Next-Generation Sequencing — MCQs
Which is the correct sequence of steps in isolating desirable protein using recombinant DNA technology? 1. Expression of protein and lysis of the bacterial cell 2. Incorporation of genes into bacteria 3. SDS PAGE 4. Protein elution 5. Column chromatography
Steps of PCR in sequence are?
Mutations are due to changes in:
Which of the following techniques is used for the detection of variations in DNA sequence and gene expression?
Statement 1 - A 59-year-old patient presents with flaccid bullae. Histopathology shows a suprabasal acantholytic split. Statement 2 - The row of tombstones appearance is diagnostic of Pemphigus vulgaris.
What is the term used to describe the process where complementary single-stranded DNA molecules bind together to form a double helix in molecular biological methods?
All of the following statements are TRUE about second generation antihistaminic agents EXCEPT:
Agarose gel electrophoresis from DNA of a population of cells as seen under ultraviolet light is shown below. What is the correct explanation for the finding seen in the band labeled as "C"?
Foci of granulomatous inflammation show all of the following except?
NKX3-1 immunohistochemistry is used for the diagnosis of which of the following?
Next-Generation Sequencing Indian Medical PG Practice Questions and MCQs
Question 1: Which is the correct sequence of steps in isolating desirable protein using recombinant DNA technology? 1. Expression of protein and lysis of the bacterial cell 2. Incorporation of genes into bacteria 3. SDS PAGE 4. Protein elution 5. Column chromatography
- A. 2,1,3,5,4 (Correct Answer)
- B. 2,4,5,3,1
- C. 1,2,4,3,5
- D. 1,5,2,4,3
Explanation: ***2,1,3,5,4*** - This sequence accurately reflects the typical order of operations in **recombinant protein isolation**: first, the gene is introduced into bacteria, then protein is expressed and cells lysed, followed by **SDS-PAGE as an intermediate quality check** to confirm protein expression before proceeding to purification steps (column chromatography and elution). - The process starts with gene incorporation, includes an analytical checkpoint after lysis, and ends with purified protein elution. *2,4,5,3,1* - This sequence is incorrect because **protein elution (4)** and **column chromatography (5)** are purification steps that occur *after* protein expression and cell lysis. - **Lysis (1)** cannot happen after elution, as cells must be lysed first to release the protein for purification. *1,2,4,3,5* - This sequence is incorrect because **expression and lysis (1)** must occur *after* the gene has been **incorporated into bacteria (2)** - the gene must be present before it can be expressed. - Additionally, **protein elution (4)** should follow **column chromatography (5)**, as elution is the step where protein is collected from the chromatography column. *1,5,2,4,3* - This sequence is incorrect because **incorporation of genes (2)** must be the first step - the gene needs to be in the bacteria before any expression, lysis, or purification can occur. - Starting with **expression and lysis (1)** before gene incorporation is impossible.
Question 2: Steps of PCR in sequence are?
- A. Denature DNA, Extend DNA, Anneal Primers
- B. Anneal Primers, Extend DNA, Denature DNA
- C. Extend DNA, Anneal Primers, Denature DNA
- D. Denature DNA, Anneal Primers, Extend DNA (Correct Answer)
Explanation: ***Denature DNA, Anneal Primers, Extend DNA*** - This sequence represents the three fundamental steps of each PCR cycle, ensuring accurate and efficient **DNA amplification**. - **Denaturation** separates the double-stranded DNA template, **annealing** allows primers to bind to specific sequences, and **extension** synthesizes new DNA strands. *Denature DNA, Extend DNA, Anneal Primers* - This order is incorrect because **primer annealing** must occur before DNA extension can begin. - Primers provide the necessary starting points for the **DNA polymerase** to synthesize the new strands. *Anneal Primers, Extend DNA, Denature DNA* - This sequence is incorrect as the **template DNA** must first be denatured to separate the strands before primers can anneal to them. - If the DNA is not denatured, the primers cannot access their target sequences. *Extend DNA, Anneal Primers, Denature DNA* - This order is incorrect because **DNA extension** is the final step, occurring only after denaturation and primer annealing. - The polymerase requires both a denatured template and bound primers to initiate synthesis.
Question 3: Mutations are due to changes in:
- A. DNA nucleotide sequence (Correct Answer)
- B. RNA nucleotide sequence
- C. Amino acid sequence of ribonuclease
- D. Cell membrane
Explanation: ***DNA nucleotide sequence*** - **Mutations** are defined as changes in the **genetic material**, which is primarily composed of **DNA**. - These changes in the **nucleotide sequence** of DNA can alter the genetic code, leading to changes in **protein structure and function**. *RNA nucleotide sequence* - While RNA can have its nucleotide sequence altered, these changes are generally not considered true **mutations** in the heritable sense for most organisms. - RNA is typically a temporary molecule, and changes to its sequence are usually not passed down to subsequent generations. *Amino acid sequence of ribonuclease* - An altered **amino acid sequence** in a protein like ribonuclease is a consequence of a **mutation in the DNA**, not the mutation itself. - **Ribonucleases** are enzymes that catalyze the degradation of RNA, and their structure is determined by the **DNA sequence**. *Cell membrane* - The cell membrane is a **lipid bilayer** with embedded proteins that regulates cellular transport and communication. - While its components can be affected by genetic mutations, alterations in the cell membrane itself do not constitute the primary definition of a **mutation**.
Question 4: Which of the following techniques is used for the detection of variations in DNA sequence and gene expression?
- A. Southern blot
- B. Western blot
- C. Microarray (Correct Answer)
- D. Northern blot
Explanation: ***Microarray*** - **Microarrays** are designed to detect thousands of DNA or RNA sequences simultaneously, making them ideal for analyzing **gene expression profiles** and identifying **sequence variations** like SNPs. - They involve hybridizing labeled sample DNA/RNA to probes fixed on a solid surface, with the intensity of hybridization indicating the presence or abundance of specific sequences. *Northern blot* - The **Northern blot** technique is primarily used to study **gene expression** by detecting specific **RNA sequences** in a sample. - It does not directly analyze DNA sequence variations. *Southern blot* - The **Southern blot** is a molecular biology method used to detect specific **DNA sequences** in DNA samples. - While it can identify large-scale DNA rearrangements or deletions, it is not optimized for simultaneous detection of multiple gene expression levels or subtle sequence variations. *Western blot* - The **Western blot** is used to detect specific **proteins** in a sample. - It analyzes protein expression levels and modifications and is not designed for the detection of DNA sequence variations or gene expression at the RNA level.
Question 5: Statement 1 - A 59-year-old patient presents with flaccid bullae. Histopathology shows a suprabasal acantholytic split. Statement 2 - The row of tombstones appearance is diagnostic of Pemphigus vulgaris.
- A. Statements 1 & 2 are correct, 2 is not explaining 1 (Correct Answer)
- B. Statements 1 and 2 are correct and 2 is the correct explanation for 1
- C. Statements 1 and 2 are incorrect
- D. Statement 1 is incorrect
Explanation: ***Correct: Statements 1 & 2 are correct, 2 is not explaining 1*** **Analysis of Statement 1:** - A 59-year-old patient with **flaccid bullae** and **suprabasal acantholytic split** on histopathology is the classic presentation of **Pemphigus vulgaris** - The flaccid (easily ruptured) nature of bullae distinguishes it from tense bullae seen in bullous pemphigoid - The suprabasal location of the split (just above the basal layer) with acantholysis (loss of cell-to-cell adhesion) is pathognomonic - **Statement 1 is CORRECT** ✓ **Analysis of Statement 2:** - The **"row of tombstones" or "tombstone appearance"** is indeed a diagnostic histopathological feature of Pemphigus vulgaris - This appearance results from basal keratinocytes remaining attached to the basement membrane while suprabasal cells separate due to acantholysis - The intact basal cells standing upright resemble a row of tombstones - **Statement 2 is CORRECT** ✓ **Does Statement 2 explain Statement 1?** - Statement 2 describes a **histopathological appearance** (tombstone pattern) that is a **consequence** of the suprabasal split - However, it does NOT explain the **underlying cause** of the flaccid bullae or the suprabasal split - The true explanation involves **IgG autoantibodies against desmoglein 3 (and desmoglein 1)**, which attack intercellular adhesion structures (desmosomes), causing **acantholysis** - Therefore, **Statement 2 does NOT explain Statement 1** ✗ *Incorrect: Statement 2 is the correct explanation for Statement 1* - While both statements describe features of Pemphigus vulgaris, the tombstone appearance is a descriptive finding, not an explanatory mechanism *Incorrect: Statements 1 and 2 are incorrect* - Both statements are medically accurate descriptions of Pemphigus vulgaris features *Incorrect: Statement 1 is incorrect* - Statement 1 correctly describes the cardinal clinical and histopathological features of Pemphigus vulgaris
Question 6: What is the term used to describe the process where complementary single-stranded DNA molecules bind together to form a double helix in molecular biological methods?
- A. DNA-DNA hybridization
- B. DNA annealing (Correct Answer)
- C. DNA-DNA ligation
- D. DNA denaturation
Explanation: ***DNA annealing*** - **Annealing** is the specific term used in molecular biology to describe the process where two **complementary single-stranded DNA** molecules bind together through base pairing to form a double helix. - This term is commonly used in techniques like **PCR** (polymerase chain reaction), where primers anneal to template DNA during the annealing step. - The process involves **controlled cooling** that allows complementary sequences to recognize and bind to each other through hydrogen bonding. *DNA-DNA hybridization* - **Hybridization** is a broader, more general term that encompasses any pairing of complementary nucleic acid strands (DNA-DNA, DNA-RNA, or RNA-RNA). - While annealing is a type of hybridization, in molecular biology practice, "annealing" specifically refers to the binding step in enzymatic reactions like PCR. - Hybridization is more commonly used to describe probe-target binding in techniques like Southern blotting or in situ hybridization. *DNA-DNA ligation* - **Ligation** is an entirely different process involving the enzymatic joining of two **DNA fragments** using **DNA ligase**. - This creates a **phosphodiester bond** between the 3'-hydroxyl and 5'-phosphate ends of adjacent DNA strands, not base pairing between complementary strands. *DNA denaturation* - **Denaturation** (or melting) is the **opposite process** where a double-stranded DNA molecule separates into two single strands by breaking hydrogen bonds between base pairs. - This typically occurs through **heating** or chemical treatment and is the step that precedes annealing in PCR cycles.
Question 7: All of the following statements are TRUE about second generation antihistaminic agents EXCEPT:
- A. These may possess additional antiallergic mechanisms
- B. These do not impair psychomotor performance
- C. These lack anticholinergic actions
- D. These possess high anti-motion sickness activity (Correct Answer)
Explanation: ***These possess high anti-motion sickness activity*** - Second-generation antihistamines have **poor penetration** into the central nervous system (CNS), making them ineffective for treating **motion sickness**. - **First-generation antihistamines**, which readily cross the blood-brain barrier and have **anticholinergic activity**, are typically used for motion sickness. *These may possess additional antiallergic mechanisms* - Many second-generation antihistamines, such as **cetirizine** and **loratadine**, have additional anti-inflammatory and **antiallergic properties** beyond H1 receptor blockade. - These mechanisms can include inhibiting the release of inflammatory mediators and **stabilizing mast cells**. *These do not impair psychomotor performance* - Second-generation antihistamines are **non-sedating** because they have limited ability to cross the **blood-brain barrier** and thus do not significantly affect CNS function. - This characteristic makes them suitable for use without causing **drowsiness** or impairing activities like driving. *These lack anticholinergic actions* - Unlike first-generation antihistamines, second-generation agents have **minimal to no affinity** for muscarinic acetylcholine receptors. - This lack of **anticholinergic activity** means they do not cause side effects such as **dry mouth**, blurred vision, or urinary retention.
Question 8: Agarose gel electrophoresis from DNA of a population of cells as seen under ultraviolet light is shown below. What is the correct explanation for the finding seen in the band labeled as "C"?
- A. Predominantly necrotic cells
- B. Mixed population of normal and apoptotic cells
- C. A population of viable cells
- D. Apoptotic cells (Correct Answer)
Explanation: ***Apoptotic cells*** - Band C shows a characteristic **DNA ladder pattern** with discrete bands representing fragmentation into nucleosome-sized units (multiples of ~180-200 base pairs), which is pathognomonic of **apoptosis**. [1] - During apoptosis, **endonucleases** cleave DNA at internucleosomal linker regions, creating uniform fragments that migrate as distinct bands on agarose gel electrophoresis. *Predominantly necrotic cells* - **Necrotic cell DNA** undergoes random, non-specific degradation by cellular enzymes, resulting in a continuous **smear pattern** rather than discrete bands. - The **smear appearance** reflects DNA fragments of varying sizes distributed throughout the gel, unlike the organized pattern seen in band C. *Mixed population of normal and apoptotic cells* - A mixed population would show both **intact high molecular weight DNA** (remaining near the gel origin) and the **apoptotic ladder pattern** superimposed. - Band C displays a pure ladder pattern without evidence of intact DNA, indicating a homogeneous apoptotic population. *A population of viable cells* - **Viable cells** maintain intact genomic DNA that remains as a single **high molecular weight band** near the top of the gel. - The **fragmented ladder pattern** in band C is incompatible with viable cell DNA, which should show minimal degradation. **References:** [1] Kumar V, Abbas AK, et al.. Robbins and Cotran Pathologic Basis of Disease. 9th ed. Cellular Responses to Stress and Toxic Insults: Adaptation, Injury, and Death, pp. 63-64.
Question 9: 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.
Question 10: NKX3-1 immunohistochemistry is used for the diagnosis of which of the following?
- A. Colorectal carcinoma
- B. Pancreatic carcinoma
- C. Prostate carcinoma (Correct Answer)
- D. Renal cell carcinoma
Explanation: **Explanation:** **NKX3.1** is a prostate-specific androgen-regulated homeobox gene located on chromosome 8p21. It plays a critical role in normal prostate development and the differentiation of prostatic epithelial cells. 1. **Why Prostate Carcinoma is Correct:** NKX3.1 is currently considered one of the most sensitive and specific immunohistochemical (IHC) markers for **Prostatic Adenocarcinoma**. Its primary clinical utility lies in identifying the prostatic origin of a metastatic tumor, especially when the tumor is high-grade or poorly differentiated [1]. While PSA (Prostate-Specific Antigen) and PSAP (Prostate-Specific Acid Phosphatase) are traditional markers, NKX3.1 often maintains expression in cases where PSA might be focal or negative. 2. **Why Other Options are Incorrect:** * **Colorectal Carcinoma:** The characteristic IHC profile includes **CDX2**, CK20 (+), and CK7 (-). * **Pancreatic Carcinoma:** Typically expresses **CA19-9**, CK7, and CK19. * **Renal Cell Carcinoma (RCC):** The most specific markers for RCC are **PAX8** and **RCC antigen** (CD10 is also frequently positive). **High-Yield Clinical Pearls for NEET-PG:** * **Sensitivity:** NKX3.1 is highly sensitive (>95%) for both primary and metastatic prostate cancer. * **Nuclear Staining:** Unlike PSA (which is cytoplasmic), NKX3.1 shows a distinct **nuclear staining** pattern, making it easier to interpret in small biopsies. * **Differential Diagnosis:** In the context of a "Small Round Blue Cell Tumor" in the testis/prostate region, NKX3.1 helps distinguish prostate alveolar rhabdomyosarcoma (negative) from metastatic prostate cancer (positive). * **Loss of Expression:** Deletion of the 8p21 locus (where NKX3.1 resides) is one of the earliest genetic events in prostate carcinogenesis, often associated with the transition from PIN (Prostatic Intraepithelial Neoplasia) to invasive cancer [1]. **References:** [1] Kumar V, Abbas AK, et al.. Robbins and Cotran Pathologic Basis of Disease. 9th ed. The Lower Urinary Tract and Male Genital System, pp. 988-994.
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