PCR Technology — MCQs
Steps of PCR in sequence are?
What is the primary function of the sigma subunit of prokaryotic RNA polymerase?
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 by all, except:
The Takayama test is primarily used for what purpose?
DNA fingerprinting is based on possessing what in DNA?
Latte's crust of blood stain is used to detect which of the following?
For which of the following anticoagulants are blood samples transported for DNA fingerprinting?
What is a confirmatory test for species of origin?
PCR Technology Indian Medical PG Practice Questions and MCQs
Question 1: 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 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: 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 4: 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 5: 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 6: The Takayama test is primarily used for what purpose?
- A. To determine the crystalline structure of a stain. (Correct Answer)
- B. To identify the species of origin of a stain.
- C. To perform blood grouping.
- D. None of the above.
Explanation: The **Takayama test** (also known as the **Haemochromogen crystal test**) is a microcrystalline test used for the confirmatory identification of blood. ### Why Option A is Correct The test involves treating a suspected bloodstain with Takayama reagent (containing glucose, sodium hydroxide, and pyridine). When heated, the pyridine reacts with the heme group of hemoglobin to form **pink, feathery, needle-shaped crystals** of pyridine haemochromogen. Therefore, the test's primary mechanism is the formation and observation of a specific **crystalline structure**, confirming the presence of hemoglobin. ### Why Other Options are Incorrect * **Option B:** Species of origin is determined using **serological tests** like the **Precipitin test** or Electrophoresis (e.g., Counter-immunoelectrophoresis), which rely on antigen-antibody reactions. * **Option C:** Blood grouping is typically performed using the **Absorption-Elution method** (Siracusa method) or the **Lattes Crust method** to identify ABO antigens or antibodies in dried stains. ### High-Yield Pearls for NEET-PG * **Confirmatory vs. Screening:** The Takayama test and Teichmann test are **confirmatory** for blood. Benzidine, Phenolphthalein (Kastle-Meyer), and Luminol are **presumptive/screening** tests. * **Takayama vs. Teichmann:** The Takayama test is generally preferred over the Teichmann (Haemin) test because it is more reliable, works better on old/weathered stains, and requires less heat. * **Reagent Components:** Remember the "Pyridine" in Takayama reagent; it is the key chemical that produces the characteristic pink crystals.
Question 7: DNA fingerprinting is based on possessing what in DNA?
- A. Constant tandem repeat
- B. Variable number tandem repeat (Correct Answer)
- C. Non-repetitive sequence
- D. Exon
Explanation: **Explanation:** DNA fingerprinting (DNA profiling) relies on the fact that while 99.9% of human DNA is identical, certain regions of non-coding DNA are highly polymorphic. The correct answer is **Variable Number Tandem Repeats (VNTRs)**, also known as minisatellites. These are short sequences of DNA (10–100 base pairs) that are repeated head-to-tail. The number of repeats varies significantly between individuals, creating a unique genetic "barcode" used for identification. **Analysis of Options:** * **B. Variable Number Tandem Repeat (Correct):** These serve as the molecular basis for RFLP (Restriction Fragment Length Polymorphism) and traditional DNA profiling because the length of these repetitive segments is inherited and unique to every individual (except monozygotic twins). * **A. Constant Tandem Repeat:** If repeats were constant across the population, they would provide no discriminatory power for forensic identification. * **C. Non-repetitive Sequence:** Most of the human genome consists of unique, non-repetitive sequences (genes). These do not show the high level of variation required to distinguish between two individuals. * **D. Exon:** Exons are the coding regions of DNA. They are highly conserved to maintain protein function; therefore, they lack the polymorphism needed for forensic profiling. **NEET-PG High-Yield Pearls:** * **Father of DNA Fingerprinting:** Sir Alec Jeffreys (World); Dr. Lalji Singh (India). * **STRs (Short Tandem Repeats):** Currently the "Gold Standard" in forensics. These are microsatellites (2–6 bp) that are easier to amplify via PCR than VNTRs. * **Mitochondrial DNA (mtDNA):** Used for maternal lineage and when samples are highly degraded (e.g., old bones/hair shafts). * **Specimen Choice:** Any nucleated cell can be used (Blood, Semen, Hair follicle, Skin). Mature RBCs cannot be used as they lack a nucleus.
Question 8: Latte's crust of blood stain is used to detect which of the following?
- A. Nature of the stain
- B. Detection species
- C. Blood group (Correct Answer)
- D. Secretor status
Explanation: **Explanation:** **Lattes’ Crust Method** is a classic serological technique used in forensic medicine for the determination of the **ABO blood group** from dried bloodstains. **Why the correct answer is right:** The principle behind this method is the detection of **iso-antibodies (agglutinins)** present in the dried crust of the bloodstain. When the blood dries, the antibodies (Anti-A and Anti-B) are preserved. In this test, the crust is exposed to known A, B, and O red blood cells. Agglutination of the known cells indicates the presence of the corresponding antibody in the stain, allowing the forensic expert to deduce the blood group (e.g., if the stain agglutinates B cells, it contains Anti-B, indicating Group A). **Analysis of incorrect options:** * **Nature of the stain:** This is determined by preliminary (presumptive) tests like the Phenolphthalein (Kastle-Meyer) test or confirmatory tests like the Teichmann or Takayama crystal tests. * **Detection of species:** This is determined by the **Precipitin test**, which identifies whether the blood is of human or animal origin. * **Secretor status:** This refers to the presence of blood group antigens in other body fluids (saliva, semen). It is typically detected using the **Absorption-Elution** or **Absorption-Inhibition** methods, not the Lattes’ Crust method. **High-Yield Pearls for NEET-PG:** * **Lattes’ Crust Method:** Detects **Antibodies** (Agglutinins). * **Absorption-Elution Method:** Detects **Antigens** (Agglutinogens); it is much more sensitive than the Lattes method and is the gold standard for old/dried stains. * **Takayama Test:** Produces pink, feathery, salmon-colored **Hemochromogen** crystals (Confirmatory for blood). * **Teichmann Test:** Produces rhombic, dark brown **Haemin** crystals.
Question 9: For which of the following anticoagulants are blood samples transported for DNA fingerprinting?
- A. Saline
- B. EDTA (Correct Answer)
- C. NaF
- D. Thymol
Explanation: **Explanation:** **1. Why EDTA is the Correct Answer:** EDTA (Ethylenediaminetetraacetic acid) is the preferred anticoagulant for DNA fingerprinting/profiling because it is a potent **chelating agent**. It binds to divalent metal ions, specifically **Magnesium (Mg²⁺)**. Since **DNase enzymes** (which degrade DNA) require Mg²⁺ as a cofactor to function, EDTA effectively inhibits these enzymes, preserving the structural integrity of the DNA for high-molecular-weight analysis. Furthermore, EDTA does not interfere with the **Polymerase Chain Reaction (PCR)**, which is the standard technique used in forensic DNA analysis. **2. Why the Other Options are Incorrect:** * **Saline (A):** This is an isotonic solution, not an anticoagulant. While it can be used to moisten swabs, it does not prevent clotting or inhibit DNase activity. * **Sodium Fluoride (NaF) (C):** NaF is an antiglycolytic agent used for blood glucose estimation. It inhibits the enzyme enolase. In forensics, it is used for **toxicology** (e.g., alcohol levels) but is avoided for DNA work as it can interfere with PCR amplification. * **Thymol (D):** This is a preservative used primarily for urine samples to prevent bacterial growth. It has no role in preserving DNA in blood samples. **3. High-Yield Clinical Pearls for NEET-PG:** * **Color Code:** EDTA tubes have **Lavender/Purple** tops. * **Sample Volume:** For forensic DNA profiling, 2–5 ml of whole blood is typically required. * **Storage:** If immediate transport is not possible, EDTA blood should be refrigerated at **4°C**, not frozen (to avoid cell lysis before processing). * **Alternative:** If blood is dry, it should be collected on sterile gauze and kept dry; moisture leads to fungal growth which destroys DNA.
Question 10: What is a confirmatory test for species of origin?
- A. Origin Test
- B. Ouchterlony Test (Correct Answer)
- C. Olympus Test
- D. SOO Test
Explanation: ### Explanation The **Ouchterlony Test** (Double Immunodiffusion) is the gold standard confirmatory test for determining the **species of origin** in forensic biology. **1. Why Ouchterlony Test is Correct:** When a biological sample (like a bloodstain) is found at a crime scene, forensic experts must determine if it is human or animal. The Ouchterlony test uses the principle of **antigen-antibody reaction** in an agar gel. An extract of the unknown sample (antigen) and specific anti-sera (antibodies, e.g., anti-human serum) are placed in separate wells. If the sample belongs to that species, the antigens and antibodies diffuse toward each other, meeting to form a visible **precipitin line**. This confirms the species identity with high specificity. **2. Analysis of Incorrect Options:** * **A. Origin Test:** This is a generic term and not a specific forensic laboratory technique. * **C. Olympus Test:** This is a distractor; Olympus is a well-known brand of microscopes and optical equipment, not a serological test. * **D. SOO Test:** This is an invented acronym (likely standing for "Species of Origin") used to confuse candidates; no such standardized test exists in forensic practice. **3. High-Yield Clinical Pearls for NEET-PG:** * **Precipitin Test:** The broader category of tests for species origin; Ouchterlony is the most common variant. * **Kastle-Meyer Test:** A common screening (presumptive) test for the *presence* of blood (turns pink), but it does not determine species. * **Teichmann and Takayama Tests:** Confirmatory tests for the *presence* of blood (crystal tests), not species. * **Species Identification:** Essential in hit-and-run cases or animal poaching investigations to differentiate human blood from animal blood.
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