Biochemical Techniques — MCQs

Biochemical Techniques Indian Medical PG Practice Questions and MCQs

Question 121: Gel filtration chromatography separates proteins based on which property?

A. Polarity
B. Hydrophobicity
C. Stokes radius (Correct Answer)
D. pH

Explanation: ### Explanation **Correct Answer: C. Stokes radius** **Mechanism of Action:** Gel filtration chromatography, also known as **Size-Exclusion Chromatography (SEC)**, separates molecules based on their size and shape. The stationary phase consists of porous beads (e.g., Sephadex). * **Small molecules** enter the pores of the beads, increasing their path length and slowing their movement. * **Large molecules** are excluded from the pores and travel only through the void volume between beads, eluting **first**. The effective size of a molecule in solution, which determines its ability to enter these pores, is termed the **Stokes radius**. While molecular weight is often used as a proxy, the Stokes radius accounts for the hydration shell and the 3D shape of the protein. **Why Other Options are Incorrect:** * **A. Polarity:** Separation based on polarity is the principle of **Normal Phase or Partition Chromatography**. * **B. Hydrophobicity:** This is the basis for **Hydrophobic Interaction Chromatography (HIC)** or Reverse-Phase HPLC. * **D. pH:** While pH affects a protein's charge, separation based on charge (isoelectric point) is the principle of **Ion Exchange Chromatography** or Isoelectric Focusing. **High-Yield NEET-PG Pearls:** 1. **Elution Order:** In Gel Filtration, the **largest** proteins elute first, and the **smallest** elute last. 2. **Applications:** It is commonly used for **desalting** a protein solution (removing small salt ions) and determining the quaternary structure (molecular weight) of proteins. 3. **Void Volume ($V_0$):** The volume of the mobile phase outside the gel beads. Large molecules elute at $V_0$. 4. **Comparison:** Do not confuse this with **SDS-PAGE**, where smaller proteins move faster; in Gel Filtration, larger proteins move faster.

Question 122: Two proteins with the same charge can be separated by which of the following methods?

A. Agarose gel electrophoresis
B. DEAE-Cellulose chromatography
C. Sephadex gel filtration chromatography (Correct Answer)
D. None of the above

Explanation: ### Explanation The separation of proteins depends on exploiting differences in their physical and chemical properties, such as size, net charge, or binding affinity. **Why Sephadex gel filtration chromatography is correct:** Sephadex gel filtration (also known as **Size-Exclusion Chromatography**) separates proteins based on their **molecular weight and size (hydrodynamic radius)**, not their charge. The stationary phase consists of porous beads. Large molecules cannot enter the pores and elute first (void volume), while smaller molecules enter the pores, follow a longer path, and elute later. Therefore, if two proteins have the same charge but different sizes, this technique will effectively separate them. **Why the other options are incorrect:** * **Agarose gel electrophoresis:** This technique separates molecules primarily based on their **charge-to-mass ratio**. If two proteins have the same net charge and similar shapes, they are unlikely to separate effectively in a standard electrophoretic field. * **DEAE-Cellulose chromatography:** This is a type of **Anion Exchange Chromatography**. It separates proteins based on their **net surface charge**. Proteins with the same charge would have similar binding affinities to the positively charged DEAE matrix and would elute together, making separation impossible. **High-Yield NEET-PG Pearls:** * **Molecular Sieving:** Another name for Gel Filtration. * **Elution Order:** In Gel Filtration, **Large** proteins elute **First**; in SDS-PAGE (electrophoresis), **Small** proteins migrate **Fastest**. * **SDS-PAGE:** Unlike native electrophoresis, SDS-PAGE separates proteins solely by **mass** because the detergent SDS coats proteins with a uniform negative charge, masking their intrinsic charge. * **Isoelectric Focusing (IEF):** Separates proteins based on their **isoelectric point (pI)**.

Question 123: Which cation is commonly used in PCR?

A. Calcium
B. Lithium
C. Magnesium (Correct Answer)
D. Sodium

Explanation: **Explanation:** **1. Why Magnesium ($Mg^{2+}$) is the Correct Answer:** Magnesium ions (usually added as $MgCl_2$) are an essential **cofactor** for **Taq DNA polymerase**. The enzyme requires divalent cations to function. $Mg^{2+}$ ions facilitate the formation of the phosphodiester bond by stabilizing the transition state of the enzyme-substrate complex and neutralizing the negative charges on the phosphate backbone of the DNA and the incoming dNTPs. This reduces electrostatic repulsion, allowing the primer to anneal to the template and the polymerase to catalyze DNA synthesis. **2. Why the Other Options are Incorrect:** * **Calcium ($Ca^{2+}$):** While a divalent cation, $Ca^{2+}$ actually inhibits DNA polymerase activity by competing with $Mg^{2+}$ for the binding site, leading to reduced efficiency. * **Lithium ($Li^+$) and Sodium ($Na^+$):** These are monovalent cations. While monovalent cations (like $K^+$ or $Na^+$) are sometimes used in PCR buffers to neutralize the DNA backbone and affect the melting temperature ($T_m$), they cannot replace the catalytic role of a divalent cofactor like Magnesium. **3. NEET-PG High-Yield Facts & Clinical Pearls:** * **Concentration Matters:** Too little $Mg^{2+}$ results in low yield or no product; too much $Mg^{2+}$ decreases the specificity of the reaction, leading to **non-specific amplification** (spurious bands). * **Chelating Agents:** If a sample contains **EDTA** (a chelator), it can sequester $Mg^{2+}$ and inhibit the PCR reaction. This is why DNA for PCR is often stored in low-EDTA buffers. * **Other Components:** Remember the "Ingredients" of PCR: Template DNA, Primers (forward and reverse), dNTPs, Taq Polymerase, and $Mg^{2+}$ buffer. * **Taq Polymerase:** Derived from the thermophilic bacterium *Thermus aquaticus*; it is heat-stable, which is crucial for the denaturation step ($94\text{--}96^\circ\text{C}$).

Question 124: Which of the following enzymes is present inside the given organelle?

A. Glucokinase
B. Isocitrate dehydrogenase
C. Glucose-6-phosphatase (Correct Answer)
D. Fructose 2, 6 bisphosphatase

Explanation: ***Glucose-6-phosphatase*** - This enzyme is specifically localized to the **endoplasmic reticulum (ER)** membrane and plays a crucial role in **gluconeogenesis** and **glycogenolysis**. - It catalyzes the final step in glucose production by converting **glucose-6-phosphate** to free glucose, which can then be released into the bloodstream. *Glucokinase* - This enzyme is primarily found in the **cytosol** of hepatocytes and pancreatic beta cells, not in the ER. - It functions as a **glucose sensor** and catalyzes the phosphorylation of glucose to glucose-6-phosphate during **glycolysis**. *Isocitrate dehydrogenase* - This enzyme is located in the **mitochondrial matrix** as part of the **citric acid cycle (Krebs cycle)**. - It catalyzes the oxidative decarboxylation of **isocitrate** to **α-ketoglutarate**, generating **NADH** and **CO₂**. *Fructose 2, 6 bisphosphatase* - This enzyme is found in the **cytosol** and is involved in regulating **glycolysis** and **gluconeogenesis**. - It hydrolyzes **fructose-2,6-bisphosphate** to **fructose-6-phosphate**, acting as a key regulatory enzyme in glucose metabolism.

Question 125: The Southern blot test is used for the detection of which of the following?

A. Antibodies against viral proteins
B. DNA (Correct Answer)
C. RNA
D. Carbohydrate epitopes

Explanation: **Explanation:** The **Southern blot** is a fundamental molecular biology technique used to detect specific **DNA** sequences within a complex mixture of genomic DNA. The process involves four key steps: (1) Digestion of DNA using restriction endonucleases, (2) Separation of fragments by size via gel electrophoresis, (3) Transfer (blotting) of the DNA onto a nitrocellulose or nylon membrane, and (4) Hybridization with a labeled, sequence-specific DNA probe for visualization. **Analysis of Options:** * **Option B (DNA):** This is the correct answer. Named after Edwin Southern, this technique is the gold standard for identifying gene mutations, deletions, or insertions (e.g., detecting Sickle Cell Anemia or Fragile X syndrome). * **Option C (RNA):** RNA is detected using the **Northern blot**. This technique measures gene expression levels by identifying specific mRNA sequences. * **Option A (Antibodies/Proteins):** Proteins (including antibodies) are detected using the **Western blot**. This is clinically significant as a confirmatory test for HIV (detecting antibodies against viral proteins like p24 or gp120). * **Option D (Carbohydrate epitopes):** While less common, the detection of carbohydrates or lipids is sometimes referred to as **Eastern blotting** (though this term is also used for post-translational modifications of proteins). **High-Yield Clinical Pearls for NEET-PG:** * **Mnemonic (SNOW DROP):** * **S**outhern = **D**NA * **N**orthern = **R**NA * **O** = **O** (No technique) * **W**estern = **P**rotein * **Southwestern Blot:** Used specifically to detect **DNA-binding proteins** (e.g., transcription factors like c-Jun or c-Fos). * **Application:** Southern blotting is used in **RFLP (Restriction Fragment Length Polymorphism)** analysis for forensic "DNA fingerprinting" and prenatal diagnosis of genetic disorders.

Question 126: Which ion is essential in PCR?

A. Ca+2
B. Cu+2
C. Mg+2 (Correct Answer)
D. Zn+2

Explanation: **Explanation:** **Why Mg+2 is the Correct Answer:** Magnesium ions ($Mg^{2+}$) act as an essential **cofactor** for **Taq DNA polymerase**, the enzyme responsible for synthesizing new DNA strands in a Polymerase Chain Reaction (PCR). $Mg^{2+}$ functions by coordinating with the phosphate groups of the deoxynucleotide triphosphates (dNTPs) and the enzyme's active site, facilitating the formation of phosphodiester bonds. Furthermore, $Mg^{2+}$ helps stabilize the primer-template complex. The concentration of $Mg^{2+}$ is critical: too little results in low yield, while too much can lead to non-specific amplification (mispriming). **Why Other Options are Incorrect:** * **Ca+2 (Calcium):** While vital for cell signaling and blood coagulation, $Ca^{2+}$ is not a cofactor for DNA polymerase. In fact, high concentrations of calcium can inhibit PCR. * **Cu+2 (Copper):** Copper is a cofactor for enzymes like Cytochrome c oxidase and Superoxide dismutase, but it is not involved in DNA replication and can be toxic to the PCR process. * **Zn+2 (Zinc):** Zinc is a structural component of "Zinc finger" transcription factors and a cofactor for Carbonic anhydrase, but it does not facilitate the catalytic activity of Taq polymerase. **High-Yield Clinical Pearls for NEET-PG:** * **Taq Polymerase:** Derived from the thermophilic bacterium *Thermus aquaticus*; it is heat-stable, allowing it to survive the denaturation step ($94$-$95^\circ C$). * **Chelating Agents:** EDTA (found in purple-top vacutainers) inhibits PCR because it chelates $Mg^{2+}$, removing the necessary cofactor for the enzyme. * **PCR Steps:** Denaturation ($95^\circ C$) $\rightarrow$ Annealing ($50$-$65^\circ C$) $\rightarrow$ Extension ($72^\circ C$). * **RT-PCR:** Used to detect RNA viruses (like SARS-CoV-2) by first converting RNA to cDNA using Reverse Transcriptase.

Question 127: Which blotting technique is used for RNA analysis?

A. Western blot
B. Northern blot (Correct Answer)
C. Southern blot
D. None of the above

Explanation: **Explanation:** Blotting techniques are fundamental laboratory procedures used to identify specific biological molecules within a complex mixture. The correct answer is **Northern blot**, which is specifically designed for the detection and analysis of **RNA** sequences. **1. Why Northern Blot is Correct:** In Northern blotting, RNA fragments are separated by size via gel electrophoresis and then transferred to a membrane (nitrocellulose or nylon). These fragments are then identified using a labeled nucleic acid probe that is complementary to the target RNA sequence. This technique is crucial for studying **gene expression** by measuring the amount of mRNA in a tissue sample. **2. Analysis of Incorrect Options:** * **Southern Blot (Option C):** Named after Edwin Southern, this technique is used for **DNA** analysis. It involves the detection of specific DNA sequences and is used in applications like DNA fingerprinting and detecting genetic mutations. * **Western Blot (Option A):** This technique is used for **Protein** analysis. It uses specific antibodies to identify target proteins and is clinically significant as a confirmatory test for HIV (detecting anti-HIV antibodies). **3. High-Yield Clinical Pearls for NEET-PG:** To remember these easily, use the mnemonic **SNOW DROP**: * **S**outhern = **D**NA * **N**orthern = **R**NA * **O** = (nothing) * **W**estern = **P**rotein * **Southwestern Blot:** A hybrid technique used to identify **DNA-binding proteins** (e.g., transcription factors like c-Jun or c-Fos). * **Eastern Blot:** Used to analyze post-translational modifications of proteins (e.g., carbohydrates or lipids). * **ELISA vs. Western:** While both detect proteins/antibodies, Western blot is more specific and serves as the "gold standard" for confirmation.

Question 128: All of the following assays are used for estimation of total serum protein, except?

A. Biuret
B. Bradford
C. Lowry's
D. Bromocresol green (Correct Answer)

Explanation: ### Explanation The estimation of serum proteins is a fundamental biochemical analysis. The key to this question lies in distinguishing between methods that measure **total protein** versus those specific to **albumin**. **Why Bromocresol Green (BCG) is the correct answer:** Bromocresol green is a dye-binding technique specifically used for the **quantitation of Serum Albumin**, not total protein. At a slightly acidic pH, albumin acts as a cation and binds to the anionic BCG dye, causing a color shift from yellow-green to blue-green. It is the most common automated method for albumin due to its speed and specificity. **Analysis of incorrect options (Total Protein Methods):** * **Biuret Method (Option A):** The gold standard and most common method for **Total Protein**. It relies on copper ions ($Cu^{2+}$) forming a violet-colored complex with peptide bonds in an alkaline medium. It requires at least two peptide bonds to react. * **Lowry’s Method (Option C):** A highly sensitive method combining the Biuret reaction with the reduction of the **Folin-Ciocalteu reagent** by aromatic amino acids (Tyrosine and Tryptophan). It is used for dilute protein solutions. * **Bradford Assay (Option B):** A rapid method based on the binding of **Coomassie Brilliant Blue G-250** dye to proteins. The dye shifts its absorption maximum from 465 nm to 595 nm upon binding to protein (primarily basic and aromatic residues). **Clinical Pearls for NEET-PG:** * **A/G Ratio:** Total Protein minus Albumin gives the **Globulin** fraction. A reversed A/G ratio (normal 1.2:1 to 2:1) is seen in Multiple Myeloma and Chronic Liver Disease. * **Kjeldahl Method:** The reference method for total protein based on nitrogen content, though too cumbersome for routine clinical use. * **Bence-Jones Proteins:** Detected via heat precipitation or electrophoresis, not standard Biuret/BCG methods.

Question 129: Concentration of DNA is measured by:

A. Absorption at 260 nm (Correct Answer)
B. D-oxyribose estimation
C. Infrared examination
D. Ultrasound examination

Explanation: **Explanation:** **1. Why Option A is Correct:** The concentration and purity of DNA are most commonly measured using **UV Spectrophotometry**. DNA contains nitrogenous bases (purines and pyrimidines) with conjugated double bonds that naturally absorb ultraviolet light. The maximum absorption peak for nucleic acids occurs at **260 nm**. According to the **Beer-Lambert Law**, the amount of light absorbed is directly proportional to the concentration of the DNA in the sample. * *High-yield fact:* An Optical Density (OD) of 1.0 at 260 nm corresponds to approximately 50 µg/mL of double-stranded DNA. **2. Why Other Options are Incorrect:** * **B. D-oxyribose estimation:** While DNA contains deoxyribose, measuring the sugar component (e.g., via the Dische diphenylamine test) is a colorimetric chemical assay. It is less precise, more destructive to the sample, and not the standard method for rapid quantification compared to spectrophotometry. * **C. Infrared examination:** IR spectroscopy is used to identify functional groups and molecular structures (vibrational modes) rather than quantifying the concentration of nucleic acids in a clinical or research setting. * **D. Ultrasound examination:** This is a diagnostic imaging modality used to visualize internal body structures; it has no application in measuring molecular concentrations of DNA. **Clinical Pearls for NEET-PG:** * **Purity Check (A260/A280 Ratio):** To check for protein contamination, the ratio of absorbance at 260 nm to 280 nm is calculated. * **Pure DNA:** Ratio of **~1.8**. * **Pure RNA:** Ratio of **~2.0**. * A lower ratio indicates protein or phenol contamination. * **Hyperchromicity:** When DNA is denatured (melting), its absorbance at 260 nm **increases**. This is known as the hyperchromic effect.

Question 130: Which of the following methods is used for the analysis of the C-terminal end of a polypeptide?

A. Sanger's method
B. Edman's degradation method
C. Akabori method (Correct Answer)
D. None of the above

Explanation: **Explanation:** Protein sequencing involves identifying the specific order of amino acids in a polypeptide chain. This is typically achieved by identifying the N-terminal (amino) and C-terminal (carboxyl) ends. **1. Why Akabori Method is Correct:** The **Akabori method (Hydrazinolysis)** is a chemical technique used for **C-terminal analysis**. In this process, the polypeptide is heated with hydrazine ($NH_2-NH_2$). Hydrazine cleaves all peptide bonds and converts every amino acid into an amino acid hydrazide, **except for the C-terminal amino acid**. Since the C-terminal residue has a free carboxyl group (not involved in a peptide bond), it remains as a free amino acid, which can then be isolated and identified via chromatography. **2. Analysis of Incorrect Options:** * **Sanger’s Method (1-fluoro-2,4-dinitrobenzene / FDNB):** This reagent reacts with the free $\alpha$-amino group of the **N-terminal** amino acid to form a yellow DNP-amino acid derivative. It is used for N-terminal analysis. * **Edman’s Degradation (Phenylisothiocyanate / PITC):** This is the most common method for **N-terminal** sequencing. It removes one amino acid at a time from the N-terminus without hydrolyzing the rest of the peptide chain, allowing for automated sequencing (Sequenator). **High-Yield Clinical Pearls for NEET-PG:** * **N-terminal Reagents:** Sanger’s (FDNB), Edman’s (PITC), and Dansyl Chloride. * **C-terminal Reagents:** Akabori method (Hydrazinolysis) and **Carboxypeptidases** (enzymatic method). * **Cyanogen Bromide (CNBr):** Specifically cleaves peptide bonds at the carboxyl side of **Methionine** residues. * **Trypsin:** Cleaves at the carboxyl side of **Lysine and Arginine** (basic amino acids).

Practice by Chapter

Spectrophotometry and Colorimetry

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Chromatography Techniques

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Electrophoresis and Applications

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Centrifugation and Ultracentrifugation

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Radioisotope Techniques

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Enzyme-Linked Immunosorbent Assay (ELISA)

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Polymerase Chain Reaction (PCR)

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Blotting Techniques: Southern, Northern, Western

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Mass Spectrometry in Biochemistry

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Recombinant DNA Technology

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DNA Sequencing

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Proteomics and Metabolomics

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