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BB3701 Genomic Medicine – SB leader: Dr Predrag Slijepcevic

BB3701 Genomic Medicine

BB3701 Genomic Medicine

1.  Learning outcomes assessed (from Assessment Block Outline)

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

2.  Description of assessment task ( for academic staff: please copy the coursework instruction here)

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. (Practical session 1)
  • To introduce you to practical skills required for setting up and performing FISH. (Practical session 2)

 

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.

LY-R Chromosome number Expected    number of telomeres Observed number of telomeres
Cell 1
Cell 2
LY-S
Cell 1
Cell 2
  1. 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

  1. 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.

  1. 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

A number of software packages exist on the internet that can be used to measure telomere fluorescence such as TFL Telo http://www.flintbox.com/public/project/502/ 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

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.

  1. 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

  1. 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

  1. 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

 

Explain your classification using logical arguments. Please use a separate4 page for this answer.

 

200 words of text allowed excluding legend for the graph

 

3.  Data set or relevant protocol for the practical related to the coursework (for academic staff please copy related information, eg practical protocol here)

(Below are details of protocol for the practical session, which the procedures will help you for some aspect of your coursework required) :

Practical schedule (AT practical)

You will receive a set of four microscope slides containing chromosome preparations from fibroblast cell lines established from two patients. One patient is a healthy volunteer (cell line 08399) and the other patient is an AT patient (cell line AT1). We exposed above cell lines to a chemical called Bleomycin (BLM). This is a chemical used in tumor therapy and it causes the same effects on cells as IR (this type of chemical is called radiomimetic). The concentration of BLM used was 4 mg/ml. Therefore, the set of four samples that you will receive include:

  • Untreated cells from a healthy patient
  • Cells from a healthy patient treated with 4 mg/ml BLM
  • Untreated cells from an AT patient
  • Cells from an AT patient treated with 4 mg/ml BLM

For your information cell lines have been prepared according to the following protocol:

 

  • Frozen cell samples from a healthy patient and an AT patient were taken from liquid nitrogen and transferred to cell culture flasks with growth medium called DMEM (Dulbecco’s Modified Eagle Medium) and 10% foetal calf serum. The content of each tube was transferred to a plastic flask and cells were allowed to grow at 37oC in an atmosphere of 10% CO2. Please note that fibroblasts attach to the bottom of the cellculture flask and the growth of cells can be monitored using contrast-phase microscopy.
  • Cells were allowed to reach confluence. The confluence is defined as the stage of cell growth when the cell density is maximal i.e. there is no space between individual cells. At this stage fibroblasts usually stop growing due to what is known as “contact inhibition”. Confluent cells were trypsinized to detach them from the bottom of cell culture flasks and cell suspension was transferred to two new
  • Cells were again allowed to grow and reach
  • One flask of fully confluent 08399 cells and one flask of fully confluent AT1 cells were exposed to 4 mg/ml BLM for 1 h. The other two flasks were not exposed to BLM and therefore served as negative
  • BLM was washed out from the cells and these were subcultured into a new flask at a ratio 1:2 and allowed to grow for 30 h. The same procedure was applied to untreated flasks.
  • A chemical called colchicine was added to each of the four flasks. This chemical arrests the cells in metaphase, a phase of mitosis in which chromosomes are most clearly
  • Following 6 h colchicines tretment cells were trypsinized to detach them from bottoms of cell culture dishes, centrifuged and treated with hypotonic solution for 15 min. Hypotonic solution causes swelling of the nucleus which will result in the release of
  • Cells were centrifuged, supernatant removed and cell suspension exposed to a fixative solution consisting of glacial acetic acid and methanol (ratio 1:3).
  • Fixation procedure was repeated 3 times and few drops of cell suspension were placed on to pre-cleaned microscope
  • Slides were allowed to dry and stained with 4% Giemsa for 10

 

Your task

You will receive four slides marked as:

  • Slide 1
  • Slide 2
  • Slide 3
  • Slide 4

Your task will be to determine identity of slides by quantifying CAs. Untreated cells from a healthy patient and untreated cells from an AT patient might have some spontaneous CAs but these are expected to be rare and perhaps slightly higher in AT cells. Cells from a healthy individual treated with BLM are expected to have some CAs. Cells from an AT patient treated with BLM are expected to have the highest number of CAs. Therefore, at the end of this exercise you should be able to classify your samples into at least three categories:

  • Category 1 – Samples with no CAs or extremely low number of
  • Category 2 – A sample with multiple CAs the frequency of which exceeds that of the Category 1

Category 3 – A sample with the highest number of CAs i.e. it outnumbers those in the category 2 sample.

 

Practical schedule (FISH practical)

In this practical you will perform FISH using a telomeric oligonucleotide as a probe, labelled with the fluorescence dye FITC which produces green fluorescence. This oligonucleotide is single stranded and there is no need to denature it. Also, this nucleotide is not a classical DNA sequence but rather a chemically modified form of DNA called peptide nucleic acid (PNA). The oligonucleotide has been synthesized by a specialized biotech company and directly labelled with the above fluorescence dye. The composition of the oligonucleotide is as follows:

 

CCTAAA CCTAAA CCCTAA –00- FITC or Cy3

 

In the above formula 0 represents a chemical linker and two linkers are required between a nucleotide and fluorescence label.

 

You will receive an aliquot of the PNA oligonucleotide (probe) dissolved in the appropriate buffer containing 70% formamide. Formamide is a highly alkaline reagent which is capable of denaturing DNA. The telomeric PNA probe is prepared as follows:

 

700 ml of deionized 100% formamide 5 ml of blocking reagents

10 ml of 1 M TRIS ph 7.2 50 ml of MgCl2 buffer

83 ml of PNA probe solution (concentration 6 ml/ml) 152 ml H20

————–

1,000 ml total volume

 

An amount of 8 ml / microscope slide is sufficient for hybridization and you will receive this amount/slide in a single plastic tube.

 

You will also receive a set of two microscope slides containing chromosome spreads from two mouse cell lines, LY-R and LY-S. Chromosome spreads were prepared in a conventional way (growing cells arrested in metaphase by colchicine, hypotonic treatment performed to release chromosomes and chromosomes fixed chemically and spread on slides). One cell line has long telomeres and the other cell line has short telomeres. You will receive pre-washed chromosome preparations so that you can use them directly for hybridization. For your information chromosome preparations on slides have been washed using following reagents:

 

  • PBS (phosphate buffer saline)
  • A buffer containing the enzyme pepsine, which will remove proteins from chromosomes that interfere with in situ hybridization
  • A series of different concentrations of ethanol (70%, 90% and 100%), which will dehydrate the material on the microscope

 

Protocol for FISH Step 1

Put gloves on. Prepare the box and wrap coupling jar with foil. Measure 8 ml of telomeric PNA probe and transfer this amount between pencil marks on side of each slide. Please note that telomeric PNA probe is light-sensitive, please keep the alu foil on the tube all the time.

 

Step 2

Cover the drop with a coverslip by simply placing the coverslip on top of the drop located between pencil marks.

 

Step 3

Carry slides using moist box. Place microscope slides on a heating block, which has been preheated to 70oC. Combination of 70% formamide in the probe solution and heat will denature DNA in chromosomes. Leave the slides for 2 minutes at the heating block. Remove the slides from heating block.

 

Step 4

Place the slides in humidified plastic boxes and incubate them 45 min at room temperature. This will allow hybridization of telomeric PNA oligonucleotide to mouse chromosomes.

 

Step 5

Remove coverslips from slides (in fume cupboard) by dipping slides in formamide solution and gently tapping them. Discard coverslips in sharps container. Wash 1 x 10 minutes in 70% formamide solution in fume cupboard.

 

 

Step 6

Collect the formamide wash in fume cupboard (ask technician/demonstrator for help). Wash slides in PBS 3 X 2 minutes. Drain slides, dip them in jars with 70% ethanol, followed by 90% and 100% ethanol. Let slides dry.

 

Step 7

Demonstrator will add 8 ml DAPI to each slide (this will stain DNA in blue). Cover with a new coverslip.

 

Step 8

Go to fluorescence microscope room and acquire images of metaphase cells using instruction given by Dr. Slijepcevic. Save images as TIFF files on your memory sticks. (Please bring your own memory sticks).

4.  Student support

Academic Support and ASK

 

You are supported to be successful in this assessment by various formative activities you undertake in the practical sessions of your study block so attention to and engagement in these will be important for you to do well in this assessment. Pay attention and engage with the lecture material presented in your study block as this will provide you with the background information to the case study. However, it is important that you manage your time and studies in order to do your best work in this assessment.

 

The academic skills (ASK) services can help you improve your writing and presentation skills. If you think you need help, don’t hesitate to ask them! They also have a large number of really good online resources, including video tutorials, useful resources on the internet, quick guides and slides from their workshops. You can find it all here: http://www.brunel.ac.uk/study/beec/academic- skills/Academic-Skills

 

Writing Fellows are professional writers who offer individual appointments to improve the writing of Brunel students. These appointments are free, independent and confidential sessions where they can advise you about your literature review. Karin Altenberg, Julian Birkett and John Harrison are based in Room 104 Heinz-Wolff, Monday to Friday during term time from 10am to 4pm.

 

Student Support and Welfare Team

 

The Student Support and Welfare Team are available to offer support and guidance on a range of personal, welfare and financial issues. They are here to help you access support that is right for you; you can make appointments through this team to access the Counselling and Mental Wellbeing Service, Disability and Dyslexia Service and budgeting/money advice sessions.

5.  Grading of the assessment

The assignment will be marked with reference to the guidance below, which describes our expectations in relation to the assignment. A single mark will be derived from a holistic judgement about the assignment. For more information, please read Brunel University Undergraduate Grade Descriptors (http://www.brunel.ac.uk/about/quality-assurance/documents/pdf/grade-descriptors- undergraduate.pdf#search=grade%20descriptors).

6.  Additional Information

None.

 

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