Biochemical Techniques Practice Questions

Q: Proteins can be separated by the following methods except?

Gas-liquid Chromatography. **Explanation:** The separation of proteins is based on their physical and chemical properties, such as size, charge, solubility, and binding affinity. **Why Gas-liquid Chromatography (GLC) is the correct answer:** GLC is primarily used for the separation of **volatile compounds** or substances that can be vaporized without decomposition (e.g., fatty acids, cholesterol, and drugs). Proteins are large, heat-labile macromolecules that denature at high temperatures and lack volatility; therefore, they cannot be analyzed using GLC. **Analysis of other options:** * **Electrophoresis:** This is the most common method for protein separation based on their **charge-to-mass ratio** in an electric field (e.g., Serum Protein Electrophoresis, SDS-PAGE). * **Ultra-centrifugation:** This technique separates proteins based on their **molecular weight and density** (sedimentation coefficient). It is often used to separate lipoprotein fractions (VLDL, LDL, HDL). * **Salt separation (Salting out):** This relies on protein **solubility**. Increasing the salt concentration (e.g., using Ammonium sulfate) removes the hydration shell from proteins, causing them to precipitate. This is a classic method for purifying bulk proteins like albumin and globulins. **High-Yield Clinical Pearls for NEET-PG:** * **SDS-PAGE:** Separates proteins strictly by **mass** because the detergent SDS confers a uniform negative charge to all proteins. * **Isoelectric Focusing (IEF):** Separates proteins based on their **pI (isoelectric point)**. * **Dialysis:** A technique used to separate proteins from small crystalloids (like salts) using a semi-permeable membrane. * **Affinity Chromatography:** The most specific method, utilizing the biological affinity of a protein for a ligand (e.g., enzyme-substrate or antigen-antibody).

Q: Which technique is best suited for identifying the substances present in gall bladder or kidney stones?

X-ray diffraction. **Explanation:** **Why X-ray Diffraction (XRD) is the correct answer:** X-ray diffraction is the gold standard technique for the analysis of crystalline substances. Gallstones and kidney stones (calculi) are primarily composed of crystalline materials, such as calcium oxalate, uric acid, or cholesterol. When X-rays strike these crystalline structures, they scatter in specific patterns based on the atomic arrangement. By measuring these patterns, XRD can precisely identify the chemical composition and crystalline phase of the stone, which is crucial for determining the underlying metabolic cause and preventing recurrence. **Analysis of Incorrect Options:** * **Fluorescence spectroscopy:** This technique measures the light emitted by a substance after it has absorbed electromagnetic radiation. It is used for detecting specific proteins or drugs but cannot determine the complex crystalline structure of a solid stone. * **Electron microscopy:** While excellent for visualizing the surface morphology or internal ultrastructure of cells and tissues at high magnification, it does not provide the chemical or crystallographic identification required for stone analysis. * **Nuclear magnetic resonance (NMR):** NMR is primarily used to determine the structure of organic molecules in solution or to visualize soft tissues (as MRI). It is not the standard tool for analyzing the solid, inorganic crystalline lattice of a calculus. **Clinical Pearls for NEET-PG:** * **XRD** is also the technique used to determine the **3D structure of proteins** (e.g., the double helix of DNA or hemoglobin). * **Most common kidney stone:** Calcium oxalate (Radiopaque). * **Most common gallstone:** Cholesterol (Radiolucent, but often mixed). * For rapid clinical screening of stone composition in a lab, **Infrared (IR) Spectroscopy** is an alternative, but XRD remains the definitive method for structural crystallography.

Q: Which of the following is used in recombinant DNA technology?

Restriction endonucleases. **Explanation** **Recombinant DNA (rDNA) technology** involves joining DNA molecules from different sources into a single host organism to produce new genetic combinations. **Why Option A is Correct:** **Restriction endonucleases** are the fundamental "molecular scissors" of rDNA technology. They recognize specific palindromic sequences and cleave the phosphodiester bonds of double-stranded DNA. This creates "sticky" or "blunt" ends, allowing foreign DNA fragments to be inserted into vectors (like plasmids). Without these enzymes, the precise cutting and splicing required to create recombinant molecules would be impossible. **Analysis of Incorrect Options:** * **Option B (PCR):** While PCR (Polymerase Chain Reaction) is used to *amplify* DNA, it is a standalone technique for making millions of copies of a specific sequence. It is often a precursor to rDNA technology but is not the defining tool for creating recombinant molecules. * **Option C (Reverse transcriptase):** This enzyme synthesizes DNA from an RNA template. It is primarily used to create cDNA libraries or in RT-PCR, but it does not perform the "recombining" step. * **Option D (FISH):** Fluorescence In Situ Hybridization is a *cytogenetic* technique used to detect and locate specific DNA sequences on chromosomes. It is a diagnostic tool, not a manipulative tool for DNA recombination. **High-Yield Clinical Pearls for NEET-PG:** * **Type II Restriction Endonucleases** are the most commonly used in labs because they cut at specific sites within the recognition sequence and do not require ATP. * **DNA Ligase** is the "molecular glue" that seals the nicks after restriction enzymes have done their work. * **Thermus aquaticus (Taq):** The source of heat-stable DNA polymerase used in PCR. * **Clinical Application:** rDNA technology is used to mass-produce human insulin (Humulin), growth hormone, and Hepatitis B vaccines.

Q: What is true about Western blot?

All of the above. Western blotting (Immunoblotting) is a core molecular technique used to detect specific proteins in a clinical or research sample. The process follows a logical sequence that validates all the given options: 1. **Separation by Mass (Option A):** Before detection, proteins must be separated. While proteins have varying intrinsic charges, the use of **SDS (Sodium Dodecyl Sulfate)** masks these charges with a uniform negative charge. This ensures that the separation during electrophoresis is strictly based on the **molecular weight (mass)** of the proteins. 2. **SDS-PAGE (Option B):** The standard method for this separation is **SDS-Polyacrylamide Gel Electrophoresis**. The polyacrylamide gel acts as a molecular sieve, allowing smaller proteins to migrate faster toward the anode than larger ones. 3. **Enzyme-linked Antibodies (Option C):** After transferring the separated proteins onto a membrane (nitrocellulose/PVDF), detection is achieved using a primary antibody specific to the target protein, followed by a **secondary antibody linked to an enzyme** (e.g., Horseradish Peroxidase). This enzyme reacts with a substrate to produce a detectable signal (chemiluminescence or color). **Clinical Pearls for NEET-PG:** * **Mnemonic (SNoW DRoP):** **S**outhern - **D**NA; **N**orthern - **R**NA; **W**estern - **P**rotein. * **Confirmatory Test:** Western blot was historically the gold standard confirmatory test for **HIV** (detecting antibodies against p24, gp41, and gp120/160) and is used for **Lyme disease**. * **Southwestern Blotting:** A variation used to detect **DNA-binding proteins** (e.g., transcription factors).

Q: Ion exchange chromatography is based on which principle?

Charge. **Explanation:** **Ion Exchange Chromatography (IEC)** is a technique used to separate molecules based on their **net surface charge**. The principle relies on the reversible electrostatic attraction between charged molecules in a sample and oppositely charged groups on a stationary phase (resin). * **Cation Exchange:** Uses a negatively charged resin to bind positively charged molecules (cations). * **Anion Exchange:** Uses a positively charged resin to bind negatively charged molecules (anions). **Analysis of Options:** * **Option B (Correct):** The separation depends on the **isoelectric point (pI)** of the protein and the pH of the buffer, which determines the molecule's net charge. * **Option A (Incorrect):** Separation based on **size** (molecular weight) is the principle of **Gel Filtration** (Size-Exclusion) Chromatography. * **Option C (Incorrect):** Separation based on **solubility** is typically seen in fractional precipitation methods (e.g., Salting out with Ammonium Sulfate). * **Option D (Incorrect):** Separation based on **polarity** (hydrophilic/hydrophobic interactions) is the hallmark of **Reverse-Phase Chromatography** or Partition Chromatography. **High-Yield Clinical Pearls for NEET-PG:** * **HbA1c Estimation:** Ion exchange chromatography is a gold-standard method for measuring glycated hemoglobin (HbA1c). * **Amino Acid Analysis:** IEC is the primary technique used in automated amino acid analyzers to diagnose Inborn Errors of Metabolism (IEM). * **Affinity Chromatography:** Uses highly specific biological interactions (e.g., Enzyme-Substrate, Antigen-Antibody) and is the most specific type of chromatography.

Q: Who discovered the polymerase chain reaction?

Kary Mullis. **Explanation:** The **Polymerase Chain Reaction (PCR)** is a revolutionary molecular biology technique used to amplify specific DNA sequences. It was discovered by **Kary Mullis** in 1983, for which he was awarded the Nobel Prize in Chemistry in 1993. The technique relies on thermal cycling, consisting of cycles of repeated heating and cooling for DNA melting and enzymatic replication of the DNA. **Analysis of Options:** * **Kary Mullis (Correct):** He conceptualized the use of Taq polymerase (a heat-stable enzyme from *Thermus aquaticus*) and primers to exponentially amplify DNA fragments. * **Frederick Sanger:** Known for developing the "Sanger Sequencing" method (dideoxy chain termination) and determining the amino acid sequence of insulin. He is a two-time Nobel laureate. * **Walter Gilbert:** Developed a chemical degradation method for DNA sequencing (Maxam-Gilbert sequencing) and shared the Nobel Prize with Sanger. * **Barbara McClintock:** Discovered "jumping genes" or **transposons** in maize, demonstrating that genes can move within the genome. **High-Yield Clinical Pearls for NEET-PG:** * **Steps of PCR:** Denaturation (94–96°C) → Annealing (50–65°C) → Extension (72°C). * **Taq Polymerase:** Derived from a thermophilic bacterium; it remains functional at high temperatures required for denaturation. * **RT-PCR:** Used for detecting RNA viruses (like SARS-CoV-2 or HIV) by first converting RNA to cDNA using Reverse Transcriptase. * **Real-Time PCR (qPCR):** Allows for the quantification of DNA in real-time using fluorescent probes.

Q: Which of the following techniques is based on ionic charge?

Electrophoresis. **Explanation:** **1. Why Electrophoresis is the Correct Answer:** Electrophoresis is a laboratory technique used to separate macromolecules (proteins, DNA, or RNA) based on their **size and electrical charge**. The underlying principle is the migration of charged particles through a medium (like agarose or polyacrylamide gel) under the influence of an applied electric field. Positively charged ions (cations) move toward the cathode, while negatively charged ions (anions) move toward the anode. The rate of migration depends on the net charge-to-mass ratio of the molecule. **2. Why the Other Options are Incorrect:** * **Filtration:** This is a physical separation method based on **particle size**. It uses a porous medium (filter) that allows smaller molecules to pass through while retaining larger particles. * **Dialysis:** This technique separates molecules based on **size and concentration gradients** across a semi-permeable membrane. It is commonly used to remove low-molecular-weight solutes (like urea) from blood. * **Ultracentrifugation:** This method separates particles based on their **sedimentation rate**, which depends on **density, size, and shape** (mass) when subjected to high centrifugal force. **3. Clinical Pearls & High-Yield Facts for NEET-PG:** * **Serum Protein Electrophoresis (SPEP):** A vital clinical tool. A "M-spike" in the gamma-globulin region is diagnostic for **Multiple Myeloma**. * **SDS-PAGE:** A variation where the detergent SDS gives all proteins a uniform negative charge, allowing separation based **strictly on size/molecular weight**. * **Isoelectric Focusing (IEF):** Separates proteins based on their **isoelectric point (pI)**, where their net charge is zero. * **Ampholytes:** These are used in IEF to create a pH gradient.

Q: What method is used for the measurement of bilirubin in serum?

Colorimetric method. **Explanation:** The measurement of serum bilirubin is primarily performed using the **Van den Bergh reaction**, which is a **colorimetric method**. In this reaction, bilirubin reacts with diazotized sulfanilic acid (Ehrlich’s reagent) to form a purple-colored compound called **azobilirubin**. The intensity of the color produced is proportional to the concentration of bilirubin in the serum, which is then measured using a colorimeter or photometer at a specific wavelength (typically 540 nm). **Analysis of Options:** * **Electrophoresis (B):** This technique separates proteins or nucleic acids based on their charge and size in an electric field. It is used for hemoglobin typing or serum protein analysis, not for quantifying small molecules like bilirubin. * **Spectrophotometry (C):** While spectrophotometers are used to read the results of colorimetric assays, "Colorimetric method" is the more specific term for the chemical reaction involved. Direct spectrophotometry is only used in neonates (where interfering pigments are low) but is not the standard method for general serum bilirubin. * **Chromatography (D):** Techniques like HPLC are highly sensitive and used in research settings to separate bilirubin fractions, but they are too complex and expensive for routine clinical laboratory measurement. **High-Yield Clinical Pearls for NEET-PG:** * **Direct Bilirubin (Conjugated):** Reacts rapidly with the reagent (water-soluble). * **Indirect Bilirubin (Unconjugated):** Requires an "accelerator" (like methanol or caffeine) to react because it is water-insoluble. * **Biphasic Reaction:** Occurs when both conjugated and unconjugated bilirubin are present in high amounts. * **Normal Serum Bilirubin:** 0.2 – 1.2 mg/dL. Jaundice becomes clinically detectable when levels exceed **2.0 – 2.5 mg/dL**.

Question 1

Fluorescence is commonly used in the assessment of levels of which hormone?

Accepted Answer

Thyroid hormone

Answer

Steroid hormone

Answer

Catecholamines

Answer

Leutenising releasing hormone

Question 2

Proteins can be separated by the following methods except?

Accepted Answer

Gas-liquid Chromatography

Answer

Electrophoresis

Answer

Ultra-centrifugation

Answer

Salt separation

Question 3

Which technique is best suited for identifying the substances present in gall bladder or kidney stones?

Accepted Answer

X-ray diffraction

Answer

Fluorescence spectroscopy

Answer

Electron microscopy

Answer

Nuclear magnetic resonance

Question 4

Which of the following statements regarding electrophoresis is incorrect?

Accepted Answer

Electrophoretic separation primarily depends on charge, not solely on size or shape.

Answer

Isoelectric focusing utilizes ampholytes and isoelectric point (pI).

Answer

Electrophoresis can adversely affect protein structure and function.

Answer

Electrophoresis is a common method for purifying proteins.

Question 5

Which of the following is used in recombinant DNA technology?

Accepted Answer

Restriction endonucleases

Answer

PCR

Answer

Reverse transcriptase

Answer

FISH

Question 6

What is true about Western blot?

Accepted Answer

All of the above

Answer

Separation of proteins is based on mass

Answer

SDS-PAGE electrophoresis is used for separation

Answer

Enzyme-linked antibodies are used

Question 7

Ion exchange chromatography is based on which principle?

Accepted Answer

Charge

Answer

Size

Answer

Solubility

Answer

Polarity

Question 8

Who discovered the polymerase chain reaction?

Accepted Answer

Kary Mullis

Answer

Walter Gilbert

Answer

Barbara McClintock

Answer

Frederick Sanger

Question 9

Which of the following techniques is based on ionic charge?

Accepted Answer

Electrophoresis

Answer

Filtration

Answer

Dialysis

Answer

Ultracentrifugation

Question 10

What method is used for the measurement of bilirubin in serum?

Accepted Answer

Colorimetric method

Answer

Electrophoresis

Answer

Spectrophotometry

Answer

Chromatography

Biochemical Techniques — MCQs

Biochemical Techniques — MCQs

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