurately determined it helps to ensure that appropriate management strategies are implemented to maintain a sustainable catch. (2) Genome Analysis - Answers (a) Achieved: Genome analysis is about finding out the order of bases in the DNA as well as finding the location of genes and the parts of the DNA that controls the activity of the genes. (b) Achieved: describes how OR why the TWO techniques are used e.g. DNA sequencing determines the order of bases of the genome e.g. DNA chips are used as a tool to analyse the presence or absence of a gene/sequence of bases in the genome. Merit: explains how or why ONE of the two techniques are used e.g. WHY – DNA sequencing – by determining the exact sequence of the bases, scientists can identify the location of the genes and their known base sequence and use this information to screen for mutations and advantageous genes. HOW – DNA sequencing – mixture of target DNA, excess nucleotides, trace labelled nucleotide, DNA polymerase, fragments of assorted length synthesised based on incorporation of nucleotide analogues, fragments separated by electrophoresis, terminal nucleotide established through fluorescence e.g. WHY – DNA chips can be used to determine if a gene of interest is present or absent very quickly, by having the complementary sequence on the DNA chip and adding an individuals DNA (single-stranded). The fluorescent tag indicates to the scientists if the gene of interest is present or not. HOW – DNA chips – a single stranded DNA or mRNA is attached onto a grid that is divided up. The target of single-stranded DNA or mRNA, is then introduced to the grip, and once it locates a complementary sequence, base pairing occurs. A fluorescent tag lights-up/indicates when base-pairing has occurred. Excellence: discusses how AND why ONE of the two techniques is used. (c) Achieved: describes human need or demand. e.g. Sheep genome analysis enables scientists to identify sheep with the genes for greater productivity. Merit: explains human need or demand. e.g. Sheep genome analysis enables scientists to identify sheep with the genes for greater productivity. This information can then be used in breeding programmes to produce whole flocks that are carrying the genes for greater productivity. Excellence: discusses human need or demand e.g. Sheep genome analysis enables scientists to identify sheep with the genes for greater productivity. This information can then be used in breeding programmes to produce whole flocks that are carrying the genes for greater productivity. The identification of specific genes allows the improved features to be established much more quickly in a breeding programme than would be possible without the genome analysis information. (3) Transgenesis - Answers (a) Achieved: Transgenesis is when genetic material (DNA) is transferred from one organisms to another of a different species. It allows for direct modification of an organisms genome. (b) Achieved: describes how OR why the TWO techniques are used e.g. Restriction enzymes are used to cut the plasmid vector and to the cut the Chymosin gene. e.g. Ligase is used to join wanted chymosin gene to the plasmid vector. Merit: explains how or why ONE of the two techniques are used e.g. WHY – Restriction enzymes are used to cut the chymosin gene and the plasmid. The same restriction enzyme must be used to cut both the gene and the plasmid as this will produce the same sticky ends that can then be joined together by DNA ligase.. HOW – Use of specific restriction enzymes has enabled the cutting of DNA at a recognition site specific to that enzyme. For example the restriction enzyme EcoR1 from E coli has the recognition site GAATTC. It will produce a DNA fragment with the sticky end AATT at one end and TTAA at the other. During ligation of the DNA into the plasmid the AATT end will be joined to the TTAA end of the open plasmid. When both ends are joined in again a molecule of recombinant DNA is produced. e.g. WHY – DNA ligase joins together sticky ends that have been created through the use of restriction enzymes. HOW – Weak bonds between the wanted chymosin gene and the plasmid vector form when their sticky ends connect. DNA ligase moves along the two to make more permanent, stronger hydrogen bonds between the bases. Excellence: discusses how AND why ONE of the two techniques is used. (c) Achieved: describes human need or demand. e.g. Chymosin can be produced on demand in the quantities required. Merit: explains human need or demand. e.g. Chymosin can be produced on demand in the quantities required at any time of the year instead of having to wait until you have calves that are less than 10 days old. This means that it is more readily available throughout the year. Excellence: discusses human need or demand e.g. Chymosin can be produced on demand in the quantities required at any time of the year instead of having to wait until you have calves that are less than 10 days old. When compared to calves transgenic bacteria are easily housed and the culture easily maintained in vats that have the right conditions for bacterial growth and reproduction. All the materials the bacteria need to produce chymosin are supplied and the temperature can be held at the optimum level so that chymosin production is very efficient. (4) Gene Cloning - Answers (a) Achieved: Gene cloning is the process that allows us to obtain a gene, insert it into a suitable vector and then make multiple copies of the gene. (b) Achieved: describes how OR why the TWO techniques are used e.g. Restriction enzymes are used to cut the plasmid vector and to the cut the Insulin gene. e.g. Ligase is used to join wanted Insulin gene to the plasmid vector. Merit: explains how or why ONE of the two techniques are used e.g. WHY – Restriction enzymes are used to cut the insulin gene and the plasmid. The same restriction enzyme must be used to cut both the gene and the plasmid as this will produce the same sticky ends that can then be joined together by DNA ligase.. HOW – Use of specific restriction enzymes has enabled the cutting of DNA at a recognition site specific to that enzyme. For example the restriction enzyme EcoR1 from E coli has the recognition site GAATTC. It will produce a DNA fragment with the sticky end AATT at one end and TTAA at the other. During ligation of the DNA into the plasmid the AATT end will be joined to the TTAA end of the open plasmid. When both ends are joined in again a molecule of recombinant DNA is produced. e.g. WHY – DNA ligase joins together sticky ends that have been created through the use of restriction enzymes. HOW – Weak bonds between the wanted chymosin gene and the plasmid vector form when their sticky ends connect. DNA ligase moves along the two to make more permanent, stronger hydrogen bonds between the bases. Excellence: discusses how AND why ONE of the two techniques is used. (c) Achieved: describes human need or demand. e.g. Insulin can be produced on demand in the quantities and chemical structure required. Merit: explains human need or demand. e.g. Insulin can be produced in a way that has a chemical composition identical to human insulin and can also be produced on demand in the quantities required. Excellence: discusses human need or demand e.g. Insulin can be produced in a way that has a chemical composition identical to human insulin and can also be produced on demand in the quantities required. In comparison to insulin that used to be derived from bovine and porcine, the insulin produced through gene cloning will trigger the production of antibodies as it’s chemical composition will not be identified as foreign by the human immune system.