ECA on Molecular Biology: Laboratory Test and Analysis

Part I: Plasmid Extraction and Analysis

The aim of this ECA is to test conceptual understanding of both the fundamental and more advanced concepts in molecular biology along with your biomedical laboratory practical skills. You are expected to reflect, review and explore the publicly available information in the internet to complete this assignment. Throughout your answers, please ensure that you have hyperlinked all URLs.

This ECA is divided into TWO parts. Part I is a laboratory test, where you will extract DNA and obtain results that will also be used in Part II. Part II consists of questions that examine your analysis of the sample results obtained from laboratory methods taught in the course.

Part I (20 marks)

To be completed during the final week of your laboratory session.

Question 1

Objective: You are required to extract plasmid DNA from the provided culture and analyse it.

You are provided the necessary equipment and materials for a plasmid extraction on the table before you. Please ensure that you have the following:

1) 2 x 1.5 ml microfuge tubes

2) 1 x spin column

3) 1 x A1, A2, A3, W1, W2, and EB buffer on your bench

4) 1x bacterial culture

Pipettes and pipette tips

Centrifuge (to be shared)

Instructions:

You have 2 hours to complete the DNA extraction, run the gel (already cast for you), and report the amount of plasmid purified as well as the band size of the plasmid. Take a picture and identify the size of your plasmid. Include the OD readings.

(a) State the concentration of your miniprep. (3 marks)

(b) Record down the 260/280 ratio. Is it in an acceptable range? Give reasons why the extracted DNA might not fall within the acceptable range. (9 marks)

(c) Determine the total amount of DNA you have extracted (show your calculations). (3 marks)

(d) Assume that you are preparing for a sequencing reaction. You will need 1 µg of DNA in no more than 10 µl. Describe the steps you would take. Explain how you would get the final requested concentration and amount. (5 marks)

Part II (80 marks)

Question 2

Apply the mobile tools shown in the course to examine and analyse the gels and results obtained from Part I. Document each step with screenshots of the apps. You are strongly advised to read the user guides and view the relevant videos on how to use them. Note: This question should be attempted based on your results from the lab test above.

(a) Use GelApp (Android or iOS) to determine the sizes of the bands from your results. Show as your final picture the size of your band of interest. Make sure that screenshots of the steps including the final graph and result are included in your answers. (10 marks)

(b) Discuss if the band sizes were within your expectations and give reasons if they were not. (Maximum 200 words). (10 marks)

(c) Discuss the relevance of more precise and accurate detection of band sizes in gel electrophoresis, and how it can assist clinical diagnosis. (Maximum 200 words). (10 marks)

Question 3

Solve the identity and the functions of the ab1 file (to be provided closer to the ECA release). Document the steps used to identify the gene, the origin organism, and the function of the sequence. Provide screenshots to support your answers. (15 marks)

Question 4

Critically evaluate the laboratory steps to express the SARS-COV2 Spike protein. State the necessary resources and, include the steps from finding the gene of interest to laboratory purification of proteins. (15 marks)

Question 5

Evaluate in an essay not more than one page, how the research article ‘Comparison of customized spin-column and salt-precipitation finger-prick blood DNA extraction’ https://doi.org/10.1042/BSR20140105 can be used for rapid diagnostics of an inheritable genetic disease of your choice with details e.g. primer sequences. Briefly describe how you would do this based on the processes you have learnt from this module. Your essay will be assessed based on the following.

(a) Evidence of having read the paper and understanding. (5 marks)

(b) Detection method. (5 marks)

(c) Description of the molecular detection methods. (5 marks)

(d) Listing of resources. (2 marks)

(e) Selection of reasonable disease and target.