NCERT Class 11 Biotechnology Chapter 12 Answer English Medium | Tools and Technologies

  Tools and Technologies

Chapter 12

Exercises

1. The function of ethidium bromide in electrophoresis is to

(a) Track the progression of electrophoresis

(b) visualise the DNA molecules

(c) separate the DNA molecules

(d) provide charge to DNA molecules

Ans: (b) visualise the DNA molecules.

2. Match the following

3. Mass spectrometry is used to

(a) identify unknown compounds

(b) elucidate the structure of molecules

(c) quantify compounds

(d) All of the above

Ans: (d) All of the above.

4. Match the following table with reference to Antigen

5. In DNA gel electrophoresis,

I. Longer DNA fragments remain close to the well.

II. Longer DNA fragments move towards the positive end

of gel.

III. Smaller DNA fragments move close to the positive end of gel.

IV. Smaller DNA fragments remain close to the well.

Which of the above options are correct

(a) I and III

(b) II and IV

(c) Only II

(d) None of the above

Ans: (c) Only II.

6. For a resolved image of the surface of an object, which of the following microscopes would you prefer

(a) Transmission electron microscope

(b) Scanning electron microscope

(c) Phase contrast microscope

(d) Fluorescence microscope

Ans: (b) Scanning electron microscope.

7. Match the following:

8. Which of the following techniques is feasible to quantify the expression of a large number of genes

(a) Mass spectrometry

(b) Microarray

(c) FISH

(d) Agarose gel electrophoresis

Ans: (b) Microarray.

9. Differentiate between the following types of microscopy techniques

(a) Scanning electron microscopy (SEM) and transmission electron microscopy (TEM)

(b) Dark field microscopy and bright field microscopy

(c) Phase contrast microscopy and confocal microscopy

Ans: (a) Scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

10. Discuss the principle of agarose gel electrophoresis.

Ans: Agarose Gel Electrophoresis: Separating Molecules by Size and Charge

Agarose gel electrophoresis is a fundamental technique in molecular biology used to separate mixtures of DNA, RNA, or other charged molecules based on their size and charge. It's like sifting different sizes of beads through a mesh screen – smaller beads pass through more easily than larger ones.

Here's a breakdown of the principle:

1. The Gel:

*Imagine a jelly-like slab made of agarose, a natural sugar polymer. Its microscopic pores act like a size-based sieve.

2. Sample Loading:

*DNA or RNA samples are mixed with a dye and loaded into wells at one end of the gel.

3. Electrical Field:

*An electric current is applied across the gel, creating a positive pole (anode) and a negative pole (cathode).

4. Charged Molecules Migrate:

*DNA and RNA are negatively charged molecules due to their phosphate backbone.

*When the current is applied, these molecules are pulled towards the positive pole.'

5. Size Matters:

*Smaller molecules move through the gel pores more easily and travel faster towards the positive end.

*Larger molecules encounter more resistance due to their size and travel slower.

6. Separation Achieved:

*After a certain time, the electric current is turned off.

*By looking at the gel under UV light, the DNA or RNA appears as bands at different positions.

*Smaller molecules have migrated further, forming bands closer to the positive end.

*Larger molecules stay closer to the wells, forming bands near the loading point.

Key Factors Influencing Separation:

*Agarose concentration: A higher concentration creates denser pores, slowing down larger molecules.

*Electric field strength: A stronger field leads to faster migration for all molecules.

*DNA/RNA fragment size: Smaller fragments move faster due to less hindrance in the gel.

Applications of Agarose Gel Electrophoresis:

*Analyzing DNA fragments after restriction enzyme digestion.

*Visualizing PCR products to confirm amplification.

*Assessing purity and integrity of DNA or RNA samples.

*Determining the size of DNA fragments for cloning or sequencing.

11. Name a tracking dye which is used to track DNA as well as proteins during electrophoresis. What will happen if you forget to add tracking dye to your sample during electrophoresis?

Ans: Tracking Dyes in Electrophoresis: Keeping an Eye on Your Molecules

In electrophoresis, bromophenol blue (BPB) is a commonly used tracking dye for both DNA and protein samples. This versatile dye serves as a visual indicator of the progress of your sample during the run.

BPB Properties:

*Color: Blue

*Charge: Negatively charged (similar to DNA and proteins)

*Migration: Moves slightly slower than smaller DNA fragments and protein bands

Benefits of using BPB:

*Visually monitors the electrophoresis progress: You can observe the movement of the blue band to estimate how far the DNA or proteins have migrated through the gel.

*Helps align multiple samples: By ensuring all samples start at the same point relative to the BPB band, you can easily compare their migration distances and estimate sizes.

*Provides a reference point for gel documentation: When photographing or imaging the gel, the BPB band serves as a marker for orientation and scale.

What happens if you forget BPB?

Although not ideal, forgetting BPB won't completely ruin your electrophoresis run. However, you will encounter some disadvantages:

*No visual progress indicator: You won't be able to directly see how far your DNA or proteins have migrated, making it difficult to estimate their sizes or optimal run time.

*Alignment challenges: Without the BPB reference, aligning multiple samples and interpreting their migration patterns becomes more challenging.

*Documentation uncertainty: Without a clear marker, accurately interpreting and referencing gel images becomes tricky.

Alternatives to BPB:

While BPB is popular, other tracking dyes can be used for specific purposes:

*Xylene cyanol FF: Blue-green dye that migrates slower than BPB, suitable for large DNA fragments.

*Orange G: Orange dye with similar migration to BPB, often used in protein electrophoresis.

*ROX dye: Non-fluorescent dye used as an internal standard for fluorescence detection in DNA sequencing.

12. Two polyacrylamide gels A and B were prepared. Gel A had 4% acrylamide whereas Gel B had 12% acrylamide. Based on the given information answer the following

(a) Which gel is harder: A or B?

(b) Which gel offers greater friction to the proteins: A or B?

(c) Which gel (A or B) will be used to separate a mixture containing low molecular weight proteins?

(d) Which gel (A or B) will be used to separate a mixture containing both low and high molecular weight proteins?

Ans: Here's how the properties of gels A and B (with 4% and 12% acrylamide) compare:

(a) Harder Gel: Gel B (12% acrylamide) will be harder than Gel A (4% acrylamide). More acrylamide creates a denser mesh, resulting in a stiffer and less flexible gel.

(b) Friction for Proteins: Gel B (12% acrylamide) will offer greater friction to proteins. The denser mesh in Gel B presents more obstacles for proteins to navigate, slowing down their movement.

(c) Separating Low Molecular Weight Proteins: Gel A (4% acrylamide) will be used to separate a mixture containing low molecular weight proteins. With larger pores, Gel A allows small proteins to move through more easily, enabling their efficient separation.

(d) Separating Both Low and High Molecular Weight Proteins: Gel B (12% acrylamide) is more suitable for separating a mixture containing both low and high molecular weight proteins. The tighter mesh in Gel B hinders larger proteins more significantly, allowing for better resolution between them and the smaller proteins.

Here's a table summarizing the key differences:

Therefore, the choice of gel depends on the desired separation and the size of the target proteins. For smaller proteins, a looser gel like A is preferred, while for a wider range of protein sizes, a denser gel like B is more suitable.

13. What is a chromatogram? Draw a well labeled diagram of a chromatogram of a mixture containing three different solutes.

Ans: Chromatogram: Unraveling Separation Stories

A chromatogram is a visual representation of the separation of a mixture's components by chromatography. It depicts the distribution of each component within the stationary phase as it progresses through the mobile phase during the separation process.

Here's a breakdown:

Key Features of a Chromatogram:

*X-axis: Represents the retention time (tR) or retention volume (VR) of each component. Components that interact more strongly with the stationary phase spend more time there, increasing their retention time and appearing further to the right.

*Y-axis: Represents the detector signal, usually intensity or concentration, of the separated components. Higher peaks indicate higher concentrations.

*Peaks: Each component in the mixture appears as a peak on the chromatogram. Peak area, height, or width can be used to quantify the relative amount of each component.

*Baseline: The horizontal line indicating the background signal without any sample components present.

Understanding the Story:

By analyzing the chromatogram, you can learn:

*Number of components: The number of peaks indicates the number of distinct components in the mixture.

*Relative abundance: Peak heights or areas reflect the relative concentrations of each component.

*Separation efficiency: Sharp, well-resolved peaks indicate good separation, while overlapping peaks suggest insufficient separation.

*Component identity: Retention times can be compared to known standards for tentative identification of components.

Sample Chromatogram with Three Solutes:

Imagine we have a mixture containing three solutes (A, B, and C) separated by reversed-phase chromatography. Here's a possible chromatogram:

*X-axis: Retention time (tR)

*Y-axis: Detector signal (arbitrary units)

*Baseline: Horizontal line

*Peaks: A, B, and C represent the peaks for each solute

Labels:

*Injection point: Where the sample was injected onto the column.

*Detection window: Region where the detector monitors the separated components.

*Peak labels: A, B, and C for each solute peak.

14. Explain the principle of FISH. How is FISH technique applied in chromosome painting? What are the advantages of chromosome painting?

Ans: FISH: Unveiling DNA Through Fluorescence

Fluorescence In Situ Hybridization (FISH) is a powerful cytogenetic technique for visualizing specific DNA sequences on chromosomes using fluorescent probes. Imagine illuminating specific words in a book with a highlighter – FISH does the same for DNA within the complex landscape of chromosomes.

Principle of FISH:

1. Probes: Short, single-stranded DNA fragments labeled with fluorescent dyes are designed to target specific sequences of interest.

2. Denaturation: Cells are treated to partially denature the DNA, making the target sequences accessible for probe hybridization.

3. Hybridization: Fluorescent probes bind to their complementary sequences on the chromosomes.

4. Washing: Unbound probes are washed away.

5. Visualization: Cells are mounted on slides and observed under a fluorescence microscope. Specific chromosomes or chromosomal regions light up with the corresponding fluorescent color, revealing the location of the target DNA sequences.

Chromosome Painting:

In chromosome painting, multiple FISH probes are used, each targeting a different chromosome or chromosomal region. These probes are labeled with distinct fluorescent dyes, creating a "paintbrush" effect. When hybridized to chromosomes, each chromosome appears a specific color, allowing for complete visualization and differentiation of all chromosomes in a single experiment.

Advantages of Chromosome Painting:

.High Specificity: FISH probes bind only to complementary sequences, minimizing background noise and offering accurate identification of target DNA.

.Multicolor Visualization: Simultaneous visualization of multiple chromosomes with different colors provides a comprehensive picture of chromosomal arrangement.

.Wide Applications: FISH painting can be used for various purposes, including:

*Identifying chromosomal abnormalities like translocations, deletions, or duplications.

*Analyzing gene expression patterns on specific chromosomes.

*Tracking the fate of chromosomes during cell division.

*Detecting specific pathogens in infected cells.

15. Mention the various applications of spectroscopy techniques.

Ans: Spectroscopy techniques, utilizing the interaction of light with matter, have a wide range of applications across various fields. Here's a brief overview of some prominent applications:

Chemistry:

*Structure determination: Analyzing the vibrational and electronic energy levels of molecules to elucidate their structure and functional groups.

*Quantitative analysis: Determining the concentration of specific components in a mixture.

*Monitoring chemical reactions: Studying the progress and mechanism of chemical reactions by tracking changes in the spectra.

*Material characterization: Identifying and characterizing the composition and properties of materials.

Physics:

*Atomic and molecular structure: Investigating the energy levels and transitions of atoms and molecules to understand their fundamental properties.

*Plasma diagnostics: Characterizing the temperature, density, and composition of plasmas (ionized gases).

*Astrophysics: Analyzing the light emitted from stars and galaxies to understand their composition, temperature, and evolution.

Biology and Medicine:

*DNA and RNA analysis: Studying the structure and function of DNA and RNA molecules for genetic research and diagnostics.

*Protein analysis: Characterizing the structure and function of proteins for understanding biological processes and developing drugs.

*Cell and tissue analysis: Identifying and characterizing cell types and their components for medical diagnosis and research.

*Drug discovery and development: Developing new drugs by studying the interaction of drug candidates with biological molecules.

Environmental Science:

*Air and water pollution monitoring: Detecting and quantifying pollutants in air and water.

*Soil analysis: Characterizing the composition and properties of soil for agricultural and environmental monitoring.

*Remote sensing: Studying the environment from satellites and aircraft using spectroscopic techniques.

In addition to these, spectroscopy applications can be found in diverse areas like:

*Forensic science: Identifying unknown materials and analyzing evidence.

*Art and cultural heritage: Investigating the composition and history of artwork and artifacts.

*Food science and safety: Analyzing the composition and quality of food products.

*Semiconductor industry: Characterizing and optimizing the properties of semiconductor materials.

16. What are major components of UV-visible spectrophotometer? Explain each in brief.

Ans: A UV-visible spectrophotometer measures the interaction of light in the ultraviolet (UV) and visible range with matter. To achieve this, it relies on several key components:

1. Light Source:

.Provides a stable and broad spectrum of light covering both UV and visible wavelengths. Common sources include:

*Deuterium lamps: Emit UV light (190-330 nm).

*Tungsten or halogen lamps: Emit visible light (330-800 nm).

*Xenon lamps: Emit a full UV-visible spectrum, but are more expensive and have shorter lifespans.

2. Monochromator:

.Isolates a narrow band of light from the broad spectrum generated by the source. Common types include:

*Prism monochromators: Use prisms to disperse light into different wavelengths and select a specific band using slits.

*Grating monochromators: Use diffraction gratings for better resolution and wider wavelength range.

3. Sample Holder:

.Holds the sample in the path of the monochromatic light. Different types exist for liquids, solids, and gaseous samples.

4. Detector:

.Converts the light intensity transmitted through or reflected by the sample into an electrical signal. Common detectors include:

*Photomultiplier tubes (PMTs): Highly sensitive for low light levels but expensive.

*Diode array detectors (DADs): Faster and offer simultaneous recording of a wider range of wavelengths.

5. Data Processor and Recorder:

.Amplifies and processes the electrical signal from the detector, converts it into absorbance or transmittance values, and displays or records the data as a spectrum.

Optional Components:

.Beamsplitter: Divides the light beam into two paths, one for the sample and one for a reference, allowing for ratiometric measurements and background subtraction.

.Computer interface: Enables control of the instrument and analysis of spectra using dedicated software.

17. Write the major differences between the Sanger’s method and Maxam and Gilbert’s method of DNA sequencing.

Ans: Major Differences between Sanger's and Maxam-Gilbert's DNA Sequencing Methods:

Underlying principle:

*Sanger: Chain termination by incorporating dideoxynucleotides (ddNTPs) during DNA synthesis.

*Maxam-Gilbert: Chemical modification and cleavage of DNA backbone at specific bases.

Labeling:

*Sanger: Radioactive or fluorescent label at the 5' end of the primer.

*Maxam-Gilbert: Radioactive label at the 5' end of the DNA fragment.

DNA preparation:

*Sanger: Requires single-stranded DNA template (initially relied on cloning).

(Maxam-Gilbert: Works with double-stranded DNA, simplifying preparation.

Chemical specificity:

*Sanger: ddNTPs specifically block chain elongation based on their base pairing.

*Maxam-Gilbert: Chemicals react with specific bases to modify and cleave the DNA backbone.

Safety:

*Sanger: Requires less hazardous chemicals.

*Maxam-Gilbert: Uses potent and dangerous chemicals requiring careful handling.

Technical difficulty:

*Sanger: Easier to perform and automate.

*Maxam-Gilbert: More complex and prone to errors, requires highly skilled personnel.

Read length:

*Sanger: Initially shorter reads, later improved to longer lengths.

*Maxam-Gilbert: Typically shorter reads compared to later Sanger variations.

Overall:

*Sanger: Became the dominant method due to its ease, safety, and scalability, leading to the development of automated sequencing technologies.

*Maxam-Gilbert: Played an important role in early DNA sequencing but was eventually superseded by Sanger due to its limitations.

Additional Points:

*Both methods utilize gel electrophoresis to separate DNA fragments based on size.

*Both methods were groundbreaking for their time, revolutionizing the field of genetics.

*Today, next-generation sequencing technologies have overtaken both Sanger and Maxam-Gilbert for their speed and efficiency.

18. Write the principle of flow cytometry.

Ans: The principle of flow cytometry rests on three fundamental pillars: fluid dynamics, optics, and electronics. It essentially analyzes individual cells, one at a time, as they flow through a focused laser beam in a single file line. Here's a breakdown of the process:

1. Fluid Dynamics:

.A sheath fluid carries the suspended cells in a thin stream through a flow chamber.

.This creates a hydrodynamically focused stream, ensuring individual cells pass through the laser one at a time.

2. Optics:

.A focused laser beam illuminates the cells as they pass through the flow chamber.

.The interaction of the laser with the cells generates two types of light:

*Forward Scatter (FSC): Light scattered directly forward by the cell, reflecting its size and overall morphology.

*Side Scatter (SSC): Light scattered at an angle by the cell's internal complexity, such as organelle density and granularity.

.In some cases, cells are fluorescently labeled with specific antibodies or dyes.

.When excited by the laser, these labels emit fluorescence at particular wavelengths, revealing specific molecules or structures within the cell.

3. Electronics:

.Specialized detectors (photomultiplier tubes) capture the light signals (FSC, SSC, and fluorescence) from each cell.

.These signals are converted into electronic pulses and processed by a computer.

.The computer analyzes the intensity and distribution of these signals for each cell, generating a multi-parametric data set.

Analysis:

.The data is then visualized and analyzed using specialized software, allowing researchers to:

*Count the number of cells with specific characteristics.

*Distinguish different cell populations based on their size, complexity, and fluorescence intensity.

*Quantify the expression of specific proteins or molecules within individual cells.

*Sort cells with desired characteristics in real-time for further analysis.

   

Question And Answer Type By: Himashree Bora.