Nucleic Acid Extraction Methods — MCQs
DNA sequence is determined by?
What is the technique for accurate quantification of gene expression?
Which of the following statements about nucleic acid amplification tests (NAATs) for STIs is FALSE?
Which of the following is a primarily RNA based technique?
Separation of proteins based on size is done by
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 by all, except:
What is the blood form of folic acid?
Diagnosis of C. difficile infection is made by which of the following methods?
Following are true of Gram negative bacterial cell wall compared to Gram positive bacteria except:
Nucleic Acid Extraction Methods Indian Medical PG Practice Questions and MCQs
Question 1: DNA sequence is determined by?
- A. Sanger sequencing (Correct Answer)
- B. PCR
- C. FISH
- D. Gel electrophoresis
Explanation: ***Correct: Sanger sequencing*** - **Sanger sequencing** (chain-termination method) is the gold standard technique used to determine the exact order of nucleotides within a DNA molecule - It uses dideoxynucleotides (ddNTPs) to terminate DNA strand elongation at specific bases, producing fragments of varying lengths - These fragments are separated by capillary electrophoresis and the sequence is read based on the terminal fluorescent label - Directly determines DNA sequence with high accuracy *Incorrect: PCR* - **Polymerase Chain Reaction (PCR)** amplifies specific DNA segments to create millions of copies - It does NOT determine the sequence itself - it only makes copies of DNA - PCR-amplified DNA can be used as a template for subsequent sequencing, but PCR itself doesn't reveal sequence information *Incorrect: FISH* - **Fluorescence in situ hybridization (FISH)** detects and localizes specific DNA sequences on chromosomes - Used for chromosomal mapping and detecting chromosomal abnormalities - Does not determine the nucleotide sequence *Incorrect: Gel electrophoresis* - Separates DNA fragments based on size and charge - Used to analyze DNA but cannot determine the specific nucleotide sequence - Useful for visualizing DNA after amplification or restriction digestion
Question 2: 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 3: Which of the following statements about nucleic acid amplification tests (NAATs) for STIs is FALSE?
- A. They can be used for test of cure after 3 weeks
- B. They can detect dead organisms after treatment
- C. They can be used for pharyngeal gonorrhea screening
- D. They are less sensitive than culture for rectal chlamydia (Correct Answer)
Explanation: ***They are less sensitive than culture for rectal chlamydia*** - This statement is **FALSE**. NAATs are generally **more sensitive** than culture methods for detecting *Chlamydia trachomatis* in all anatomical sites, including the rectum. - The high sensitivity of NAATs allows for the detection of very low bacterial loads, making them the preferred diagnostic method for many STIs. *They can be used for test of cure after 3 weeks* - This statement is generally **true**. While a "test of cure" (TOC) is not routinely recommended for uncomplicated *Chlamydia* or *Gonorrhea* infections due to high treatment efficacy, it can be considered in specific circumstances (e.g., persistent symptoms, pregnancy, or use of alternative regimens). - If a TOC is performed, it should ideally be done **no sooner than 3 weeks post-treatment** to minimize potential false positives from detecting residual nucleic acids from dead organisms. *They can detect dead organisms after treatment* - This statement is **true**. NAATs detect the **nucleic acids (DNA or RNA)** of the target organism. - These nucleic acids can persist in the body for a period even after the organism has been killed by treatment, leading to a positive NAAT result despite successful eradication of the infection. *They can be used for pharyngeal gonorrhea screening* - This statement is **true**. NAATs are the **recommended method** for detecting *Neisseria gonorrhoeae* in extragenital sites, including the pharynx. - Pharyngeal gonorrhea is often **asymptomatic**, making screening of at-risk individuals important for public health.
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: Separation of proteins based on size is done by
- A. Affinity chromatography
- B. High performance liquid chromatography
- C. SDS-Polyacrylamide gel electrophoresis (Correct Answer)
- D. Ion exchange chromatography
Explanation: ***SDS-Polyacrylamide gel electrophoresis*** - **SDS-PAGE** separates proteins primarily based on their **molecular weight** (size). - Proteins are denatured and coated with negatively charged **SDS**, causing them to migrate through a polyacrylamide gel based on size. *Affinity chromatography* - This technique separates proteins based on their **specific binding affinity** to a ligand. - It does not directly separate based on size, but rather on **molecular recognition**. *High performance liquid chromatography* - **HPLC** is a chromatographic technique that separates molecules in a complex mixture, but the primary basis of separation depends on the column type. - While some HPLC methods (**size-exclusion HPLC**) can separate by size, it is a broader technique and not the most specific answer for protein size separation in general context. *Ion exchange chromatography* - This method separates proteins based on their **net charge** at a particular pH. - Proteins bind to a charged resin and are eluted by increasing salt concentration or changing pH, not based on size.
Question 6: 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 7: 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 8: What is the blood form of folic acid?
- A. Folinic acid
- B. Pteroglutamate
- C. Methyltetrahydrofolate (Correct Answer)
- D. None of the options
Explanation: ***Methyltetrahydrofolate*** - **5-methyltetrahydrofolate (5-MTHF)** is the **primary circulating form** of folate in the blood plasma and the most metabolically active form of folate. - It plays a crucial role in various metabolic pathways, especially in **one-carbon metabolism** for DNA synthesis and repair. *Folinic acid* - **Folinic acid** (leucovorin) is a **reduced form of folic acid** that does not require reduction by dihydrofolate reductase for activity. - It is often used as a therapeutic agent, particularly to **counteract the effects of methotrexate** toxicity, but it is not the main physiological circulating form. *Pteroglutamate* - **Pteroglutamate** is a generic term referring to compounds structurally related to folic acid, which is itself chemically known as pteroylglutamic acid. - While it describes the **general structure**, it is not the specific blood form of folic acid. *None of the options* - This option is incorrect because **methyltetrahydrofolate** is indeed the correct answer.
Question 9: Diagnosis of C. difficile infection is made by which of the following methods?
- A. Stool microscopy for pseudomembranes
- B. Culture
- C. Toxin gene detection by polymerase chain reaction (PCR) (Correct Answer)
- D. Enzyme-linked immunosorbent assay (ELISA)
Explanation: ***Toxin gene detection by polymerase chain reaction (PCR)*** - **PCR for toxin genes (tcdA and tcdB)** is the most sensitive and specific method for diagnosing **Clostridioides difficile infection (CDI)**, directly detecting the genetic material responsible for the pathology. - This method is superior because it identifies the presence of toxigenic C. difficile, which is crucial for determining clinical significance and guiding treatment. *Stool microscopy for pseudomembranes* - While **pseudomembranes** are a hallmark of severe CDI, their detection requires **endoscopy** and is not a direct diagnostic test for the pathogen itself. - Furthermore, their absence does not rule out CDI, as pseudomembranes may not form in all cases, especially milder ones. *Culture* - **Culture for C. difficile** can identify the presence of the organism, but it does not differentiate between toxigenic and non-toxigenic strains. - Many individuals can be **colonized with non-toxigenic C. difficile** without having an active infection, leading to false positives if culture alone is used for diagnosis. *Enzyme - linked immunosorbent assay (ELISA)* - ELISA tests primarily detect **C. difficile toxins A and B** or **glutamate dehydrogenase (GDH)** antigen in stool. - While rapid, ELISA for toxins A/B has **lower sensitivity** than PCR, potentially missing cases, and GDH detection alone only indicates the presence of C. difficile (toxigenic or non-toxigenic), requiring further toxin testing for confirmation.
Question 10: Following are true of Gram negative bacterial cell wall compared to Gram positive bacteria except:
- A. Thinner
- B. Presence of lipopolysaccharide
- C. Presence of outer membrane
- D. Presence of Teichoic acid (Correct Answer)
Explanation: ***Presence of Teichoic acid*** - **Teichoic acid** is a unique component of the cell wall in **Gram-positive bacteria**, playing a role in cell wall structure and antigenicity. - Its presence is **not a characteristic of Gram-negative bacteria**, making this statement the exception. *Thinner* - The cell wall of **Gram-negative bacteria** is indeed **thinner** than that of Gram-positive bacteria. - This **thin peptidoglycan layer** (2-3 nm) is much less substantial compared to the thick peptidoglycan layer (20-80 nm) of Gram-positive bacteria. *Presence of lipopolysaccharide* - **Lipopolysaccharide (LPS)**, or endotoxin, is a characteristic component of the **outer membrane** of Gram-negative bacteria. - LPS contributes to the **pathogenicity** of Gram-negative bacteria and is absent in Gram-positive bacteria. *Presence of outer membrane* - **Gram-negative bacteria** have a unique **outer membrane** that lies external to the thin peptidoglycan layer. - This outer membrane contains LPS and porins, and is a distinguishing feature **absent in Gram-positive bacteria**, which have only a single cytoplasmic membrane.
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