Polymerase Chain Reaction Applications — MCQs
Disputed maternity can be solved by using the following tests, EXCEPT:
Molecular genetic testing is used to detect all of the following except?
Which of the following is used to detect abnormal gene sequences EXCEPT?
DNA fingerprinting was first used by Alec Jeffreys in a criminal case for detecting:
DNA amplification is done in:
DNA amplification is done by all, except:
Best method to diagnose HIV in an infant?
Gene amplification is achieved through
BCR-ABL fusion gene is MOST CHARACTERISTICALLY seen in?
Foci of granulomatous inflammation show all of the following except?
Polymerase Chain Reaction Applications Indian Medical PG Practice Questions and MCQs
Question 1: Disputed maternity can be solved by using the following tests, EXCEPT:
- A. Blood grouping
- B. HLA typing
- C. DNA fingerprinting
- D. Precipitin test (Correct Answer)
Explanation: ***Precipitin test*** - The **precipitin test** is used to determine the origin of a **blood sample**, specifically whether it is **human or animal blood**, by detecting species-specific proteins. It is not used for assessing maternity. - This test is primarily employed in **forensic serology** to differentiate between blood from different animal species, making it irrelevant for paternity or maternity disputes. *Blood grouping* - **Blood grouping** (e.g., ABO and Rh systems) can be used to **exclude paternity or maternity** by comparing the blood types of the child, mother, and alleged father. - If the child's blood type is incompatible with the alleged parents based on Mendelian inheritance, one or both can be excluded. *HLA typing* - **HLA typing** (Human Leukocyte Antigen) is a more powerful genetic marker system than ABO/Rh for determining paternity or maternity. - It involves analyzing highly polymorphic genes on chromosome 6 that encode cell surface proteins, providing a more definitive means of **inclusion or exclusion**. *DNA fingerprinting* - **DNA fingerprinting** (also known as **DNA profiling**) is the **most accurate and widely accepted method** for resolving paternity and maternity disputes. - It analyzes highly variable regions of DNA unique to each individual, providing a statistically strong basis for **inclusion or exclusion** by comparing genetic profiles.
Question 2: Molecular genetic testing is used to detect all of the following except?
- A. Deletion
- B. Translocation (Correct Answer)
- C. Amplification
- D. Point mutation
Explanation: ***Translocation*** - **Translocations** are chromosomal rearrangements that were historically detected primarily by **cytogenetic methods** (karyotyping, conventional FISH), rather than by traditional molecular genetic testing methods focused on DNA sequencing [3]. - While modern molecular techniques like **RT-PCR for fusion transcripts** (e.g., BCR-ABL), **NGS-based fusion detection**, and **targeted breakpoint sequencing** can now detect translocations, the classic distinction is that translocations involve large-scale structural chromosomal changes better visualized by cytogenetics [2], [3]. - In the traditional classification, molecular genetic testing referred primarily to **sequence-based methods** (PCR, Sanger sequencing) that detect smaller-scale DNA changes rather than gross chromosomal rearrangements. *Deletion* - **Deletions** are readily detected by molecular genetic testing using PCR, Sanger sequencing, MLPA (Multiplex Ligation-dependent Probe Amplification), and NGS [5]. - These techniques identify missing DNA sequences by analyzing changes in fragment size, read depth, or absence of expected amplification products [2], [5]. *Amplification* - **Amplification** (increased gene copy number) is detected by molecular methods including **quantitative PCR (qPCR)**, **digital PCR**, and **NGS-based copy number analysis** [4]. - These techniques quantify gene copy numbers to identify amplifications like HER2 amplification in breast cancer. *Point mutation* - **Point mutations** are the primary target of classic molecular genetic testing [1]. - Detected by **Sanger sequencing**, **allele-specific PCR**, **NGS panels**, and other sequence-based methods that identify single nucleotide changes in DNA [1], [2]. **References:** [1] Kumar V, Abbas AK, et al.. Robbins and Cotran Pathologic Basis of Disease. 9th ed. Genetic Disorders, p. 185. [2] Kumar V, Abbas AK, et al.. Robbins and Cotran Pathologic Basis of Disease. 9th ed. Genetic Disorders, pp. 185-186. [3] Kumar V, Abbas AK, et al.. Robbins and Cotran Pathologic Basis of Disease. 9th ed. Neoplasia, pp. 342-343. [4] Kumar V, Abbas AK, et al.. Robbins and Cotran Pathologic Basis of Disease. 9th ed. Neoplasia, p. 344. [5] Kumar V, Abbas AK, et al.. Robbins and Cotran Pathologic Basis of Disease. 9th ed. Genetic Disorders, pp. 183-184.
Question 3: Which of the following is used to detect abnormal gene sequences EXCEPT?
- A. RFLP analysis
- B. Pyrosequencing
- C. Flow cytometry (Correct Answer)
- D. FISH
Explanation: ***Flow cytometry*** - **Flow cytometry** is primarily used to analyze **cell populations** based on their physical and biochemical characteristics (e.g., size, granularity, and protein expression) by passing them single file through a laser beam, not for direct gene sequencing. - It detects and quantifies cells labeled with **fluorescent antibodies**, making it useful for immunophenotyping, cell sorting, and DNA content analysis, but not for identifying specific gene sequences or mutations. *RFLP analysis* - **Restriction fragment length polymorphism (RFLP) analysis** detects variations in **DNA sequences** by using **restriction enzymes** to cut DNA at specific sites. - Differences in fragment lengths indicate **polymorphisms** or **mutations** within the recognition sites, thereby identifying abnormal gene sequences. *Pyrosequencing* - **Pyrosequencing** is a method of **DNA sequencing** that determines the sequence of nucleotides by detecting the release of pyrophosphate during DNA synthesis. - It is used to identify **single nucleotide polymorphisms (SNPs)** and **short genetic variations**, making it suitable for detecting abnormal gene sequences. *FISH* - **Fluorescence in situ hybridization (FISH)** uses **fluorescently labeled DNA probes** that bind to specific complementary **DNA sequences** on chromosomes. - It is a powerful cytogenetic technique for detecting **chromosomal abnormalities**, such as deletions, translocations, and amplifications, thereby identifying abnormal gene sequences.
Question 4: DNA fingerprinting was first used by Alec Jeffreys in a criminal case for detecting:
- A. Immigration purpose
- B. Disputed paternity
- C. Murder
- D. Rape (Correct Answer)
Explanation: ***Rape*** - **Alec Jeffreys** first applied DNA fingerprinting in 1986 to solve the **Narborough murders case** in Leicestershire, UK. - The technique was used to analyze **semen samples** from two rape-murder victims (1983 and 1986), linking them to a single perpetrator. - The **DNA evidence from semen** (sexual assault evidence) was the key forensic material that demonstrated the power of DNA fingerprinting in criminal investigation. - This led to the conviction of **Colin Pitchfork** in 1988, marking the first use of DNA profiling to solve a criminal case. *Immigration purpose* - While DNA fingerprinting is used for immigration cases to confirm family relationships, this was **not its initial application** by Jeffreys. - Its use in immigration came later, after its breakthrough in criminal forensics. *Disputed paternity* - Paternity testing is a common application of DNA fingerprinting, but it was **not the first criminal case** where Jeffreys demonstrated its utility. - The technique's power in establishing biological relationships was recognized after its initial use in criminal investigations. *Murder* - While the Narborough case did involve murders, the question focuses on what was **detected through DNA evidence**. - The DNA profiling was performed on **semen samples** (rape evidence), not on evidence directly proving murder. - The forensic breakthrough was in linking the sexual assault evidence to the perpetrator, which then solved the murder cases.
Question 5: DNA amplification is done in:
- A. PCR (Correct Answer)
- B. Ligase chain reactions
- C. NASBA (Nucleic Acid Sequence-Based Amplification)
- D. All of the options
Explanation: ***Correct: PCR*** - **Polymerase Chain Reaction (PCR)** is the gold standard technique for DNA amplification in molecular diagnostics and research - Uses **DNA polymerase** (typically Taq polymerase) to exponentially amplify specific DNA sequences through thermal cycling - Involves repeated cycles of **denaturation, annealing, and extension** using primers, dNTPs, and thermostable polymerase - Can generate **millions of copies** from a single DNA template in hours *Incorrect: Ligase chain reactions* - **Ligase Chain Reaction (LCR)** is a technique that uses **DNA ligase** to join adjacent oligonucleotide probes that hybridize to a target sequence - While LCR can amplify DNA through exponential ligation cycles, it is primarily used for **detection of known point mutations** and SNPs rather than general DNA amplification - Requires **four primers** (two for each strand) and perfect complementarity at ligation junctions - Less commonly used than PCR for routine DNA amplification in clinical practice *Incorrect: NASBA (Nucleic Acid Sequence-Based Amplification)* - **NASBA** is an **isothermal RNA amplification** technique that operates at a constant temperature (41°C) - Specifically designed to amplify **RNA targets** using reverse transcriptase, RNase H, and T7 RNA polymerase - Produces single-stranded RNA products, not double-stranded DNA like PCR - Used primarily for **RNA virus detection** (HIV, HCV) and gene expression analysis, not for DNA amplification *Incorrect: All of the options* - While LCR technically can amplify DNA, **PCR is the standard method** for DNA amplification in molecular biology - NASBA is designed for RNA, not DNA amplification - In the context of medical education and clinical practice, **PCR is the definitive answer** for DNA amplification
Question 6: DNA amplification is done by all, except:
- A. DNA sequencing (Correct Answer)
- B. Loop-mediated isothermal amplification (LAMP)
- C. Ligase chain reaction
- D. Polymerase chain reaction
Explanation: ***DNA sequencing*** - **DNA sequencing** determines the **nucleotide base order** in a DNA molecule but does not increase the amount of DNA. - While requiring a DNA template, it is an **analytical technique** rather than an amplification method. *Loop-mediated isothermal amplification (LAMP)* - **LAMP** is an **isothermal DNA amplification** technique that amplifies target DNA sequences at a constant temperature (60-65°C). - It uses a DNA polymerase with strand displacement activity and 4-6 primers to produce large amounts of DNA rapidly. *Ligase chain reaction* - **LCR** is an amplification method that detects specific **DNA sequences** by ligating adjacent probes. - It amplifies the signal from a target DNA sequence rather than the DNA itself by creating many copies of joined probes. *Polymerase chain reaction* - **PCR** is a widely used technique for **amplifying** a specific segment of DNA to produce many copies. - It involves cycles of **denaturation**, **annealing**, and **extension** using a DNA polymerase.
Question 7: Best method to diagnose HIV in an infant?
- A. ELISA
- B. PCR (Correct Answer)
- C. Western blot
- D. All of the options
Explanation: ***PCR*** - **Polymerase Chain Reaction (PCR)** detects **HIV nucleic acids** (DNA or RNA) directly, which is crucial for infants because maternal antibodies can persist for up to 18 months, interfering with antibody-based tests. - PCR allows for early diagnosis, often within the first few weeks or months of life, facilitating timely intervention. *ELISA* - **Enzyme-linked immunosorbent assay (ELISA)** detects HIV antibodies. - In infants, ELISA can be misleading due to the presence of **maternal HIV antibodies** transferred across the placenta, making it unreliable for diagnosing active infection. *Western blot* - **Western blot** is used to confirm positive ELISA results in adults by detecting specific HIV proteins. - Like ELISA, it relies on the detection of **antibodies** and is therefore not reliable in infants due to maternally transmitted antibodies. *All of the options* - This option is incorrect because **ELISA** and **Western blot** are antibody-based tests that are unreliable in infants due to the presence of **maternal antibodies**. - Only **PCR** directly detects the virus itself, making it the preferred diagnostic method in this age group.
Question 8: Gene amplification is achieved through
- A. Polymerase Chain Reaction (Correct Answer)
- B. DNA strand hybridization
- C. In situ DNA hybridization
- D. Ligase chain reaction (LCR)
Explanation: ***Polymerase Chain Reaction*** - **PCR** is the **gold standard** molecular biology technique that generates **millions to billions of copies** of a specific DNA segment over a short period. - It utilizes a cyclical process of **denaturation**, **annealing**, and **extension** with **thermostable DNA polymerase** to achieve exponential amplification. - **Most widely used** method for gene amplification in research and diagnostics. *DNA strand hybridization* - **DNA strand hybridization** is the process where two complementary single-stranded DNA molecules bind together to form a **double-stranded molecule**. - This process is fundamental to many molecular techniques but does not, in itself, achieve **amplification**; rather, it is a **binding event**. *In situ DNA hybridization* - **In situ hybridization** is a technique that localizes and detects specific **nucleic acid sequences** (DNA or RNA) within cells or tissues directly on a slide. - While it uses **hybridization**, its primary purpose is **detection and localization**, not the **amplification** of DNA sequences. *Ligase chain reaction (LCR)* - **LCR** is a molecular technique that does amplify DNA sequences exponentially using **DNA ligase** to join adjacent oligonucleotide probes. - However, it is **less commonly used** than PCR, has more **stringent requirements** (requires knowledge of both strands), and is primarily used for detecting **known point mutations** rather than general gene amplification. - **PCR remains the standard** technique when the question refers to gene amplification without additional qualifiers.
Question 9: BCR-ABL fusion gene is MOST CHARACTERISTICALLY seen in?
- A. CML (Correct Answer)
- B. AML
- C. Chronic Lymphocytic Leukemia (CLL)
- D. Acute Lymphoblastic Leukemia (ALL)
Explanation: ***CML*** - The **BCR-ABL gene mutation** is characteristic of **Chronic Myeloid Leukemia (CML)**, resulting from a translocation between chromosomes 9 and 22 [1]. - This mutation leads to the production of the **BCR-ABL fusion protein**, which promotes cell proliferation and inhibits apoptosis [1]. *AML* - Acute Myeloid Leukemia (AML) does not typically exhibit the **BCR-ABL fusion gene**; rather, it is associated with various other genetic mutations. - Key features of AML include **myeloblast proliferation** and it presents with different cytogenetic abnormalities like **FLT3 or NPM1 mutations**. *CLL* - Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of **mature lymphocytes**, not the **BCR-ABL mutation**. - It is often associated with mutations such as **TP53** and **NOTCH1**, distinct from myeloid malignancies. *ALL* - Acute Lymphoblastic Leukemia (ALL) is primarily linked with **chromosomal translocations** involving **the TCF3** gene or others, but not specifically with **BCR-ABL**. - In ALL, **lymphoid progenitor cells** proliferate, whereas CML is primarily a **myeloid process** associated with the BCR-ABL gene [1]. **References:** [1] Kumar V, Abbas AK, et al.. Robbins and Cotran Pathologic Basis of Disease. 9th ed. Diseases of White Blood Cells, Lymph Nodes, Spleen, and Thymus, pp. 624-625.
Question 10: Foci of granulomatous inflammation show all of the following except?
- A. Eosinophils (Correct Answer)
- B. Epithelioid cells
- C. Fibrosis
- D. Lymphocytes
Explanation: ### Explanation A **granuloma** is a distinctive pattern of chronic inflammation characterized by a focal collection of activated macrophages, often surrounded by a rim of lymphocytes and sometimes a peripheral zone of fibrosis [1]. **Why Eosinophils (Option A) is the correct answer:** Eosinophils are typically associated with **Type I hypersensitivity reactions** (allergic diseases) or **parasitic infections** [2]. While they may occasionally be seen in specific types of granulomatous diseases (like Churg-Strauss syndrome or certain fungal infections), they are **not** a defining or universal component of the classic granulomatous inflammatory focus. **Analysis of Incorrect Options:** * **Epithelioid cells (Option B):** These are the hallmark of granulomas. They are activated macrophages that have developed abundant pink cytoplasm, resembling epithelial cells [1]. They are induced by IFN-γ secreted by T-cells. * **Fibrosis (Option C):** In older granulomas, a rim of fibroblasts and connective tissue (fibrosis) often develops due to the secretion of growth factors like TGF-β [1]. This is the body’s attempt to "wall off" the offending agent. * **Lymphocytes (Option D):** Granulomatous inflammation is a form of **Type IV (delayed-type) hypersensitivity**. T-lymphocytes (specifically CD4+ Th1 cells) are essential for activating macrophages into epithelioid cells via cytokine signaling [1]. **NEET-PG High-Yield Pearls:** * **The "Signature" Cell:** The epithelioid cell is the diagnostic cell of a granuloma. * **Giant Cells:** Formed by the fusion of epithelioid cells [1]. Examples include **Langhans giant cells** (peripheral nuclei; seen in TB) and **Foreign body giant cells** (disorganized nuclei). * **Caseation:** Central "cheese-like" necrosis is highly suggestive of *Mycobacterium tuberculosis*. * **Non-caseating granulomas:** Classically seen in Sarcoidosis, Crohn’s disease, and Berylliosis. **References:** [1] Kumar V, Abbas AK, et al.. Robbins and Cotran Pathologic Basis of Disease. 9th ed. Inflammation and Repair, pp. 107-109. [2] Cross SS. Underwood's Pathology: A Clinical Approach. 6th ed. (Basic Pathology) introduces the student to key general principles of pathology, both as a medical science and as a clinical activity with a vital role in patient care. Part 2 (Disease Mechanisms) provides fundamental knowledge about the cellular and molecular processes involved in diseases, providing the rationale for their treatment. Part 3 (Systematic Pathology) deals in detail with specific diseases, with emphasis on the clinically important aspects., pp. 195-196.
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