Al-Zaytoonah University of Jordan ‫جـامعـة الـزيتـونــــة األردنيــة‬

Exam Question Form - Teaching Follow-up procedures/ Faculty of Pharmacy QF02/2611-3.0E

Course Name Pharmaceutical Course Code 0201764

Biotechnology
Exam Date 22-12-2022 (First, Second, Final) Exam Midterm
Exam Time 4-6pm Exam Place 2204
Maximum mark 30 Exam duration 2 hrs.
Semester First Academic Year 2022-2023

To be filled by the Student

Student Name University ID
Instructor Name Section Number

Exam Instructions:-
1. Bring your University ID

2. Don't borrow things from others

3. Answer with (blue or black) pen

4. Mobile phones are forbidden

5. Cheating is forbidden

6. Don't enter the exam room after the pass of one fourth of exam duration

7. Don't leave the exam room before the pass of one fourth of exam duration

Question
Maximum mark Student mark
.No
1 5
2 5
3 5
4 5
5 5
6 5
Total 30

QF02/2611– page 1/14

 Al-Zaytoonah University of Jordan ‫جـامعـة الـزيتـونــــة األردنيــة‬

Exam Question Form - Teaching Follow-up procedures/ Faculty of Pharmacy QF02/2611-3.0E

:Dear student, please answer the following questions

First Question: Grade
A researcher running microarray experiment, has labeled the extracted RNA from a cancerous sample in
his study with a Cy3 dye and has labeled RNA from a paired normal sample with a Cy5 dye. His genes
of interest, labeled A through E gave the following results after scanning:

GENE ARRAY SPOT COLOR

A red
B green
C yellow
D green
E black

Assuming there was no experimental error, and these results are repeatable, what do they suggest about
expression of each of these genes in the two samples? What experiments are needed to follow to verify
the levels of expression of these genes.

Normal cell……...Cy5……red.
Cancer cell………Cy3……green.

 We saw that spot A is red, this indicates that the gene is more active
in the normal cell than in cancer cells.
 In spots B and D, we saw that are green, which means that the genes
are active in cancer cells more than in the normal cell.
 In spot C, we saw that the gene in normal and cancer cells is equal
(present in both cells).
 Spot E, is black which means the gene is not found in both cells (no
expression gene in both cells).

A gene's level of expression can be determined by RT- PCR.

As following:
the RNA sample is first converted into complementary DNA
(cDNA). with reverse transcriptase the complementary DNA
(cDNA), at least one set of targeted primers, a
deoxyribonucleotide, a suitable buffer solution, and DNA
polymerase are employed in the same way as in traditional PCR to
amplify amounts of DNA. (Master mix contains SYBR green or
TaqMan probes.
QF02/2611– page 2/14
 Al-Zaytoonah University of Jordan ‫جـامعـة الـزيتـونــــة األردنيــة‬

Exam Question Form - Teaching Follow-up procedures/ Faculty of Pharmacy QF02/2611-3.0E

Use thermocycler, The cycles typically consist of three stages: the

first (Denaturation) around 95°C allows for double-strand
separation of the DNA, the second (Annealing) around 50-60°C
allows for primer binding to a DNA template, and the third
(Extension) between 68-72°C allows for polymerization by DNA
polymerase.
The primers initiate the polymerization of a new strand of DNA,
and once the polymerase reaches the probe, it degrades the probe,
physically separating the fluorescent reporter from the Quencher,
and resulting in an increase in fluorescence.
This makes it possible to evaluate how quickly amplified products
are produced throughout each PCR cycle. The software can
evaluate the data to calculate the relative gene expression.
A positive reaction in a real-time PCR assay is detected by the
accumulation of a fluorescent signal. Ct levels are inversely
proportional to the target amount.
The lower the Ct level, the more target nucleic acid is present in
the sample. Real-time assays are amplification cycled 40 times.
Cts 29 are strong positive reactions that indicate a high
concentration of target nucleic acid in the sample.
Positive reactions with Cts of 30-37 indicate moderate amounts of
target nucleic acid.
Cts of 38-40 is weak reactions indicating low levels of target
nucleic acid, which could indicate an infection or environmental
contamination.
To confirm the RT- PCR result we use the housekeeping gene.

QF02/2611– page 3/14

 Al-Zaytoonah University of Jordan ‫جـامعـة الـزيتـونــــة األردنيــة‬

Exam Question Form - Teaching Follow-up procedures/ Faculty of Pharmacy QF02/2611-3.0E

Second Question: Grade

You were handed new novel hypoglycemic compounds. In your laboratory you have access to cell
culture facility, animal house and all molecular techniques and equipment.
Design experiments to determine their activity and their possible mechanism of action.

The drug can be tested in vitro and in vivo to determine its effects
and MOA. In-vitro studies can be carried out to investigate the
direct effects on cell proliferation and phenotype. In-vivo studies
can be conducted to determine the qualitative and quantitative
effects. As a result, it is important to select an appropriate animal

QF02/2611– page 4/14

 Al-Zaytoonah University of Jordan ‫جـامعـة الـزيتـونــــة األردنيــة‬

Exam Question Form - Teaching Follow-up procedures/ Faculty of Pharmacy QF02/2611-3.0E

for research.
In this experiment, first, the concentration of the drug must be
determined, (the concentration that kills diseased cells and does
not affect healthy cells), using the MTT test.

When determining the concentration of a new drug, we use an

animal model (Mice or rats).
We bring four groups of mice:
Group 1…... control
Group 2…... we give them new drugs.
Group 3……we give them Alloxan (induced hyperglycemia (.
Group 4……we give them (Alloxan + our drug).
Blood glucose concentrations are the most commonly measured
when testing therapies in animal models of diabetes. we take a
blood sample from each group and do a test. The result is:
Group 1…. …...normal blood sugar.
Group 2….……lower blood sugar.
Group 3 …....... higher blood sugar.
Group 4………??
To determine the drug's effect, we extract RNA from the muscle
cells of mice from all groups, convert it to cDNA, and then make an
RT-PCR with a primer specific to the Insulin gene, to determine
whether the gene has been down or up-regulated.

QF02/2611– page 5/14

 Al-Zaytoonah University of Jordan ‫جـامعـة الـزيتـونــــة األردنيــة‬

Exam Question Form - Teaching Follow-up procedures/ Faculty of Pharmacy QF02/2611-3.0E

Third Question: Grade

Techniques for Separation
Isoelectric Focusing in 2D-GE
• Protein separation based on isoelectric point (1st dimension) •
Protein migration through pH gradient until the overall charge is
neutral
• IEF strip soaked in the buffer to impart a large negative charge
to all proteins (for next step).
QF02/2611– page 6/14
 Al-Zaytoonah University of Jordan ‫جـامعـة الـزيتـونــــة األردنيــة‬

Exam Question Form - Teaching Follow-up procedures/ Faculty of Pharmacy QF02/2611-3.0E

• Proteins are then visualized by staining with dye, in the second

dimension (SDS-PAGE separation according to size).
Techniques for Separation Liquid Chromatography
Proteins washed through capillary columns • Separates
based on specific properties • Charge • Size • Hydrophobicity
• Depends on column matrix/eluent • Usually 2 (or more)
columns used (MDLC) (offline)
Separation Methods Mass spectrometry
An analytical technique for determining the mass: charge ratio
(m/z) of ions
• Mass spectrometers are made up of three parts:
• An ion source • A mass analyzer • A detector system •
In proteomics, only certain types of Mass Spec are used
• MALDI, SELDI, or Electrospray ion sources
• Time of Flight, Quadrupole, or Fourier Analysis • Mass
spectrometers that can mass spec whole proteins, but usually
only peptides
Protein Recognition
Mass Peptide Fingerprinting
Once the proteins of interest have been separated, create
proteolytic peptide fragments by digesting the protein into
peptides (trypsin cuts peptide bonds after lysine and arginine).
Peptide masses are determined using MS—the peptides are
"weighed" in a mass spectrometer.
Peptide masses are then compared to databases of protein or
nucleotide sequences.
Compare the data to known proteins to see if there is a match.
After that, we compare it with healthy cells, and we know
whether this protein is present or not.
We need to confirm by using western blot.
QF02/2611– page 7/14
 Al-Zaytoonah University of Jordan ‫جـامعـة الـزيتـونــــة األردنيــة‬

Exam Question Form - Teaching Follow-up procedures/ Faculty of Pharmacy QF02/2611-3.0E

 If there is a common protein in all patient samples and not

detected in normal people it could be a biomarker.
 Or if there is a common protein missing in the patient and
detected in normal also it could be a biomarker.

Fourth Question: Grade

In October 2022, European Medicines Agency (EMA) based on their Pharmacovigilance Risk
Assessment Committee (PRAC) issued new recommendations for the use of JAK inhibitors in many
inflammatory diseases. These recommendations were recently adapted by JFDA.
Discuss the mechanism of action of these inhibitors.
Discuss why cardiovascular side effects and cancer might develop in patients using JAK inhibitors.
Discuss what experiment you might design to verify the pathways of possible side effects development.

JAK inhibitors:
Competitively binds to JAK's adenosine triphosphate-binding
site and inhibits JAK enzyme activity, thereby suppressing
cytokine signal transduction and action.
The attachment of cytokines to these receptors is
prevented.
When cytokines lack a binding site, the immune system
produces less inflammation, reducing the severity of a
person's symptoms.

QF02/2611– page 8/14

 Al-Zaytoonah University of Jordan ‫جـامعـة الـزيتـونــــة األردنيــة‬

Exam Question Form - Teaching Follow-up procedures/ Faculty of Pharmacy QF02/2611-3.0E

We can search for a common pathway between JAK inhibiter

and the possible side effect (Cardiovascular or cancer).
From the literature (for example common pathway IL-6) to
study the JAK inhibitor in this pathway we can do:
Use 2 groups of animal models (mouse)by injecting animals
(group 1) with the JAK inhibitor drug after that inject
animals with IL-6(for example).
Group 2 (control).
We can do: -flow cytometry.
-Elisa.
-PCR.
To determine the expression level or gene level and protein
level.
If there is a defect in the expression of the gene, there will
be side effects.

QF02/2611– page 9/14

 Al-Zaytoonah University of Jordan ‫جـامعـة الـزيتـونــــة األردنيــة‬

Exam Question Form - Teaching Follow-up procedures/ Faculty of Pharmacy QF02/2611-3.0E

Fifth Question: Grade

From your understanding of epigenetics: A clinical trial is being conducted in the USA to compare the
epigenetic changes in patients diagnosed with COVID19 infections and those who received mRNA
vaccines in comparison to their baseline tests.
QF02/2611– page 10/14
 Al-Zaytoonah University of Jordan ‫جـامعـة الـزيتـونــــة األردنيــة‬

Exam Question Form - Teaching Follow-up procedures/ Faculty of Pharmacy QF02/2611-3.0E

a. Do you think modifications on the epigenome would be seen due to the infection or the vaccine?
Explain.

I think it is from infection because the vaccine does not enter

the nucleus of the cell where our DNA (genetic material) is
located, so it cannot change or influence our genes.

b. What best method of epigenome determination would be used from the different tools discussed
in the class? Explain

We bring two people infected with Coronavirus

I find one who took the vaccination and one who did not take
vaccinated, we take a blood sample from each one and then do a
DNA extraction after that we do sequencing to detect mutation
by the Next generation.
The Basics of NGS Chemistry
In principle, the concept behind NGS technology is similar to
sequencing.
Random fragmentation of the DNA or cDNA sample is followed
by 5′ and 3′ adapter ligation to create the sequencing library.
The templates are ready for sequencing once the cluster
generation is complete.
Illumina SBS technology detects single bases using a proprietary
reversible terminator-based method. Natural competition
reduces incorporation bias and raw error rates significantly
when compared to other technologies. As a result, base-by-base
sequencing is extremely accurate, virtually eliminating context-
specific errors.

QF02/2611– page 11/14

 Al-Zaytoonah University of Jordan ‫جـامعـة الـزيتـونــــة األردنيــة‬

Exam Question Form - Teaching Follow-up procedures/ Faculty of Pharmacy QF02/2611-3.0E

Sixth Question: Grade

β-thalassemias are group of disorders characterized by the lack of expression of functional β-
hemoglobin chains. Although most of these disorders are due to mutations or deletions in the β-globin
gene, some are not. You were asked to analyze DNA from a sample of a β-thalassemic patient and you
find that the β-globin gene is intact with no mutations. Where else would you look for an alteration?
Discuss how you can apply proper molecular tools to validate your answer.

Epigenetics is the study of gene expression regulation that

occurs independently of changes in DNA sequence.
QF02/2611– page 12/14
 Al-Zaytoonah University of Jordan ‫جـامعـة الـزيتـونــــة األردنيــة‬

Exam Question Form - Teaching Follow-up procedures/ Faculty of Pharmacy QF02/2611-3.0E

The majority of changes are reversible and are caused by

chemical modifications such as DNA methylation/demethylation,
histone tail modifications (e.g., acetylation, deacetylation,
phosphorylation, methylation, ubiquitination), chromatin
remodeling, and noncoding RNA regulation.
mediated by large complexes made up of transcription factors
and their associated cofactors (coactivators and corepressors),
which bind to cis elements in DNA such as promoters and
enhancers.
the molecular mechanisms underlying the quantitative reduction
in-globin production.
Nearly 300 B-thalassemia alleles have now been identified.
In contrast to the majority of B-thalassemic mutations, which
are caused by deletions in the gene cluster, the vast majority
of B-thalassemic mutations are caused by mutations involving
one (or a limited number of nucleotides) within the gene or its
immediate flanking regions.
They include single base substitutions, small insertions, or
deletions within the gene or its immediate flanking sequences,
and they affect nearly every stage of gene expression known.
Except for the -101 C!T mutation, which has been observed
fairly frequently in the Mediterranean region and interacts with
a variety of more severe B-thalassemia mutations to produce
milder forms of B-thalassemia, "silent" B-thalassemia al-levels
are uncommon.

hemoglobin electrophoresis is a type of blood test that detects

various types of hemoglobin. The test can detect hemoglobin,
which is linked to sickle cell disease, as well as other abnormal
QF02/2611– page 13/14
 Al-Zaytoonah University of Jordan ‫جـامعـة الـزيتـونــــة األردنيــة‬

Exam Question Form - Teaching Follow-up procedures/ Faculty of Pharmacy QF02/2611-3.0E

hemoglobin types, such as hemoglobin C. It can also be used to

look into thalassemia, which is a blood disorder caused by
faulty hemoglobin production.

Wish you all the best

QF02/2611– page 14/14