Genetics and Molecular Pathology

Keep the below table and add the requested information if you have attended the lab session. Only add your P Number if you did not attend any lab session. Your group number Your P number Lab partner P number Lab partner P number Lab partner P number Lab partner P number NOTE: The use of any artificial intelligence (AI) tool is NOT permitted for this assignment. Using the output produced by AI tools as if it was your own would be in direct contravention of the DMU Academic Integrity and Misconduct Policy and would result in a referral to the Academic Practice Office. This includes, but is not limited to:  - generative AI such as ChatGPT - paraphrasing software, such as Quillbot, Grammarly. As part of your coursework work answer all the following 6 questions: Question 1: Materials and Methods Section (10 marks): Write a structured and concise "Materials and Methods" section suitable for a research journal, based on the laboratory work you conducted. Clearly describe the procedures, materials, equipment, and reagents used, ensuring sufficient detail for others to replicate the experiment. Where appropriate, use tables to organize data for clarity and readability. Answer Question 1: Materials and Methods Materials and Reagents * MyTaq™ Red Mix (Bioline): contains buffer, MgCl₂, MyTaq DNA Polymerase, dNTPs, and red dye, used as a PCR reaction mixture. * Allele-Specific Primers: Forward primer (10 µM) and reverse primer (10 µM) specific to the SNP region of interest. * Template cDNA: Samples from patients (P1, P2, P3, P4) and control samples (positive control, negative control, and blank control). * Distilled Water (DW): Used to adjust reaction volumes and serve as a blank control. * Agarose (2% concentration for gel preparation). * SYBRsafe: DNA stain for visualization under UV light. * EasyLadder I (Bioline): DNA ladder for size reference during gel electrophoresis. Equipment * Micropipettes and sterile pipette tips: For precise measurement and transfer of reagents. * 0.2 ml PCR Tubes: For PCR reactions. * Thermocycler: For PCR amplification, set to perform denaturation, annealing, and extension steps. * Gel Electrophoresis Apparatus: For separation and visualization of PCR products. * UV Transilluminator: For gel visualization after electrophoresis. * Protective Equipment: UV-protective eyewear, gloves, and lab coats. Methods 1 PCR Master Mix Preparation The PCR master mix was prepared by combining reagents to achieve the final concentrations specified in Table 1. For each reaction, 12.5 µl of MyTaq™ Red Mix, 0.5 µl of forward primer (0.2 µM final concentration), 0.5 µl of reverse primer (0.2 µM final concentration), and distilled water (DW) were added to a total reaction volume of 25 µl. A total of 192 µl of master mix was prepared to accommodate eight reactions. Table 1: PCR Master Mix Composition Reagent Starting Concentration Final Concentration Quantity (µl) for 1 Reaction Quantity (µl) for 8 Reactions MyTaq™ Red Mix (Bioline) 2X 1X 12.5 µl 100 µl Forward Primer 10 µM 0.2 µM 0.5 µl 4 µl Reverse Primer 10 µM 0.2 µM 0.5 µl 4 µl Distilled Water (DW) - - Adjusted to 25 µl total 84 µl 2 PCR Amplification Each PCR tube received 24 µl of the master mix and 1 µl of cDNA or control sample, including positive control (PC), negative control (NC), and blank control (BC) using DW. The tubes were placed in a thermocycler with the following settings: * Denaturation: 94°C for 15 seconds * Annealing: 62°C for 15 seconds * Extension: 72°C for 10 seconds * Cycles: Repeated 34 times 3 Gel Preparation and Electrophoresis A 2% agarose gel was prepared by dissolving 2g of agarose in 100 ml of 1X TBE buffer. The mixture was heated, and 3 µl of SYBRsafe was added for DNA visualization. The gel was cast in a gel tray and allowed to set. After setting, 10 µl of each PCR product and 3 µl of EasyLadder I were loaded into respective wells. The gel was run at 110V for 20 minutes in 1X TBE buffer. 4. Gel Visualization After electrophoresis, the gel was placed on a UV transilluminator to capture images of the. The order of samples was as follows: * Lanes 8-14: Patient and control samples processed with allele-specific primers, with lanes labeled according to Table 2. Table 2: Gel Sample Loading Order Lane Sample ID 8 Patient 1 (P1) 9 Patient 3 (P3) 10 Patient 2 (P2) 11 Patient 4 (P4) 12 Positive Control 13 Negative Control 14 Blank Control L EasyLadder I (size marker) All procedures were conducted following appropriate safety protocols to prevent contamination and ensure accuracy. Question 2: Results Section (20 marks): Develop a detailed "Results" section that refers to Figure 1 (in handbook). Ensure that the figure is properly labelled and includes a comprehensive figure caption. Report your findings clearly and factually, without interpretation or speculation. Answer Question 2: PCR products were amplified by DNA samples from four patients (P1, P2, P3, and P4), and control samples were separated by gel electrophoresis and are presented in Figure 1. The DNA ladder is the leftmost lane, showing a series of bright bands, which will be used to estimate the size of PCR products in the subsequent lanes. Each patient and control sample was loaded in two different gel wells and could easily be compared. A few lanes show weak bands near the wells, which can be considered primer dimers and represent small non-specific products of primer self-annealing. The MT CRC patient lanes exhibit no discernible target DNA product; no apparent bands of DNA exist in the regions that should have been amplified SNP within the genome. The first perturbation observed resulted from positive and negative controls for the second primer set being in the same tube. This control lane is shown in the figure but is Observation: not easily discernable as having bands present for a positive or negative result. The blank control (BC) represents the total lane with no visible bands. Question 3: Discussion (25 marks): (Use at least 3 references with in-text citations, following DMU Harvard Cite Them Right style. Ensure originality to avoid plagiarism.) A. Explain the fundamental principles of the methodology used in the practical session and compare these with alternative PCR techniques that could have been applied. B. Identify and describe a post-PCR technique that can be used to detect the presence of Single Nucleotide Polymorphisms (SNPs). C. Discuss and interpret the results shown in Figure 1 (in handbook). In your discussion, address the following: ...