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MIC11107 Research Skills - Molecular Analysis Assignment - Edinburgh Napier University, UK

Assessment - Report: Experimental Design report

Experimental design and sequence analysis report - You work at a biological testing facility and have been asked to look into the following three problems;

Part 1 -

A new compound SK234 has been identified and purified from a common soil-based fungus, which may have potential as a new anticancer drug to treat breast cancer. You have been tasked with investigating this problem and to design a series of experiments to test this theory.

a) Identify five appropriate scientific questions based on this information and design a suitable hypothesis for testing one of these questions.

b) Design an experimental strategy to test your hypothesis. Indicate sufficient detail so that this can be fully critiqued by your boss before you will be allowed to begin work in this area. You will need to identify suitable controls, any sources of potential experimental error, sample sizes, any potential confounding variables and how you plan to analyse and present the data at the end of this process. Your experimental strategy should be justified using appropriate literature.

Part 2 -

In your second case a whole family have been referred to you for immediate genetic analyses.

A male patient was on holiday with his family and he has now developed a fever and anaemia. A range of diagnostic genetic tests using a sample of his blood have been conducted using polymerase chain reaction (PCR) and DNA sequencing to determine the potential cause. All the tests come back as normal apart from one, in the battery of pathogen detection tests, an unusual result arose (see attached figure one).

Once the result for the tests were confirmed, the hospital immediately contacted the patients' family and called them in for testing.

a) Identify the sequence in Figure 1 by BLAST analysis. What infection has the patient contracted? Ensure that you demonstrate use of bioinformatics tools by including screen shots (with legends) of your analyses and an appropriate explanation of the data.

b) Identify the sequences in Figures 2-5 by BLAST analysis. Provide the following information: sequence name, gene name, database accession number, chromosomal location, size (bp) and exon count of the (full) gene. Ensure that you demonstrate use of bioinformatics tools by including screen shots of your analyses and appropriate explanations of the data.

Hint: You only need to provide an exemplar screen shot of one BLAST analysis, but all data should be discussed in the explanation.

c) As you are interested in the potential genetic differences so you further analyse the sequence data. Use an online alignment tool to align all the nucleotide sequences from Figures 2-4 alongside the database-derived sequence in figure 5. Provide a picture of your alignment, then identify and fully explain any differences between the sequences.

d) It is important to verify if any sequence abnormalities that occur at a nucleotide level will affect the peptide sequence. Use an online translation tool and translate all the nucleotide sequences from figures 2-5 into amino acid sequences. Then align amino acid sequences using an online alignment tool. Provide a picture of your alignment and identify and explain any differences between the sequences and explain how the function of the protein might be affected.

e) You are interested in the results you have obtained and need to prepare a report to explain to the family the results of the genetic tests and what this means for them. To develop this report, undertake a literature search to establish any links between the gene you have identified and the associated mutations, do the mutations you have identified have an impact, if so what might this be? Is there any scientific literature, which links the gene/protein and associated mutations with susceptibility to the infection? Investigate the evidence and illustrate concepts with figures where necessary. You should ensure that you reference your work appropriately using the school guidelines, APA 6th edition available on Moodle.

Part 3 -

Your supervisor has asked you to thoroughly analyse the following qRT-PCR data and present the findings to the rest of the group.

Table one. Data generated from qRT-PCR analysis of a selection of genes initially identified by next generation sequence analysis. The housekeeper value is the geometric mean of a panel of stable housekeeping genes, given in parentheses.

Gene of interest	Healthy subject	Disease patient
Gene 1	21.87 (10.47)	20.54 (10.27)
Gene 2	15.34 (10.31)	21.72 (10.86)
Gene 3	20.98 (10.21)	21.47 (11.01)
Gene 4	16.79 (10.58)	19.01 (10.89)
Gene 5	19.34 (10.08)	21.57 (11.3)

Calculate the relative levels of expression for each gene using the formula 2^-(ddCt), ensure you show all your calculations. In this case, the healthy patient should be used as the control expression level. Define the result for each gene as a percentage increase or decrease in the disease patient as compared to healthy subject. Explain the data fully.

Attachment:- Research Skills - Molecular Analysis Assignment File.rar