San Lorenzo Ruiz College of Ormoc, Inc.

College of Medical Laboratory Science

MLS 28: MOLECULAR BIOLOGY AND DIAGNOSTICS

MOLECULAR DIAGNOSTICS: STUDY QUESTIONS

General Concepts and Techniques

1. What is molecular diagnostics, and how does it differ from traditional diagnostic
methods?

Molecular diagnostics use molecular biology in testing nucleic acids (DNA & RNA) or
proteins to identify mutations and diseases. It provides faster, more sensitive and
accurate results in detecting infectious diseases compared to traditional diagnostic
methods that uses broader and longer techniques such as culturing bacteria and
serological tests that may take several hours or days to detect pathogens. Molecular
diagnostics uses advanced testing methods unlike in traditional diagnostics that cannot
detect other pathogens.

2. Name and briefly describe three common molecular diagnostic techniques.

•Southern Blotting - is used in sickle cell anemia and Fragile X syndrome. This
technique is used to identify DNA sequence. It is performed by fragmenting DNA and
separating it via electrophoresis. The denatured DNA is then transferred onto a
membrane with a labeled probed for hybridization.

•Polymerase Chain Reaction (PCR) - PCR is commonly used for detecting bacteria and
viruses. It is a highly sensitive molecular diagnostic technique that amplify or make
millions of copies of specific DNA and RNA sequences. PCR (DNA) and Reverse
Transcription PCR (RNA) is useful in detecting HPV and COVID-19.

•Fluorescence In Situ Hybridization (FISH) - is used to detect gene amplifications in

diagnosing cancer and abnormalities in the chromosome. This technique use
fluorescent labeled DNA or RNA probe to bind specific sequence on chromosomes.

3. What are the key principles of Polymerase Chain Reaction?

The key principles of PCR are Denaturation, Annealing and Elongation.

4. How does real-time PCR differ from traditional PCR, and what are the advantages of
real-time PCR?
Real-time PCR use fluorescent dyes or probes in monitoring DNA amplification in real
time that helps reduce the detection time because it does not use gel electrophoresis
while Traditional PCR method amplify DNA sequence by performing repeat cycles
(Denaturation, Annealing and Elongation), it commonly use agarose gels which are not
precise and can only detect after the reaction is complete. The advantage of Real-time
PCR is that it is more faster, precise, sensitive and risk of contamination is reduced due
to its close-tube system.

5. Explain the concept of hybridization and its role in molecular diagnostics.

Hybridization is a process in which a single-stranded DNA or RNA bind together via

hydrogen bonding between their nitrogenous bases to form double stranded molecules.
In molecular diagnostics, hybridization is useful in detection of specific nucleic acid
sequences. Hybridization is important in PCR, FISH and in Southern and Northern
Blotting.

6. What are some limitations of molecular diagnostic techniques?

Molecular diagnostic’s laboratory equipment and reagents are expensive, performing in

the laboratory require skilled laboratory technicians since complex results or data will be
interpreted. There are also risks in contamination and molecular diagnostic is
dependent on target specificity and sample quality.

7. How can molecular diagnostics be used to detect infectious diseases, genetic

Disorders, and cancers?

Molecular diagnostics technique like PCR can identify pathogens to detect infectious
disease. It can also diagnose genetic disorders by detecting abnormalities and
mutations by performing microarray analysis. Several types of cancer can be diagnosed
by analyzing genetic changes using Fluorescence In Situ Hybridization (FISH) and
Next-Generation Sequencing (NGS).

8. What are some considerations for sample collection and storage for molecular
testing?

To ensure and maintain sample integrity for molecular testing, it is important to select
appropriate samples and ensure proper labelling of the collected samples. The
laboratory technicians must collect samples using sterile equipment and techniques to
reduce contamination, and should observe proper temperature for storage and transport
of the samples.

9. How do you ensure the quality and reliability of molecular diagnostic results?

Collected samples must be handled and stored properly to avoid contamination

maintain sample integrity, the laboratory technician must follow proper testing
procedures and techniques, equipment and machines in the laboratory should be
regularly check and well-maintained to ensure that it is still working and in good
condition, and it is also important to double check the results and repeat the test to
confirm the results and findings.

Specific Applications
1. How can molecular diagnostics be used in the diagnosis and management of
Infectious diseases?

Molecular diagnostic technique like PCR use rapid and precise identification and
diagnosis of infectious diseases because it can amplify specific RNA and DNA
sequence of the pathogens. It is also useful in tracking how the diseases spread,
and provide detailed characteristic of the bacteria or viruses which can be helpful
to select appropriate medications to improve patient’s treatment .

2. What are some examples of genetic disorders that can be diagnosed using
Molecular techniques?

•Down syndrome – can be diagnosed using FISH

•Cystic Fibrosis – can be diagnosed using DNA sequencing and PCR
•Sickle cell anemia – can be diagnosed using RFLP
•Beta Thalassemia – can be diagnosed by identifying mutation in the hemoglobin

3. How can molecular diagnostics be used to identify and characterize cancer?

Molecular diagnostics can identify cancer by detecting genetic mutations, genetic

rearrangements and changes in the DNA molecule. Molecular diagnostic
techniques like FISH, NGS and PCR can identify specific alterations in the genes
that cause cancer.

4. What are some examples of molecular biomarkers used in cancer diagnosis

and Prognosis?

•BRCA1/BRCA2 – biomarker breast for breast and ovarian cancer

•CA-125 – biomarker for ovarian cancer
•PD-L1 – biomarker for lung cancer
•BRAF – mutation in BRAF gene is linked to colorectal cancer and melanoma

5. How can molecular diagnostics be used to personalize treatment decisions?

Molecular diagnostics be used to personalize treatment decisions because it can

detect biomarkers and genetic mutations which is helpful in choosing appropriate
medications to target the specific gene alterations. Molecular diagnostics can
 also be useful in testing the patient for drug sensitivity and resistance to check
how the patient is responding to the treatment.

6. What are some emerging applications of molecular diagnostics?

Application molecular diagnostic techniques like mass spectrometry is helpful in

early diagnosis and personalized treatment because this technique is fast,
sensitive and effective in detecting diseases biomarkers. Another is CRISPR-
Based Diagnostics, this technology is used is precise and rapid detection of
infectious disease and different types of cancer. Profiling diseased and identifying
genetic mutations using NGS can prevent the spread of drug-resistant infections.

7. How can Molecular diagnostics be used in pharmacogenomics?

Molecular diagnostics is useful in pharmacogenomics because identifying genetic

variations using genotyping and identification of DNA sequence can help assess
drug efficacy

Interpretation and Clinical Relevance

1. How should molecular diagnostic results be interpreted in the context of a
patient’s clinical history and other diagnostic findings?

Molecular diagnostic results must be interpreted in relation to a patient’s health

history or background, patient’s symptoms and other diagnostic tests. Monitoring
with an expert can help ensure precise interpretations of results.

2. What are some potential pitfalls in the interpretation of molecular diagnostic

results?

Some potential pitfalls in the interpretation of molecular diagnostic results are

errors that may cause false positive or false negative results, testing of incorrect
specimens, risk of contamination of the collected samples and misinterpretation
of results.

3. How can molecular diagnostics contribute to improved patient outcomes?

Molecular diagnostics contribute in improving patient outcomes by precise and
early disease detection allowing for personalized treatment for the patient,
monitoring on how well the treatment is working on the patient, all of these can
be helpful for the physicians in providing effective treatment for the patients.

4. What are some ethical considerations related to the use of molecular

diagnostics?

Some ethical considerations in molecular diagnostics include obtaining informed

consent from the patient before testing, protecting patient’s privacy and
confidentiality to prevent misuse and unauthorized access of data, managing and
addressing unexpected results and findings is also important.

5. How can molecular diagnostics be used in public health surveillance and

outbreak response?

Molecular diagnostics can be used in public health surveillance and outbreak

response by rapid and precise detection of infectious pathogens, tracing disease
transmission, monitoring drug resistance patterns of pathogens, and supporting
vaccine development for disease outbreaks.