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: 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: 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**.

Q: Which of the following blotting techniques is used to detect DNA?

Southern blot. **Explanation:** Blotting techniques are fundamental molecular biology tools used to identify specific macromolecules (DNA, RNA, or proteins) within a complex mixture. **1. Why Southern Blot is Correct:** The **Southern blot** is the gold standard technique for detecting specific **DNA** sequences. It was developed by Edwin Southern in 1975. The process involves digesting DNA with restriction endonucleases, separating the fragments by gel electrophoresis, transferring (blotting) them onto a nitrocellulose membrane, and finally hybridizing them with a labeled DNA probe complementary to the target sequence. **2. Analysis of Incorrect Options:** * **Western Blot:** Used to detect specific **Proteins**. It utilizes antibodies (primary and secondary) to identify target proteins after they have been separated by SDS-PAGE. * **Northern Blot:** Used to detect **RNA** (specifically mRNA) to study gene expression. It follows a similar principle to Southern blotting but does not require restriction digestion as RNA molecules are already short. * **Eastern Blot:** A specialized technique used to detect **post-translational modifications** of proteins, such as lipids, phosphates, or glycoconjugates. **3. NEET-PG High-Yield Pearls:** To remember these easily, use the mnemonic **SNOW DROP**: * **S**outhern — **D**NA * **N**orthern — **R**NA * **O** — **O** (No match) * **W**estern — **P**rotein * **Southwestern Blot:** A hybrid technique used to detect **DNA-binding proteins** (e.g., transcription factors). * **Clinical Application:** Southern blotting is clinically used in DNA fingerprinting, detecting gene mutations (like deletions or insertions), and diagnosing certain genetic disorders or viral infections.

Q: How is supercoiled DNA separated from relaxed DNA?

Gel electrophoresis. **Explanation:** The separation of DNA molecules based on their physical properties is a fundamental technique in molecular biology. **Why Gel Electrophoresis is correct:** Gel electrophoresis (typically using agarose) separates DNA fragments based on two primary factors: **size** and **conformation (shape)**. Even if two DNA molecules have the same number of base pairs, their shape dictates how easily they move through the gel matrix. * **Supercoiled DNA** is highly compact and "wound up," allowing it to snake through the pores of the gel very quickly. * **Relaxed (circular or nicked) DNA** is bulky and floppy, encountering more resistance. Consequently, supercoiled DNA migrates **faster** and further toward the anode than relaxed DNA of the same molecular weight. **Why other options are incorrect:** * **ELISA:** A serological technique used to detect and quantify proteins, antibodies, or hormones; it is not used for nucleic acid separation. * **DNA Footprinting:** A method used to identify the specific site where a protein (like a transcription factor) binds to a DNA sequence. * **DNA Fingerprinting:** A technique used for forensic identification or paternity testing by analyzing Variable Number Tandem Repeats (VNTRs) or Short Tandem Repeats (STRs). **High-Yield Clinical Pearls for NEET-PG:** * **Ethidium Bromide (EtBr):** The most common intercalating agent used to visualize DNA in gels; it fluoresces under UV light. * **Topoisomerases:** These are the enzymes responsible for converting supercoiled DNA into relaxed DNA (and vice versa) in vivo. Fluoroquinolones (e.g., Ciprofloxacin) act by inhibiting DNA Gyrase (Topoisomerase II). * **Migration Rule:** In standard electrophoresis, the rate of migration is: **Supercoiled > Linear > Relaxed Circular.**

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

Ion exchange chromatography is based on which principle?

Accepted Answer

Charge

Answer

Size

Answer

Solubility

Answer

Polarity

Question 6

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

Accepted Answer

Colorimetric method

Answer

Electrophoresis

Answer

Spectrophotometry

Answer

Chromatography

Question 7

Which of the following blotting techniques is used to detect DNA?

Accepted Answer

Southern blot

Answer

Western blot

Answer

Eastern blot

Answer

Northern blot

Question 8

Which of the following techniques for purification of proteins can be made specific for a given protein?

Accepted Answer

Affinity chromatography

Answer

Dialysis

Answer

Gel filtration chromatography

Answer

Ion exchange chromatography

Question 9

For the measurement of long DNA molecules (50-100 KB), which of the following techniques is used?

Accepted Answer

RFLP

Answer

Chromosome walking

Answer

NICH

Answer

SSLP

Question 10

How is supercoiled DNA separated from relaxed DNA?

Accepted Answer

Gel electrophoresis

Answer

ELISA

Answer

DNA footprinting

Answer

DNA fingerprinting

Biochemical Techniques — MCQs

Biochemical Techniques — MCQs

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