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MOD621S - MOLECULAR DIAGNOSTICS - 1ST OPP - NOVEMBER 2023 |
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nAm I BIA UnlVERSITY
OF SCIEnCE AnDTECHnOLOGY
Faculty of Health, Natural
Resources and Applied
Sciences
School of Health Sciences
Department of Clinical
Health Sciences
13 Jackson Kaujeua Street
Private Bag 73388
Windhoek
NAMIBIA
T: +264 61 207 2970
F: +264 61 207 9970
E: dchs@nust.na
W: www.nust.na
QUALIFICATION: BACHELOR of MEDICAL LABORATORY SCIENCES
QUALIFICATION CODE: 07BMLS
LEVEL: 6
COURSE:MOLECULAR DIAGNOSTICS
COURSECODE: MOD 621S
DATE: NOVEMBER 2023
SESSION: 1
DURATION: 3 HOURS
MARKS: 100
EXAMINER:
MODERATOR:
FIRST OPPORTUNITY: QUESTION PAPER
Ms Vanessa Tjijenda
Ms Cara Mia Dunaiski
INSTRUCTIONS:
1. Answer all questions on the separate answer sheet.
2. Please write neatly and legibly.
3. Do not use the left side margin of the exam paper. This must be allowed for the
examiner.
4. No books, notes and other additional aids are allowed.
5. Mark all answers clearly with their respective question numbers.
PERMISSIBLE MATERIALS:
1. Non-Programmable Calculator
ATTACHEMENTS
1. None
This paper consists of 7 pages including this front page
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SECTION A: MULTIPLE CHOICE
[10 MARKS)
QUESTION 1:
[10 MARKS]
Evaluate the statements in each numbered section and select the most appropriate answer or
phrase from the given possibilities. Fill in the appropriate letter next to the number of the
correct statement/phrase on your ANSWERSHEET.
(10]
1.1 The following enzyme is used to add 32P to the 5' end of the DNA fragment.
A. Sl Nuclease
(1)
B. RNaseH
C. T4 Polynucleotide Kinase
D. Terminal Deoxypolynucleotidyl Transferase
1.2 Which molecule is detected during Southern blotting.
(1)
A. Total RNA
B. DNA
C. Proteins
D. mRNA
1.3 How do you ensure that electrophoresis is running?
(1)
A. By setting the correct voltage
B. By observing bubbles
C. By placing DNA closer to the negative electrode
D. Switch on the electrophoresis box
1.4 Which assay is used to measure multiple gene expression?
(1)
A. Northern blotting
B. Reverse transcriptase PCR
C. Multiplex PCR
D. Microarray
1.5 The following is incorrect during nucleic acid extraction purity check:
(1)
A. DNA 260/280 ratio of 2 is normal
B. Phenol absorbs at 260 nm
C. Proteins absorbs at 260 nm
D. RNA 260/230 ratio of 2.2 is normal
1.6 Calculate the annealing temperature of the following sequence: CGGAGATTCTAGACCTCCT(G1):
A. 66°C
B. 62 °C
c. 57°c
D. 58°C
Molecular Diagnostics (MOD621S)
1st opportunity November 2023
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1.7 Which reagent is used to detect DNA during PCR?
(1)
A. SYBRGreen
B. Loading dye
C. TAE
D. Ethidium bromide
1.8 In preparing 250ml of a 0.8 % agarose gel, how much agarose is dissolved in 250ml TAE
buffer?
(1)
A. 0.8g
B. 2g
C. lg
D. 0.9 g
1.9 During PCR, primers bind during the _____
phase.
(1)
A. Denaturation
B. Extension
C. Holding temperature
D. Annealing
1.10 The following method is useful in identifying novel mutations:
(1)
A. Real time PCR
B. Variable Number Tandem Repeats.
C. Fluorescent in Situ Hybridization
D. Sanger Sequencing
Molecular Diagnostics (MOD621S)
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SECTION B: SHORT QUESTIONS
Please answer ALL of the questions in this section.
[60 MARKS]
QUESTION 2:
[27)
2.1 Define gel electrophoresis.
(2)
2.2 Discussthe consideration when hooking up electrical current and running the gel
(3)
2.3 Explain why we add loading dye to our DNA during gel electrophoresis?
(2)
2.4 A postgraduate student is interested in determining the genes responsible for causing
drugs resistance in bacteria (Klebsiella pneumonia and Escherichia coli (E. coli)). She runs a
Multiplex PCR. The results are shown below:
b
btam,
blasHv
bla C'TX-M. -
/r-1 bfoox.ift...
b.laax.M-9-
'!>,,i..,.
.."- _,.,~,_,
·"" "'<,'' -
,.<..:t
I\\~
.,..~.....
' .~ ,.............
"<' ..
"<'"
:?c,
"<'
.(..
-~
Figure 1: Lane Mis the DNA ladder; Lane 2 is a Klebsiella pneumonia strain KP796; lane 3 is an E coli strain EC90;
Lane 4 is an E coli strain EC63; Lane 5 as an E coli strain EB175; lanes 6,7 & 8 are Klebsiella pneumonia strain
KP195, KP79 and KP840 respectively. Lane 9 is the negative control (NC). Bia- TEM, SHV, CTM-Ml, OXA-1 and
CTX-M9 are genes that are known to confer resistance in bacteria.
2.4.1 Briefly define "Multiplex PCR".
(3)
2.4.2 Interpret the results for E.coli strain EC63{land 4).
(2)
2.4.3 Lane 9 contains the "Negative Control". Explain the purpose of adding a negative
control to your PCR.
(1)
2.5 List the ingredients used in a qPCRand briefly explain their purposes.
(14)
Molecular Diagnostics (MOD621S)
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QUESTION 3:
[12]
Use the information proved below to answer the following question:
5' GAATTCTCGTACATAGGATCGAATGGCTAGACGAATTAGACTTACGTATAACGGGGTAGACAGA3'
3' CTTAAGAGCATGTATCCTAGCTATCGCATCTGCTTAATCTGAATGCATATTGCCCCATCTGTCT5'
Primer 1: 5' TCGTACATAGGATCG3'
Primer 2: 5' CTACCCCGTTATACG3'
fcoRI recognition site: 5' GAATTC 3'
3' CTTAAG 5'
3.1 Using the DNA template shown above, underline the template sequence where the two
primers will anneal. Label the underlined sequence "Primer 1" and Primer 2".
(4)
3.2 Using the DNA template and the primers shown above, write the sequence of the
resulting PCRproduct after 30 cycles.
(4)
3.3 Define "restriction enzymes".
(2)
3.4
Draw a box around the site(s) on the DNA that would be recognized and cleaved
by fcoRI.
(2)
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QUESTION 4:
{10)
4.1 Analysis of the spread and transmission of TB strains, using molecular methods,
has been reported. Molecular techniques have been used in reliably differentiating
M. tuberculosis isolates. Namibia has a high prevalence of HIV/ AIDS, a cofactor for TB.
The study done in Khomas region identified different TB strains circulating in the Khomas
region. The most common strain was the Beijing, followed by the Harlem strain with the
LAM strain being the least common. The below figure show some of the results.
S 12 13 14 15 S 16 17 18 19 S 20 21 22 S
Figure 2: TBstrains isolatedfrom patients in Khomas.S= DNAladder, 12- 22 patient TB strains
4.1.1 Identify the method used.
(1)
4.1.2 Use the results in Figure 2 to corroborate the information provided above.
(6)
4.2 Briefly discuss the principle of MALDI-TOF.
(3)
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QUESTION 5:
[10]
5.1 Define DNA sequencing.
(1)
5.2 Complete the following table by comparing the three assays.
(9)
Sanger Sequencing
5.2.1 Principle
5.2.2 length of DNA
fragments sequenced
Shot gun sequencing
Next Generation
Sequencing
SECTION C: LONG ANSWER QUESTIONS
Please answer ALL of the questions in this section.
[30 MARKS]
QUESTION 6:
6.1 Explain the steps in Ion Torrent Sequencing.
(10)
6.2 Discussthe steps involved in Northern blotting.
(10)
6.3 Generate the gel electrophoresis profile of the following sequence using the Maxam
Gilbert chemical method: 5' ACTGACTGAA 3'
(10)
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END OF QUESTION PAPER
Molecular Diagnostics (MOD621S)
istopportunity November 2023
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