PCR (polymerase chain reaction) and agarose gel electrophoresis Overview of epidemiology of specified common pathologies Cellular changes in diseases – cellular responses to injury, acute inflammation, healing and repair, chronic inflammation, infections of histological importance, arteriosclerosis, thrombosis and embolism and infarction. Histopathology- pathogenesis and clinical presentations of common disease of major organs, i.e. heart, lung, liver, kidney, gastrointestinal tract, brain and muscles, cellular changes in disease including aplasia, amyloidosis, dysplasia, hyperplasia, hypertrophy, metaplasia and neoplasia including haematological malignancies. Cytopathology- pathogenesis and clinical presentation of cervical cancer. Techniques used for screening cervical neoplasia. Test for non-cervical gynaecological malignancy. Pathogenesis and clinical presentations and cytopathological diagnosis of cancer of respiratory tract, urinary tract and serous cavities. Sampling and preparation techniques used in non- gynaecological cytology. Particular emphasis will be placed on the use of a range of molecular pathology tests in assisting with diagnosis and prognosis of neoplastic and non-neoplastic diseases. Principles of immunohistochemistry,, including immunohistochemical methods used in identifying tumour- associated antigens. Learning Outcomes On completion of the module, the successful student will be able to: 13 1. Discuss at an advanced level pathogenesis of neoplastic and non-neoplastic disorders. 2. Critically evaluate practices and techniques and review the current frontiers of cellular and molecular pathology research. 3. Critically evaluate the value of histopathology and cytopathology in the diagnosis and treatment of diseases. 4. Demonstrate good laboratory practice related to cellular and molecular pathology procedures and techniques. Recommended books Cook, D.J. (2006) Cellular Pathology. Scion Publishing Ltd William B Coleman & Gregory J Tsongalis. Molecular pathology (2009) 1st Edition, Elsevier Inc – Recommended Bancroft JD, Gamble M (2002) Theory and Practice of Histological Techniques. Churchill Livingstone. Kierszenbaum A (2007) Histology and Cell Biology. An Introduction to Pathology (2nd Edition). London, Mosby. McKinnell, R et al (2006). The biological basis of cancer (2nd edition). Cambridge University Press.Shambayati, B. (2011) Cytopathology (Biomedical science). Oxford University Press. Stevens A, Lowe J,(2005) Human histology (3rd edition)> London, Elsevier Mosby Young B et al (2006) Wheater’s functional histology (5th edition). London, Churchill Livingstone. Journal references Nature, Nature Medicine, Science, Journal of pathology, Journal of clinical pathology, Journal of Cellular pathology, Histopathology, Cytopathology, Oncology research, International Journal of cancer. Practical 1: Polymerase Chain Reaction (PCR) to amplify HPV E6 genes of Hela cells following arsenic trioxide treatment As the most common technique for molecular biology, Polymerase Chain Reaction (PCR) has been widely used for medical diagnostics where it helps to detect the presence and the changes of DNA levels from various patientsâ€™ samples. PCR has many applications in diagnostics, forensics, criminology and biotechnology therefore it is very important to understand principles of this technique and to further use this technique for different applications. Aim of this session: This practical is based on the laboratory skills which you have developed from BMS2225 Molecular genetics module. You have confirmed that Hela cells contain HPV 18 E6 DNA sequences. In this practical, you will be given extracted DNA samples from both normal Hela cells (untreated ones) and those which have been treated by a drug called arsenic trioxide (ATO). ATO is a drug which has been used to treat acute pro-myelocytic leukemia and now it is under investigation to see whether it can also be used to treat solid cancers. In this practical, we have treated Hela cells for two days using 2uM and 5uM ATO and DNA samples were extracted from both untreated Hela cells and treated Hela cells. The concentrations of extracted DNA samples have been tested. The task for you today is to help a research student, Jill, to test the extracted DNA samples to see whether ATO can affect HPV 18 E6 DNA levels of treated Hela cells. Jill has prepared the following PCR reaction reagents and she has also extracted the DNAs which were labeled as H-C (without treatment), H-T1 (2uM ATO treated) and H-T2 (5uM ATO treated). â€¢ ï€ Enzyme: Taq DNA polymeraseï€ â€¢ ï€ dNTPs (mix of four deoxy-nucleotides dATP, dTTP, dGTP, dCTP)ï€ â€¢ ï€ buffer with MgCl2+ï€ â€¢ ï€ nuclease free waterï€ â€¢ ï€ forward primer (F) binding to E6 of HPV18ï€ â€¢ ï€ reverse primer (R) binding to specific sequence of a target gene/s which is E6 ofï€ HPV18Please do discuss within your group, If you are happy and you think that you have all the reagents/samples you need, please go ahead to prepare your samples. It is very important to make sure that you have labeled your samples and your group ID in order to identify your PCR product when they are ready for next lab session! So, now you need to help Jill to set up the PCR reagents and labels the tubes! If you have spotted any error for the experiment design, please do come to speak to one of us before you proceed with the work! We will see if we can help you. When your samples are prepared and labeled, you can load them for PCR reaction using the thermocycler prepared for you! In the next lab session, you will conduct gel electrophoresis to find out whether the drug has changed the levels of DNA! Jill pipetting several PCR reactions. She decided to start from here: 1. Label the tops/sides of seven small PCR tubes with numbers 1 â€“ x and last tube as NTC (Non Template Control). are these reactions? Please read before pipetting. PCR reaction 1: Hela control (H-C) DNA sample extracted from HeLa cells used as a template in PCR reactions with HPV18 primers/E6 gene PCR reaction 2: DNA sample extracted from ATO 2uM treated HeLa cells (H-T1) used as a template in PCR reactions with HPV18 primers/E6 gene PCR reaction 3: DNA sample extracted from ATO 5uM treated HeLa cells (HT2) used as a template in PCR reactions with HPV18 primers/E6 gene Last PCR reaction is called NTC: this is the control with no DNA template in the PCR mix but with HPV18 primers/E6 gene. If you are still not sure what you should do to get it started, please see the table below (Jill has prepared) for guidance! Have Jill done a good job? Are there any mistakes or anything missing from her preparation? Jill has pipetted different reagents required for the PCR reaction into the four different tubes as indicated in Table 1. Follow the order of the table from top to bottom and take utmost care not to cross contaminate reagents or samples and to include the correct amount of all reage Table 1: Components of PCR reaction mixturesClose PCR tubes and place them into the thermocycler (PCR machine) or keep on ice if you need to wait for others before loading the thermocycler. 3. Jill took 2.0 ÂµL of â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.DNA, this amount corresponds to â€¦â€¦â€¦â€¦â€¦..ng. 4. Check your PCR programme on the machine and write down the complete settings: The thermocycler has been programmed as below The PCR will take approximately 2 h to finish. We will collect your tubes and store them frozen at -20 Â°C. In next practical (4) you will analyse your PCR products by gel electrophoresis. Reagent: 1 2 3 4 5 6 NTC Nuclease free water 8.5 ÂµL 8.5 ÂµL 8.5 ÂµL 8.5 ÂµL 8.5 ÂµL 8.5 ÂµL (H20) 8. 8.5 ÂµL 2Ã— PCRBIO Taq Mix 12.5 ÂµL 12.5 ÂµL 12.5 ÂµL 12.5 ÂµL 12.5 ÂµL Red (2x) 12.5 ÂµL 10ÂµM primer F and R mixed 2.0 ÂµL 2.0 ÂµL 2.0 ÂµL – – – 2.0 ÂµL E6/HPV18 (H) 10ÂµM primer F and R mixed – – – 2 2 2 – gene = â€¦â€¦â€¦.? Nuclease free water – – – – 2.0 ÂµL (H20) – – – Control untreated DNA – Hela (250ng) (H-C) 2.0 ÂµL – 2 – – 2uM ATO treated DNA – (H-T1) (250 ng) – 2.0 ÂµL – – – – 5uM ATO treated DNA (H-T2) (250 ng) – – – – 2 – 2 ÂµL Total volume per each reaction is 25.0 ÂµL Name of step Temperature Number of Time (how long per times/cycles each cycle) Initial denaturation 95 c I cycle 1.00 Denaturation 95 c Stage 40 0.0015 Primer annealing 60 c 0.0015 Extension/elongation 72 c 00.30 Final extension 4 c Stop/cooling 0Before leaving the lab make sure that you have: – written down your PCR programme and calculated the amount of DNA used in a reaction, – taken note of your group number so that you can identify your PCR samples for your next practical – and tidied up your bench! Questions: 1. Please state what are the essential components within a PCR reaction mix? What are the purposes of these components in the reaction? 2. What is the mistake made by Jill in designing the experiment?? What consequences will have if she proceeds with the original design? 3. Research the genome structure of HPV. How many subtypes of HPV have been identified so far? Which subtypes of HPV are associated with the development of cancers? Practical 2 :Agarose gel electrophoresis on examining changes of Hela HPV E6 DNA levels following arsenic trioxide treatment Introduction: This laboratory is a continuous study based on the DNA samples you have amplified from the last lab session using polymerase chain reaction (PCR). From the last lab session, you have amplified DNA samples which have been extracted from HeLa cell lines after the cells have been treated with arsenic trioxide (ATO), a potential anti-cancer drug. You were looking for the presence of HPV oncogene E6 and any changes of this virus after ATO treatment through detection of the HPV oncogene E6 by PCR. As many of you have spotted, Jill has forgotten to amplify a housekeeping gene, GAPDH, therefore, you have added three extra DNA samples in the experiment by using a pair of primers which are specific for GAPDH. Today you will use gel electrophoresis to separate and visualize your PCR products from the last lab session. Jill made a fundamental mistake by forgetting to include a housekeeping gene in the experiment which will devalue her results dramatically as you are not sure the same amount of DNA were loaded in order to compare the effects of ATO on HPV E6 oncogene levels (upregulating, downregulating or no effect)! By examining the amplified DNA samples on the gel you should be able to visualise the products (bands) of housekeeping gene and HPV E6 oncogene. You need also include a DNA ladder to estimate their sizes of the DNA samples you amplified by PCR. You should be able to compare the DNA levels among three samples from untreated and two treated HeLa cells and to evaluate whether ATO treatment have any effect on HPV E6 oncogenes. Learning objectives for this session: 1. To further develop skills of gel electrophoresis2. To detect the presence of HPV E6 DNA from samples after PCR amplification from the last lab practical. 3. To examine and to compare the levels of HPV E6 DNA product before and after arsenic trioxide treatment 4. To discuss the benefits and limitations of agarose gel electrophoresis and suggest other alternative methods Laboratory procedures: Part 1: Collect your PCR tubes. Please join in the same group which you have been in the last lab practical (PCR lab) and make sure that you identify the correct check group number and take the samples of your own group NOT another group! H-C H-T1 H-T2 H-C-G H-T1-G H-T2-G NTPYou are also provided with the following: â€¢ Tube labelled â€˜Lâ€™â€“ contains DNA ladder (PCRBIO Ladder IV) in loading buffer (ready to use)ï€ Think how many samples will you load in the wells? Part 2: Preparing the agarose gel. The gels have been poured and left set for you due to the time restriction. You have learnt this from your last year lab session but we will demonstrate the process again. 1% agarose gel will be used for a rapid separation of small DNA fragments (consider the association between the percentage of gel and the size of the DNA you are testing). SafeView reagent which is a safe nucleic acid stain for the detection and visualization of double-stranded DNA has been added in the agarose gel. SafeView emits green fluorescence when it binds to dsDNA (or ssDNA and RNA) while exposed to UV light (ultraviolet transilluminator), or blue light (a dark reader transilluminator). Each group will have one gel and one tank to use and each gel contains 8 sample wells. 1. Check to make sure that the gel is set before you remove the comb from the gel. 2. Change the position of the gel with its tray from casting position to running position. Think about the correct orientation: which direction will DNA move in an electric field? 3. Pour the gel electrophoresis buffer (1x TAE buffer) into the tank ensuring that the gel is fully covered. Do not exceed the â€œMaxâ€ level indicated on the tank. Part 3: Loading the gel. 1. Check if the gel is covered by buffer and the sample wells are intact. Before loading the samples, make sure that the tank is close enough to the power pack to allow you to connect the cables. Do not move the tank around once samples have been loaded (Why?) 2. All PCR samples are ready to load; they include a red dye for tracking during agarose electrophoresis which also helps to load samples into the wells (due to the glycerol that the dye contains, which pulls the samples down into the wells) 3. Load your samples one by one into eight sample wells, please do not forget to include DNA ladder! Please keep a good record of the order for loaded samples: L (ladder) â€“ sample 1 â€“ sample 2 â€“ sample 3 â€“ sample 4 â€“ sample 5 â€“ sample 6 â€“ sample 7 L H-C H-T1 H-T2 H-C-G H-T1-G H-T2-G NTC Load 5 ÂµL of the ladder and 10 ÂµL of your PCR reactions. Pipetting the samples into the wells is a tricky business. Avoid damaging the wells with the pipette tip and try not to spill the sample out of the well by pipetting it too quickly orby producing air bubbles. Your tutors will demonstrate this and help you and a practice gel will be available. 4. Place the lid onto the tank and connect the power cables to the power supply. 5. A member of staff will check the power unit is set up correctly, set the unit to 90 V and switch the power on. Take note of the settings and start time. This should take at 30 mins. Part 4: Analysis. 1. The red dye in the PCR mix and in the ladder will form bands moving towards the anode visible to the eye, which is a useful indication of the process. Your tutors will advise you how far these bands should progress for the electrophoresis to be completed. 2. Switch off the power pack when instructed to do so, disconnect the power cables from the power pack and only then carefully remove the lid from the tank. 3. Remove the tray with the gel from the electrophoresis tank and place it onto a piece of tissue paper on your bench. The gels have been pre-mixed with a fluorescent dye as mentioned in the part 2 introduction that binds to DNA and is visible under UV light (SafeView). Make sure that you have gloves while touching the gel and transporting to the UV machine. 4. Proceed to the UV/Dark Reader transilluminator and carefully slide your gel from the tray onto the transilluminator. 5. Only switch the UV/Blue light on whilst the lid of the transilluminator is closed, do not look into the UV/Blue light without eye protection and do not expose your skin to UV/Blue light. Take a photo of the gel using your mobile phone and this will be used for your summative report (please make sure that you remove your gloves before you take images!) 6. Turn the transilluminator off, remove your gel and take it back to your bench or dispose of it as instructed. Wipe the surface of the transilluminator with tissue paper. 7. Before leaving the class draw a schematic of your gel recording all bands (use the template below). Estimate the size of all bands (in bp) visible in the lanes containing your PCR samples using the ladder. Check to see whether the bands from all samples migrated at a same distance? Do any bands shown have a difference in their intensities? If they do, what does this mean? Discuss the result within your group. Figure 1. The image below shows the size of the bands in the ladder we have used. You need use the ladder as a guidance to work out the size of PCR product of the samples tested.Figure title: _____________________________________________________________________ Figure 1: DNA ladder separated into 11 bands (100 â€“ 1500 bp) on a 1% agarose gel.Figure 1: shows HPV PCR for Model Result No. Sample 1 HPV18 â€“ HeLa Untreated 2 HPV18 â€“ HeLa ATO 2 ÂµM 3 HPV18 â€“ HeLa ATO 5 ÂµM 4 GAPDH â€“ HeLa Untreated 5 GAPDH â€“ HeLa ATO 2 ÂµM 6 GAPDH â€“ HeLa ATO 5 ÂµM 7 GAPDH â€“ NTC (H2O) 8 PCRBio Ladder IV HPV18: 419bp â†’ ïƒŸ GAPDH: 452bp 1 2 3 4 5 6 7 Ladderfigure 2: is my result, which shows me wrong result .so I need to compare it the Model Result which is above. Need to add figure legend. HPV PCR and DNA gel electrophoresis to investigate the presence of HPV oncogene E6. Ladder is PCRBIO DNA ladder, lane 1 is Lane 2 is lane 3 is lane 4 is lane 5 is lane 6 is lane 7 is. lane 8 is missing Ladder 1 2 3 4 5 6 7In this lab report need to talk about Practical Polymerase Chain Reaction (PCR) to amplify HPV E6 genes and Agarose gel electrophoresis on examining changes of Hela HPV E6 DNA levels following arsenic trioxide treatment of Hela cells following arsenic trioxide treatment. Guidance for writing a short communication report 1. Title: it should be concise and informative. 2. Introduction itâ€™s about PCR and agarose gel electrophoresis (200 words): This should â€œset the sceneâ€ for the report including: â€¢ relevant background information and concepts about the subject of interestï€ â€¢ focusing on the context of the study and why it is worthwhileï€ ï€ â€¢ introducing a background on techniques usedï€ â€¢ state the aim(s) of the experiment(s) â€“ these should be neutral and not ï€ pre-judge the results.ï€ â€¢ appropriate citationï€ ï€ ï€ 3. Methodology (150 words): briefly and precisely introduce the procedures. This section records what was done and should contain: â€¢ a logical description of the methods you usedï€ ï€ â€¢ an account of what was done and not a set of instructionsï€ â€¢ sufficient detail such that the experiment can be repeated without ï€ ambiguity by another person following your procedureï€ â€¢ copying the protocol from the lab handbook is not sufficient – in your ï€ experiment, things may have been changed from the original protocolï€ â€¢ methods must be written in the third person e.g.ï€ ï€ ï€ 4. Result (100 words): it should include the data and observations from the experiment together with a short descriptive text This includes: â€¢ an opening statement to lead in to the resultsï€ ï€ â€¢ results from your experimentï€ â€¢ a descriptive text highlighting main occurrences or trends observed ï€ without interpreting the resultsï€ ï€ â€¢ displaying the data using tables, graphs and diagrams as appropriateï€ â€¢ all tables/graphs/diagrams should be labelled, numbered and include a descriptive caption.ï€ ï€ ï€ 5. Discussion: (500 words): This section should include: â€¢ an interpretation of results: what do the results mean and how can they be explained? or in other words what does the data indicate and what can be ï€ deduced from the experimental results?ï€ â€¢ a consideration of your results in the context of the theory of the subject with reference to relevant literatureï€ â€¢ comments with explanation on whether the results obtained were as expected or not. If yes, what will be the next step forward? If not, what could ï€ be the possible reason(s)? What will you do next?ï€ â€¢ a discussion on any experimental errors, limitations and how the ï€ experiment could be improvedï€ â€¢ citation of relevant literature to support your arguments throughoutï€ ï€ ï€ 6. Conclusion (50 words) This should: â€¢ be one paragraphï€ â€¢ bring together main findings relating to the aims(s)ï€ ï€ ï€ 7. References: It should follow Harvard referencing style. The word limit is 1000+-10%. Title and subtitle, figure legends and references are not included in the word count.
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