Problem Solving and Data Analysis Assignment - Genomic Medicine

Learning outcomes -

Knowledge -

1. Demonstrate in depth knowledge of subject learnt from the relevant study block.

2. Understand the complexity and interrelationships of different scientific disciplines.

Cognitive skills -

1. Demonstrate independent thinking, analytical and problem solving skills.

2. Effective use of literature to support explanations and interpretations.

Other skills -

1. Ability to reflect on learning.

2. Effective written communication, with appropriate referencing of the literature.

Description of assessment task -

Aims:

• To introduce you to practical skills required to diagnose Ataxia telangiectasia (AT) using a test that detects chromosomal sensitivity to chemicals that mimic ionizing radiation (IR) effects. IR sensitivity is a hallmark of AT.

 • To introduce you to practical skills required for setting up and performing FISH.

Objectives -

At the end of AT practical (Practical session 1) you should be able to:

• Recognize major chromosomal aberrations (CAs) induced by ionizing radiation in human cells.
• Quantify CAs in multiple samples.
• Understand the logic behind radiosensitivity of AT patients.

At the end of FISH practical (Practical session 2) you should be able to:

• Prepare a sample for FISH.
• Perform FISH on your own.
• Understand steps involved in FISH.
• Acquire digital images of cells and assess the quality of your FISH experiment.

Your report -

Please note the report combines results of the FISH practical (questions 1-5) and the AT practical (question 6).

During the FISH practical you will generate 2 images of LY-R and 2 images of LY-S mouse metaphase cells.

1. Your first task is to count chromosomes and telomeres in each image. You also need to calculate expected number of telomeres. Please fill in the table below after you finish counting. You are also required to provide images of your cells. Please print images on a separate page and submit them with your report.

2. Do the numbers of expected and observed telomeres match each other?

• Yes
• No

If the answer is no, please provide an explanation as to why this may be the case and indicate relevant cases by arrows in your images of cells provided on a separate page(s). 100 words allowed.

3. Which cell line (LY-R or LY-S) has stronger telomeric signal? Circle one

• LY-R
• LY-S

Could you express the difference in telomere signal intensity/size between the cell lines in quantitative terms? In other words can you assess how many times signal in one line is stronger/larger than the signal in the other cell line (i.e. 1.5 X, 2 X 3 X etc)?

Please answer the question in one of the two ways listed below.

a) Print large images of cells (A4 format) and cut out at least 10 chromosomes from each of 4 images so that telomeres remain intact. This will give you a total of 160 telomeres (one chromosome has 4 telomeres). Half of telomeres will be from one line and the other half from the other line. Calculate the area of each telomeric signal by measuring its diameter followed by calculating the total surface of the signal. Provide the evidence that you have done this kind of analysis by including cut-outs of telomeric signals, size comparison and potential calculation.

b) A number of software packages exist on the internet that can be used to measure telomere fluorescence such as TFL Telo. You can download this software and use instructions as to how to import your images.

Once you import the images of your four cells in to the software please use the software instructions to measure telomere fluorescence in at least 10 chromosomes / cell. This should give you a total of 160 telomeres, half of which will be from LY-R cells and the other half from LY-S cells. Provide evidence that you have done the analysis including appropriate images and tables with results from TFL Telo.

Presentation of results: Irrespective of which method you use you will have values of telomere fluorescence for exactly 160 telomeres. Present your results in tables or graphs containing:

• mean telomere fluorescence for each cell.
• mean telomere fluorescence for each cell line.
• standard deviation.

You should also carry out a statistical analysis to check if the values of mean telomere fluorescence differ between cell lines using the t-test. Provide evidence of t-test analysis.

Please provide the answer to this question on separate pages. Explain the principles of your measurements i.e. how the software method works, or how your manual method was conducted. Explain differences in telomere fluorescence intensity between cell lines taking account of statistical analysis. A total number of words allowed: 500. Figure legends, statistical information etc. do not count towards the word count.

4. In the case of FISH with the DNA molecule as a probe, hybridization is usually performed for at least 15-16 h. In your practical the time allowed for hybridization was 45 min only and the molecule used was PNA. Can you explain differences between DNA and PNA which are critical for in situ hybridization and contribute to hybridization efficiency? 100 words allowed.

5. If you have clones of human DNA sequences (genes) that you wish to use for FISH in the form of plasmid, cosmid, BAC and YAC clones, please explain which clones are the best for gene mapping purposes and why? Explain briefly advantages of using interphase rather then metaphase cells for gene mapping purposes. 100 words allowed.

6. Question from the AT practical

At the end of the AT practical session the identity of samples 1-4 were revealed. If your results are correct please present a graph containing your own results. If your results are not correct please use results that will be available on the BBL.

The graph should show mean numbers of Chromosomal Aberrations (CAs) per cell for each sample plus standard deviation. You should calculate whether the differences in mean numbers of CAs between samples are statistically significant using the t-test. Please provide evidence for your t-test results.

Please interpret your results in a way to explain the identity of slides 1-4, i.e. which number you think they represent:

• Untreated cells from a healthy patient.
• Cells from a healthy patient treated with BLM.
• Untreated cells from an AT patient.
• Cells from an AT patient treated with BLM.

Explain your classification using logical arguments. Please use a separate4 page for this answer. 200 words of text allowed excluding legend for the graph.

Attachment:- Assignment Files.rar