5. A researcher is generating gene libraries for two cell populations (control a

Question

5. A researcher is generating gene libraries for two cell populations (control and treated) and looking for candidate genes which are only expressed in the treated population. (a) Which type of gene library should be constructed? (b) Which nucleic acid population should be extracted from the two cell populations? mar (1 mark) (c) Describe the advantage(s) and disadvantage(s) of using random hexamers for 1st strand cDNA (3 marks) synthesiS (d) Describe briefly how candidate genes can be identified from the two libraries by subtractive (5 marks) hybridization (e) Beside screening cDNA libraries, name two other methods which can be used to identify differentially (2 marks) expressed candidate genes in the two cell populations 6. The following question is about polymerase chain reaction (PCR) (a) Which factors can affect optimal annealing temperature in a PCR? (b) What are the advantages of using hot-start polymerase in PCR? (c) List six precautions to avoid contamination when setting up PCR. (d) Explain why amplification curve gradually plateau in a conventional PCR. (e) Distinguish between real-time PCR and conventional end-point PCR. (2 marks) (2 marks) (3 marks) (2 marks) (3 marks) 7. The following question is about gene expression regulation and genome engineering (a) Describe how gene expression is regulated at a post-transcriptional level. (3 marks) (b) Briefly explain how genetic changes outside protein coding region can result in alternation of gene(s) (3 marks) expression (c) Give and explain briefly two uses of transgenic animals (6 marks)

Explanation / Answer

5. (a) Since our main purpose is to compare the expression of certain genes in two cell populations, and are looking for candidate genes in the treated cell population, we will construct a cDNA library,

(b) The mRNA of the candidate genes should be extracted from the cell populations (as it contains only the expressed genes), which should then be converted back to a DNA template using of the enzyme reverse transcriptase, in order to form a cDNA.

(c) Advantages of using random hexamer primers for 1st strand cDNA synthesis -

Disadvantages of using random hexamer primers for 1st strand cDNA synthesis -

(d) Subtractive Hybridization refers to the technique of amplification of only those cDNA fragments that differ between the control and treated cell populations. This is done by the removal of cDNAs or gDNAs of similar abundance (by discarding dsDNA formed by the hybridization between the test and control samples). This method helps in retaining differentially expressed, or variable sequences/transcripts.

(e) Bioarrays and Southern Blotting (with streptavidin-biotin labelling) are two methods that can help identify differentially expressed candidate genes in the two population.

6. (a) Factors that affect optimum annealing temperature are -

(b) The advantages of using hot-start polymerase in PCR are -

(c) Six precautions to avoid contamination when setting up PCR are -

(d) When one of the PCR components becomes limiting, the amplification curve plateaus. This is the PCR end phase.

(e) Differences between real-time PCR and conventional end-point PCR are -

7. (a) Post-transcriptional regulation refers to regulation of gene expression at RNA level. After transcription, transcripts bind to RNA binding protein (RBP) for stability. With the help of the RNA recognition motif (RRM), RBP is key to events such as capping, splicing, RNA editing, addition of poly(A) tail, etc.

(b) Changes in non-coding regions (introns) can lead to change in gene expression in the following cases -

(c) Two uses of transgenic animals are -

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