Polymerase Chain Reaction — MCQs
Which PCR technique is best suited for identifying a syndrome with multiple causative agents?
What is the primary function of the sigma subunit of prokaryotic RNA polymerase?
What is the technique for accurate quantification of gene expression?
Which of the following is a primarily RNA based technique?
Which one of the following enzymes is obtained from Thermus aquaticus bacterium that is heat stable and used in PCR at high temperature?
DNA amplification is done in:
Which of the following statements about polymerase chain reaction (PCR) is true?
DNA amplification is done by all, except:
Gene amplification is achieved through
In which of the following conditions is viral load testing done by Real Time PCR of no role in investigative procedures?
Polymerase Chain Reaction Indian Medical PG Practice Questions and MCQs
Question 1: Which PCR technique is best suited for identifying a syndrome with multiple causative agents?
- A. RT-PCR
- B. Multiplex PCR (Correct Answer)
- C. Nested PCR
- D. Conventional PCR
Explanation: ***Multiplex PCR*** - **Multiplex PCR** allows for the simultaneous amplification of **multiple DNA targets** in a single reaction, making it ideal for identifying syndromes with numerous potential causative agents. - This method uses **multiple primer pairs** in one reaction tube, each designed to amplify a specific target sequence, thus efficiently detecting various pathogens or genetic markers. *RT-PCR* - **Reverse Transcription PCR (RT-PCR)** is used to detect **RNA targets** by first converting RNA into cDNA, which is then amplified. - While useful for RNA viruses or gene expression studies, it is not primarily designed for simultaneous detection of multiple diverse causative agents in the same way as multiplex PCR. *Nested PCR* - **Nested PCR** uses two sets of primers in sequential reactions to **increase sensitivity and specificity** by reducing non-specific binding. - This technique is generally employed to detect very low copies of a specific target or to overcome issues with non-specific amplification, rather than for identifying multiple different agents concurrently. *Conventional PCR* - **Conventional PCR** amplifies a **single specific DNA target** using one pair of primers per reaction. [1] - It requires separate reactions for each potential causative agent, making it inefficient and labor-intensive when testing for a syndrome with multiple etiologies. **References:** [1] 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. 56-57.
Question 2: What is the primary function of the sigma subunit of prokaryotic RNA polymerase?
- A. Is inhibited by α-amanitin
- B. Specifically recognizes the promoter site (Correct Answer)
- C. Is part of the core enzyme
- D. Inhibits the activity of RNA polymerase
Explanation: ***Specifically recognizes the promoter site*** - The **sigma subunit** is crucial for **transcription initiation** in prokaryotes, enabling the RNA polymerase holoenzyme to specifically bind to **promoter sequences** on the DNA. - This specific recognition ensures that transcription begins at the correct start site, making it a key component for accurate gene expression. *Inhibits the activity of RNA polymerase* - The sigma subunit does not inhibit RNA polymerase; rather, it **facilitates** its activity by guiding it to the correct transcription start sites. - After initiation, the sigma subunit often **dissociates** from the core enzyme, allowing the core polymerase to proceed with elongation. *Is inhibited by α-amanitin* - **α-amanitin** is a toxin that primarily inhibits **eukaryotic RNA polymerases**, particularly RNA polymerase II, and is not known to inhibit prokaryotic RNA polymerase or its sigma subunit. - Prokaryotic RNA polymerase has a different structure and mechanism, rendering it **insensitive** to α-amanitin. *Is part of the core enzyme* - The sigma subunit is **not considered part of the core enzyme**; the core enzyme consists of the α, β, β', and ω subunits. - Together with the core enzyme, the sigma subunit forms the **RNA polymerase holoenzyme**, which is responsible for initiating transcription.
Question 3: What is the technique for accurate quantification of gene expression?
- A. PCR
- B. Real-Time Reverse Transcriptase PCR (Correct Answer)
- C. Reverse Transcriptase PCR
- D. Northern blot
Explanation: ***Real-Time Reverse Transcriptase PCR*** - This technique allows for the **quantification of gene expression** by concurrently reverse-transcribing RNA to cDNA and amplifying it while monitoring the accumulation of DNA in real-time using fluorescent reporters. - The ** threshold cycle (Ct) value** is inversely proportional to the initial amount of target mRNA, enabling precise quantification. *Northern blot* - This method is used to detect **RNA sequences** and can provide semi-quantitative data about gene expression levels based on band intensity. - However, it is generally **less sensitive** and provides less precise quantification compared to real-time PCR. *PCR* - **Standard PCR** amplifies DNA, but it is not directly used for gene expression quantification as it starts with DNA templates. - While it can be used to detect the presence of a gene, it does not quantify its expression without further modifications or additional steps like reverse transcription and real-time monitoring. *Reverse Transcriptase PCR* - This technique involves **reverse transcribing RNA into cDNA** and then performing standard PCR to amplify the cDNA. - While it confirms the presence of mRNA and allows for cDNA amplification, it is a **qualitative or semi-quantitative** method for expression, as the endpoint detection does not accurately reflect initial mRNA concentration due to plateau effects.
Question 4: Which of the following is a primarily RNA based technique?
- A. Western blotting
- B. Northern blotting (Correct Answer)
- C. Southern blotting
- D. Sanger's technique
Explanation: ***Northern blotting*** - **Northern blotting** is a molecular biology technique used to study **gene expression** by detecting specific **RNA molecules** (mRNA) in a sample. - It involves separating RNA fragments by **gel electrophoresis**, transferring them to a membrane, and then detecting specific sequences using **labeled probes**. *Western blotting* - **Western blotting** is a technique used to detect specific **proteins** in a sample. - It involves separating proteins by **gel electrophoresis**, transferring them to a membrane, and then detecting specific proteins using labeled **antibodies**. *Southern blotting* - **Southern blotting** is a molecular biology method used for the detection of **specific DNA sequences** in DNA samples. - It involves separating **DNA fragments** by **gel electrophoresis**, transferring them to a membrane, and then hybridizing with a labeled probe. *Sanger's technique* - **Sanger sequencing**, or the **dideoxy chain-termination method**, is a widely used method for **DNA sequencing**. - It uses **dideoxynucleotides** to terminate DNA synthesis at specific bases, allowing the determination of the **DNA sequence**.
Question 5: Which one of the following enzymes is obtained from Thermus aquaticus bacterium that is heat stable and used in PCR at high temperature?
- A. DNA gyrase
- B. DNA polymerase III
- C. Taq polymerase (Correct Answer)
- D. Endonuclease
Explanation: ***Taq polymerase*** - This **heat-stable DNA polymerase** is isolated from the thermophilic bacterium *Thermus aquaticus*. - Its ability to withstand high temperatures makes it ideal for the **polymerase chain reaction (PCR)**, where DNA denaturation steps occur at elevated temperatures. *DNA gyrase* - **DNA gyrase** is a type II topoisomerase that introduces negative supercoils into DNA, which is important for DNA replication and transcription. - It is not heat-stable and is not directly used for DNA amplification in PCR. *DNA polymerase III* - **DNA polymerase III** is the primary enzyme responsible for DNA replication in *E. coli* and other bacteria. - It rapidly synthesizes DNA but is **not heat-stable** and would denature at the temperatures required for PCR. *Endonuclease* - **Endonucleases** are enzymes that cleave phosphodiester bonds within a polynucleotide chain. - While essential for processes like DNA repair and restriction mapping, they are not primarily involved in and are not heat-stable for DNA synthesis in PCR.
Question 6: 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 7: Which of the following statements about polymerase chain reaction (PCR) is true?
- A. It is used for the separation of protein fragments.
- B. It is a method for recombinant DNA synthesis.
- C. It is a method for enzymatic amplification of DNA. (Correct Answer)
- D. None of the options.
Explanation: ***It is a method for enzymatic amplification of DNA.*** - PCR is a laboratory technique used to make **millions to billions of copies** of a specific DNA segment. - This amplification is carried out enzymatically by **DNA polymerase**, which synthesizes new DNA strands. *It is a method for recombinant DNA synthesis.* - **Recombinant DNA synthesis** involves combining DNA from different sources, often for genetic engineering, which is a broader application than the core function of PCR. - While PCR can be a *tool* used within recombinant DNA technology (e.g., to amplify a gene for cloning), its primary definition is not synthesizing recombinant DNA itself. *It is used for the separation of protein fragments.* - The separation of protein fragments is typically achieved through techniques like **electrophoresis** (e.g., SDS-PAGE), which separates proteins based on size and charge. - PCR is specifically designed for manipulating and amplifying **DNA**, not proteins. *None of the options.* - This option is incorrect because the statement "It is a method for enzymatic amplification of DNA" accurately describes the fundamental function of PCR.
Question 8: 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 9: 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 10: In which of the following conditions is viral load testing done by Real Time PCR of no role in investigative procedures?
- A. Person with hepatitis B on Tenofovir therapy
- B. HSV causing temporal encephalitis (Correct Answer)
- C. CMV PCR in blood of patient of liver transplant
- D. BK virus in patient of allograft renal transplant
Explanation: ***HSV causing temporal encephalitis*** - While **HSV PCR is crucial for diagnosing HSV encephalitis** from **cerebrospinal fluid (CSF)**, **quantitative viral load testing** does not guide clinical management or predict outcomes. - The key distinction: **qualitative PCR (detecting presence of HSV DNA)** is essential for diagnosis, but **viral load quantification** has no established role in treatment monitoring or prognostication. - The focus is on confirming HSV DNA presence to initiate **antiviral therapy (Acyclovir)**, not on serial viral load measurements for treatment efficacy. *Person with hepatitis B on Tenofovir therapy* - **HBV viral load testing** via **Real-Time PCR** is **absolutely essential** for monitoring the effectiveness of antiviral therapy like Tenofovir and detecting **drug resistance**. - **Quantitative monitoring** is crucial: decreasing HBV DNA levels indicate treatment response, while persistently high levels suggest resistance or non-adherence. - Viral load determines treatment duration and endpoints. *CMV PCR in blood of patient of liver transplant* - **CMV viral load monitoring** by **Real-Time PCR** in blood is **critical** in transplant recipients to detect **CMV reactivation** and guide pre-emptive antiviral therapy. - **Quantitative thresholds** are used to decide when to initiate treatment and assess treatment response. - Rising viral loads indicate need for intervention to prevent severe CMV disease. *BK virus in patient of allograft renal transplant* - **BK virus (BKV) PCR** in plasma or urine is **vital** for detecting **BKV reactivation** and monitoring **BKV nephropathy** in renal transplant recipients. - **Serial viral load measurements** prompt reduction in immunosuppression when levels rise, preventing allograft dysfunction or loss. - Quantitative monitoring is the standard of care for BKV management.
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