APS6003- Biomolecular Analysis- Revision

Academic Year- 2022-23

Semester - Semester 2
EXAM PERIOD: ***

TIME ALLOWED: 2 hours

Learning outcomes assessed

On successful completion of this module, students will be able to:
• Demonstrate knowledge of a range of modern and novel biomolecular techniques.
• Evaluate the application and importance of current molecular biological techniques in
biomedical science.

INSTRUCTIONS TO CANDIDATES:

1. Exam Type - CLOSED BOOK

2. The paper will have four (04) questions
3. Answer two (02) questions only
4. The WRITTEN EXAM equivalent word count for EACH answer is 1000 words ± 10%.
5. The word counts are ONLY indicative of the effort required for the personal written
assessment on the day of the examination.
6. The mark awarded for EXAM1 will be a mean of the two answers provided.
7. This is an individual assessment - you must work on it on your own and not plagiarize
from another student’s effort.
8. All answers must be written in your own words.

Choose Any Two Questions

 1. Evaluate the spectrum of currently utilized DNA cloning vectors within the domain of
molecular biology, with specific emphasis on their pertinence to research endeavors.
[Discuss the features, and applications of DNA cloning vectors by providing examples. Highlight
their usefulness in cloning small to medium-sized DNA fragments. (Write about all 4 types of
vectors using one example for each) (25 x 4 = 100 marks)]

2. Conduct a thorough analysis of the benefits and limitations [40%] tied to real-time PCR
(qPCR) as a potential diagnostic tool. Draw a comparison with conventional PCR,
emphasizing its capacity to furnish quantitative data and its potential for facilitating
high-throughput analysis [60%].

Bnefits of qpcr
1. It gives a look in to the reaction that is help to decide which reactions have worked well
and which have failed.
2. The efficiency of the reaction can be precisely calculated.
3. There is no need to run the PCR product out on a gel after the reaction as the melt curve
analysis serve the purpose.
4. The real-time PCR data can be used to perform truly quantitative analysis of gene
expression.
5. Faster than normal PCR.
6. Less complexity at the quantification of sample.
7. Thus, unlike the ordinary preparative PCR, Real Time PCR allows the success of multiple
PCR reaction to be determined automatically after only a few cycles, without separate
analysis of each reaction, and avoids the problem of “false negatives”
8. Sensitivity and Specificity: qPCR is highly sensitive, capable of detecting very low amounts
of target DNA or RNA. Moreover, it offers high specificity through the use of fluorescently
labeled probes or SYBR Green, which selectively bind to the amplified target sequence.
9. qPCR platforms can process multiple samples simultaneously, making it suitable for high-
throughput applications.
10. qPCR requires small amounts of DNA or RNA, making it useful when the sample amount is
limited or precious.

Limitations of qpcr

1) Prone to Contamination: qPCR's high sensitivity can make it susceptible to contamination

issues, leading to false-positive results. Stringent laboratory practices and proper controls
are necessary to mitigate this risk.
2) Expense: The initial investment for qPCR equipment and reagents can be relatively high.
This may be a limiting factor for some research laboratories or small-scale diagnostic
facilities.
3) Assay Design Complexity: Designing qPCR assays requires careful consideration of primer
and probe sequences, specificity, and optimization. Poorly designed assays can result in
unreliable or inaccurate data.
4) Limited Multiplexing: While some qPCR platforms allow for multiplexing (detecting
multiple targets in the same reaction), the number of targets that can be simultaneously
amplified is limited.
5) Inhibitors and Variability: Some complex sample matrices, such as environmental or
clinical samples, may contain inhibitors that can interfere with qPCR reactions. Additionally,
qPCR can be sensitive to variations in sample quality and handling.
6) Limited Information on Target Structure: qPCR measures the presence and quantity of
specific sequences, but it does not provide information on target structure or alternative
splicing events. Additional techniques like RT-PCR or sequencing are required for such
analyses.
7) Dependency on Reference Genes: In gene expression studies, qPCR often relies on
reference genes for normalization. Identifying suitable reference genes can be challenging,
and their expression stability can vary between tissues and experimental conditions.
8) Cost: qPCR can be a relatively expensive technique, especially when compared to
traditional end-point PCR.

Comparison

1. Methodology:

 Conventional PCR: In conventional PCR, the target DNA is amplified through a series of
temperature cycles in a thermal cycler. These cycles involve denaturation, annealing, and
extension steps, leading to exponential amplification of the target DNA.
 qPCR: qPCR follows a similar temperature cycling process, but it incorporates fluorescently
labeled probes or DNA-binding dyes to measure the amplification in real-time. The fluorescence
intensity is recorded at each cycle, allowing for quantification and detection of the target in real-
time during the exponential phase.

2. Detection:

 Conventional PCR: Detection of PCR products in conventional PCR is usually performed post-PCR
using gel electrophoresis or other methods, which only provides qualitative information
(presence or absence of the target).
 qPCR: qPCR allows for real-time detection and quantification of the target during the PCR
reaction. The fluorescence data can be plotted against cycle number to generate amplification
curves, which provide quantitative information about the initial target concentration.

3. Sensitivity:
  Conventional PCR: Conventional PCR is generally less sensitive than qPCR. The sensitivity is
limited by the endpoint detection, where the product needs to reach a detectable concentration
before it can be visualized on a gel.
 qPCR: qPCR's real-time detection enables the quantification of low-abundance targets and
provides greater sensitivity due to the ability to measure amplification during the early
exponential phase.

4. Quantification:

 Conventional PCR: Conventional PCR is often used for qualitative assessments, such as
confirming the presence or absence of a specific target.
 qPCR: qPCR is a quantitative technique that allows accurate measurement of the initial target
concentration, making it ideal for gene expression analysis, pathogen quantification, and other
quantitative applications.

5. Speed:

 Conventional PCR: Conventional PCR requires a post-PCR analysis step (e.g., gel
electrophoresis), which adds to the overall processing time.
 qPCR: qPCR's real-time nature eliminates the need for post-PCR analysis, providing faster results
and reducing the risk of contamination.

6. Multiplexing:

 Conventional PCR: Multiplexing in conventional PCR (amplifying multiple targets in a single

reaction) can be challenging due to potential primer interactions and specificity issues.
 qPCR: qPCR can accommodate multiplexing more readily, as different targets can be labeled
with specific fluorophores, allowing simultaneous detection and quantification of multiple
targets.
 3. Examine the importance and constraints [40%] associated with DNA extraction from
human specimens, providing relevant instances. Assess the appropriateness of various
DNA extraction techniques for a minimum of 2 prevalent human specimen types
extensively utilized in research environments. [DNA isolation method for each human
specimen = 30% x 2 = 60%]

Importance of DNA extraction from human specimens:

1) Advancing medical research: DNA extraction plays a crucial role in understanding genetic
variations associated with diseases. It enables researchers to study the genetic basis of
diseases, identify potential biomarkers, and develop targeted therapies.
2) Forensic analysis: In criminal investigations, DNA extraction from human specimens such as
blood, saliva, or hair helps in identifying suspects and linking individuals to crime scenes,
thus aiding in solving criminal cases
3) Genetic testing and diagnostics: DNA extraction is essential in genetic testing for inherited
disorders, genetic predispositions, and ancestry analysis. It helps individuals and healthcare
providers make informed decisions about their health and treatment options.
4) Evolutionary studies and anthropology: DNA extraction from ancient human specimens,
like bones or teeth, offers insights into human evolution, migration patterns, and
population dynamics.
5) In paternity testing, DNA extracted from a child and potential parents is used to determine
whether the parents are biologically related.

The constraints associated with DNA extraction from human specimens include:

1. The need for specialized equipment and expertise: DNA extraction is a complex process that
requires specialized equipment and expertise.

2. Sample size and quality: The success of DNA extraction heavily depends on the quantity
and quality of the specimen. In some cases, obtaining sufficient and undamaged DNA can
be challenging, particularly when dealing with degraded or limited samples, such as ancient
DNA or forensic evidence left at crime scenes.
3. Contamination: DNA extraction requires strict precautions to prevent cross-contamination.
Even a tiny amount of external DNA can compromise the accuracy and reliability of the
results. This is especially critical in forensic analysis and ancient DNA studies.
4. Ethical considerations and consent: The extraction of DNA from human specimens raises
ethical concerns, particularly when dealing with samples collected for medical purposes or
from vulnerable populations. Researchers must ensure proper informed consent and
adhere to ethical guidelines.
5. Cost and time: DNA extraction can be a resource-intensive process, both in terms of time
and financial investment. It often involves specialized equipment and skilled personnel,
making it relatively expensive.
6. Legal and regulatory issues: The use of DNA samples for research or forensic purposes may
be subject to legal and regulatory restrictions, depending on the country or region.
Researchers must comply with relevant laws and guidelines.
 4. The role of screening gene libraries during cloning experiments holds immense
significance in genetic research. Substantiate the selection of two distinct
methodologies employed for gene library screening through an exploration of their
mechanisms [70%], applications [30%], and suitable case studies.

-2 types of botting

Western blotting techniques -mechanism (procedure)

Steps

Applications-using examples