BME356 Functional Genomics Tutor-Marked Assignment July 2025 Presentation, Singapore

TUTOR-MARKED ASSIGNMENT (TMA)

This assignment is worth 15% of the final mark for BME356 Functional Genomics.

The cut-off date for this assignment is 5^th October 2025, at 2355hrs.

Note to Students:

You are to include the following particulars in your submission: Course Code, Title of the TMA, SUSS PI No., Your Name, and Submission Date.

Question 1 – SU1: Molecular Biology and Gene Expression

(a) Propose a molecular mechanism that could explain how a single Gene X yields two protein isoforms in different tissues.

(5 marks)

(b) Develop an experiment to validate the mechanism you proposed in (a). Your answer should include the key experimental steps, appropriate controls, and an explanation of how the results would support or refute the hypothesis.

(10 marks)

Question 2 – SU2: DNA Sequencing Methods

(a) Compare Sanger sequencing and next-generation sequencing in the context of sequencing a 50 kb viral genome. In your comparison, address their throughput, cost, and practicality for this task. Conclude which method is more appropriate and justify your choice.

(5 marks)

(b) Assuming you proceed with the sequencing method selected in (a), outline the major steps in obtaining the complete genome sequence of the virus – from sample preparation to final genome assembly.

(7 marks)

(c) Once you have the assembled genome sequence, describe how you would identify and annotate genes in the viral genome and predict their possible functions.

(3 marks)

Question 3 – SU2: Gene Expression Analysis

(a) Examine the key factors and design criteria you would consider when designing a pair of primers for the qPCR assay targeting the immunoglobulin heavy chain gene. Your answer should address how these factors ensure the primer efficiency and specificity in qPCR.

(5 marks)

(b) Implement a qPCR experiment to compare gene expressions between Sample A and Sample B. Explain how you will ensure the results are quantitative and reliable (mention any controls or reference genes involved).

(5 marks)

(c) Suppose the qPCR results show that Sample A has a Ct (threshold cycle) value that is 3 cycles lower (earlier) than Sample B for the heavy chain gene. Interpret this result in terms of the relative expression levels between the samples, and describe ONE (1) additional experiment you could perform to validate this difference in expression.

(5 marks)

Question 4 – SU3: Protein Purification Strategy

(a) Illustrate a purification strategy to isolate the 40 kDa target enzyme from the E. coli crude extract. Your answer should include at least TWO (2) chromatographic techniques or separation steps, and explain the principle by which each step separates proteins.

(12 marks)

(b) After each major purification step, you run SDS-PAGE to analyze the collected fractions. Explain what information SDS-PAGE provides at each step, and how you would use this information to decide which fractions to pool for the next step.

(8 marks)

Question 5 – SU3: SDS-PAGE Data Analysis

(a) Propose TWO (2) possible explanations for the presence of an additional ~100 kDa band alongside the ~50 kDa band of your protein on the SDS-PAGE gel run under reducing conditions.

(4 marks)

(b) Illustrate how you would experimentally determine the identity or nature of the ~100 kDa band. Describe ONE (1) method and explain how it would help clarify what the band represents.

(4 marks)

(c) If the 100 kDa band is determined to be a form of your target protein, suggest ONE (1) adjustment to your purification or sample preparation protocol to eliminate or reduce this 100 kDa species in future preps.

(4 marks)

Question 6 – SU4: Protein Structure and Modeling

(a) Describe TWO (2) computational approaches you could use to predict the 3D structure of this enzyme. For each approach, state under what circumstances it is most applicable.

(8 marks)

(b) You have created a homology model of the enzyme. Assess the quality and reliability of your modeled enzyme structure. Describe at least TWO (2) methods or criteria you would use to evaluate the accuracy and validity of the model.

(6 marks)

(c) The Gly→Glu (G150E) mutation is found to abolish the enzyme’s activity. Using structural reasoning, propose possible explanations for how this mutation could disrupt enzyme function (assume Gly150 is in the enzyme’s core structure).

(6 marks)