Independent University, Bangladesh

Department of Life Sciences

Course Title: Molecular Biology and Genetic Engineering
Course Code: MCB 306
Assignment 2
Spring 2021 Total Marks: 40

1. HER2 gene amplification has been recognized for many years as an indicator of prognosis in
early breast cancer. In about 25% of breast cancers, the HER2 gene does not work correctly and
makes too many copies of itself. In this study a tumor was removed from the breast of a patient.
Genomic DNA was isolated from the tumor and normal tissue and PCR reaction was performed
using identical amounts of the two DNA samples. Reaction mixtures contained the following
components.
• DNA template (the genomic DNA samples; 50 ng each)
• Primers specific for a region of the HER2 gene
• Many copies of a TaqMan (reporter) probe
• Taq polymerase and dNTPs

Reaction mixtures were incubated in a thermocycler capable of monitoring fluorescence. The figure
shows relative fluorescence values measured after each cycle using the breast tumor (A) and normal (B)
DNA sample.

Study the figure and answer the following questions with reasons:
(2x10=20)

Write –

“A” if i>ii
“B” if ii>i
“C” if i=ii
“D” if it is not possible to deduce
a. (i) The concentration of DNA in sample A at cycle=0
(ii) The concentration of DNA in sample B at cycle=0
Ans: …………………………………………………………………..

b. (i) The number of HER2 genes in sample A

(ii) The number of HER2 genes in sample B
Ans: …………………………………………………………………..

c. (i) The number of free primer molecules in sample A after cycle 4

(ii) The number of free primer molecules in sample A after cycle 10
Ans: ……………………………………………………………………..

d. (i) The number of free primer molecules in sample A after cycle 10

(ii) The number of free primer molecules in sample B after cycle 10
Ans: …………………………………………………………………….

e. (i) The number of free TaqMan probe molecules in sample A after cycle 10
(ii) The number of free TaqMan probe molecules in sample B after cycle 10
Ans: …………………………………………………………………………………

f. (i) The number of amplified HER2 fragments in sample A after cycle 20

(ii) The number of amplified HER2 fragments in sample B after cycle 20
Ans: ………………………………………………………………………………….

g. As PCR reactions proceed, at one point fluorescence increases in both mixtures. What
process can explain this?
(i) TaqMan probe molecules are degraded
(ii) TaqMan probe molecules are released from the template as intact oligonucleotides
(iii) TaqMan probe molecules serve as primers for Taq polymerase
(iv) TaqMan probe molecules are incorporated into the newly synthesized DNA strands

h. Why there is no detectable fluorescence in the samples after the first few PCR cycles?
(i) Because Taq polymerase degrades the TaqMan probe molecules
(ii) Because there is no DNA synthesis
(iii) Because the quenchers block the fluorescence of all reporter dye molecules
(iv) Because the fluorescence detector is not sensitive enough

i. Why fluorescence does not increase after cycle 30 in either samples?

(i) Because all primer molecules have been used
(ii) Because all TaqMan probe molecules have been used
(iii) Because all template molecules have been used
(iv) Both (i) and (ii)
 j. What additional step(s) we need to do to quantify the expression levels of the HER2 genes?
(i) Just add reverse transcriptase enzyme
(ii) Add a different probe
(iii) Isolate mRNA
(iv) Both (i) and (iii)

2. To answer the following question, you need a holistic understanding of the different aspects of
recombinant DNA technology that you have acquired from this course.

With the help of a flow-chat, outline the different steps that are involved in production of a
recombinant protein. Let us say we want to industrially produce mass quantities of human
growth hormone (HGH) using a bacterial vector system. Clearly outline the different steps
involved starting from isolation of the gene to production of the protein. (20 marks).

Assignment guidelines:
a) The marks are provided to give you an idea of how much of information is required for
each question.
b) You can refer to your notes and reading materials but please refrain from copy-pasting
texts. Use your own understanding and text to answer the questions.
c) Please submit your assignment using the designated folder in the google classroom
within the deadline period.