Gene Cloning
Chapter 3
EXERCISES
1. Describe the methods used for isolation of DNA.
Ans: DNA isolation involves extracting DNA from cells or tissues, purifying it from other cellular components, and concentrating it for further analysis. Here's a brief overview of the common methods:
1. Mechanical and Enzymatic Cell Lysis:
*Mechanical Disruption: Cells are broken open using grinding, homogenization, or lysozyme treatment to release their contents.
*Enzymatic Digestion: Enzymes like proteases and nucleases digest proteins and other molecules, leaving DNA intact.
2. Separation of DNA:
*Centrifugation: Separation of cell debris and nuclei from lysate by differential centrifugation.
*Precipitation: Ethanol or isopropanol precipitation concentrates DNA while leaving impurities in solution.
3. Purification:
*Organic Extraction: Phenol-chloroform extraction removes proteins and lipids from the DNA solution.
*Column Chromatography: Different purification columns remove specific contaminants and concentrate DNA.
4. Quantification and Visualization:
*Spectrophotometry: Measuring DNA concentration by absorbance at specific wavelengths.
*Gel Electrophoresis: Analyzing DNA size and purity by separating fragments based on size and charge.
Variations and Specific Techniques:
*Miniaturized methods: Adapted for small samples or high-throughput processing.
*Plant or tissue-specific protocols: Account for diverse cell walls and complex tissues.
*Blood or forensic DNA isolation: Specialized techniques for handling blood cells and evidence samples.
Selection of Method:
The optimal method depends on several factors:
*Sample type: Cells, tissues, bacteria, etc.
*Quantity of DNA required: Large-scale or small-scale analysis.
*Purity required: Downstream applications demanding high purity.
Overall, DNA isolation is a crucial step in various research and diagnostic applications. Understanding the principles and variations in isolation methods allows researchers to choose the most effective approach for their specific needs.
2. What is the role of biological detergent in the process of isolation of nucleic acid?
Ans: Biological detergents play a crucial role in the process of nucleic acid isolation by performing several key functions:
1. Cell Lysis: Detergents disrupt cell membranes by interacting with the hydrophobic lipid bilayer. This facilitates the release of the intracellular contents, including nucleic acids, into the surrounding solution.
2. Protein Denaturation: Detergents unfold and denature proteins, preventing them from binding to and hindering the purification of nucleic acids. This includes enzymes that could degrade the isolated nucleic acids.
3. Solubilization of Cellular Components: Detergents solubilize cellular debris like membrane fragments and carbohydrates, improving the separation of nucleic acids from these contaminants during subsequent purification steps.
4. Protecting Nucleic Acids: Certain detergents shield nucleic acids from degradation by enzymes like nucleases, which can be present in the cell lysate.
5. Facilitating Precipitation: Different types of detergents can influence the precipitation of nucleic acids with alcohol or other agents, aiding in their concentration and recovery.
Choosing the Right Detergent:
The effectiveness of a detergent depends on several factors, including:
*Type of Cell: Different cell types have varying membrane compositions, necessitating specific detergent properties.
*Scale of Isolation: Large-scale isolations often require harsher detergents for efficient cell lysis.
*Downstream Applications: Some detergents may interfere with subsequent analyses like PCR or enzymatic reactions, requiring careful selection.
Commonly Used Biological Detergents:
*Sodium dodecyl sulfate (SDS)
*Triton X-100
*Cetyltrimethylammonium bromide (CTAB)
*Sarcosyl
In conclusion, biological detergents are essential tools in nucleic acid isolation, enabling efficient cell lysis, protein denaturation, and improved extraction and purification of these valuable molecules for various research and diagnostic applications.
3. How does DNA isolation from plant tissue differ from that of bacterial cell?
Ans: Isolating DNA from plant tissue presents distinct challenges compared to bacterial cells due to several key differences in their structures and compositions:
Cell Walls:
*Plant cells: Thick and rigid cell walls composed mainly of cellulose, requiring additional mechanical or enzymatic disruption steps to access the intracellular contents.
*Bacterial cells: Thin peptidoglycan walls, readily lysed by lysozyme enzymes in standard protocols.
Presence of Secondary Metabolites:
*Plant cells: May contain phenolic compounds and tannins that can bind and co-precipitate with DNA, hindering purification.
*Bacterial cells: Generally don't have these complexities in their cellular constituents.
Polysaccharides and Mucilages:
*Plant cells: Often contain abundant polysaccharides and mucilages that can clog filters and purification columns, making isolation more difficult.
*Bacterial cells: Usually lack these additional carbohydrates, simplifying the extraction process.
Methods and Adjustments:
*Mechanical disruption: Grinding with mortars and pestles or bead beating becomes crucial for effective plant cell wall digestion.
*Enzymatic digestion: Cellulase enzymes might be added to specific protocols to further break down plant cell walls.
*Purification steps: Additional filtration or centrifugation may be necessary to remove plant-specific contaminants like polysaccharides.
Alternative Techniques:
*CTAB (cetyltrimethylammonium bromide)-based methods: Particularly efficient for isolating DNA from plant tissues due to its ability to bind and precipitate DNA while leaving contaminants behind.
*Miniaturized kits: Specialized kits are available for plant DNA isolation, often involving column chromatography techniques for efficient purification.
Overall, successful isolation of DNA from plant tissues requires additional steps and considerations compared to bacterial cells. Mechanical and enzymatic disruption methods, tailored purification procedures, and sometimes modified protocols are necessary to overcome the challenges posed by plant cell walls and their unique biomolecules.
4. How many types of restriction enzymes (REs) are there? Can all REs be used in rDNA technology? Give justification.
Ans: Counting Restriction Enzymes: More Than Just Numbers
While classifying restriction enzymes (REs) can be tricky, the answer to your first
question isn't straightforward. Here's why:
1. Diversity: The exact number of REs is constantly growing as researchers discover new ones. Current estimates suggest thousands exist, with hundreds actively used in rDNA technology.
2. Classification and Subtypes: REs are traditionally categorized based on subunit composition, cleavage position, and cofactor requirements. These categories further branch into subtypes, making a definitive count challenging.
3. Sequence Variants: Many REs exist in naturally occurring variants with slightly different recognition sequences. Counting these as separate enzymes or simply variations adds another layer of complexity.
Using REs in rDNA Technology:
Not all REs are suitable for rDNA technology. Here's why:
1. Specific Recognition Sequences: REs cleave DNA at specific short sequences. If those sequences are rare or absent in the desired DNA fragment, the enzyme won't be useful.
2. Sticky Ends vs. Blunt Ends: REs generate either sticky ends (overhanging) or blunt ends (flush) upon cleavage. Some downstream applications like ligation require specific end types for efficient joining of DNA fragments.
3. Methylation Sensitivity: Some REs are sensitive to methylation, a common epigenetic modification in genomes. Such enzymes wouldn't cleave methylated DNA efficiently, limiting their applicability.
4. Star Activity: Certain REs exhibit "star activity," meaning they can cleave DNA non-specifically at low concentrations, potentially harming target DNA fragments.
Choosing the Right RE:
Selecting the appropriate RE for rDNA experiments requires careful consideration of factors like recognition sequence, activity, enzyme source, and compatibility with downstream techniques.
Therefore, while the actual number of REs isn't a fixed figure, not all of them are readily applicable in rDNA technology. Understanding the specific requirements of your experiment and the properties of available REs is key to choosing the best tool for your research.
5. What are the challenges faced during the process of nucleic acid extraction?
Ans: Extracting nucleic acids, like DNA and RNA, presents certain challenges that can affect purity, yield, and downstream success. Here are some key challenges:
1. Cell Lysis and Sample Homogenization:
*Incomplete Disruption: Inefficient cell wall or membrane breakdown can limit access to intracellular nucleic acids, reducing yield.
*Shear Stress: Excessive mechanical disruption can fragment nucleic acids, compromising their integrity and downstream applications.
*Contaminants: Incomplete removal of cellular debris, proteins, and other biomolecules can interfere with purification and analysis.
2. Nucleic Acid Loss and Degradation:
*Nucleases: Enzymatic degradation by ubiquitous nucleases present in samples or reagents can significantly reduce yield.
*Shear Stress: Similar to cell disruption, harsh handling can fragment nucleic acids.
*Chemical Degradation: Exposure to harsh chemicals or inappropriate pH can damage nucleic acid structure and functionality.
3. Purification and Contaminants:
*Co-precipitation: Non-specific binding of cellular components like polysaccharides or tannins can contaminate purified nucleic acids.
*Incomplete Removal of Contaminants: Residual proteins, salts, or organic solvents can interfere with downstream applications like PCR or enzymatic reactions.
*Low Concentration: Certain samples, like plant tissues or environmental samples, may contain inherently low amounts of nucleic acids, requiring optimization for sufficient yield.
4. Downstream Considerations:
*DNA methylation: If studying methylation patterns, harsh extraction methods can inadvertently alter these critical epigenetic modifications.
*RNA Integrity: For RNA analysis, preserving the molecule's intricate secondary structure and preventing degradation is crucial.
*Compatibility with Subsequent Techniques: Extracted nucleic acids need to be compatible with specific downstream assays for accurate and reliable results.
Strategies for Overcoming Challenges:
*Optimizing Mechanical and Enzymatic Lysis: Choosing appropriate tools and buffer conditions for efficient but gentle cell disruption.
*Inhibiting Nucleases: Including RNase inhibitors in buffers and following proper handling protocols.
*Purification Techniques: Selecting appropriate columns, filters, and precipitation methods to effectively remove contaminants.
*Downstream Specific Protocols: Tailoring extraction methods to preserve delicate modifications or structures if needed.
By understanding these challenges and adopting appropriate strategies, researchers can optimize nucleic acid extraction for improved purity, yield, and reliable downstream applications in various fields of biology and biotechnology.
6. Write the role of alkaline phosphatase, DNA ligase, terminal transferase in rDNA technology.
Ans: In rDNA technology, these three enzymes play crucial roles in manipulating and modifying DNA fragments for various applications:
1. Alkaline Phosphatase (AP):
*Role: Removes phosphate groups from the 5' ends of DNA fragments. This creates blunt ends, which are important for certain downstream techniques like ligating fragments together.
*Applications:
*Plasmid vector preparation: Dephosphorylating linearized vectors prevents self-ligation, allowing efficient insertion of foreign DNA.
*Restriction digest optimization: Dephosphorylating blunt-ended fragments generated by some REs prevents them from re-ligating, facilitating subsequent manipulations.
*cDNA library construction: Removing 5' phosphates from blunt-ended adapters prevents undesired self-ligation during library preparation.
2. DNA Ligase:
*Role: Joins two DNA fragments end-to-end by catalyzing the formation of a phosphodiester bond between the 3' phosphate end of one fragment and the 5' hydroxyl end of the other.
*Applications:
*Plasmid construction: Ligating foreign DNA fragments into linearized vectors for cloning.
*cDNA library construction: Joining adaptors to cDNA fragments for library generation.
*In vitro genome assembly: Assembling large DNA fragments for synthetic biology or gene editing applications.
3. Terminal Transferase (TdT):
*Role: Adds nucleotides to the 3' ends of single-stranded DNA molecules. This can create either homopolymeric tails (e.g., poly-A or poly-T) or specific sequences depending on the nucleotides provided.
*Applications:
*cDNA library construction: Adding poly-A tails to mRNA fragments for subsequent reverse transcription and library cloning.
*Labeling DNA fragments: Tailing with specific sequences allows for attachment of fluorescent dyes or other probes for detection and analysis.
*Blunt-end cloning: Adding complementary overhangs to blunt-ended fragments facilitates their ligation with compatible vectors or other fragments.
Overall, these enzymes represent fundamental tools in rDNA technology, facilitating efficient manipulation and joining of DNA fragments for various cloning, library construction, and other research applications.
7. Describe the role of chelating agent in the process of DNA extraction.
Ans: Chelating agents play a crucial role in several steps of DNA extraction, ensuring its purity and integrity:
1. Inactivating Metal Ions:
*DNA extraction buffers often contain chelating agents like EDTA (ethylenediaminetetraacetic acid). These molecules bind divalent metal ions like Mg2+ and Ca2+.
*Metal ions can activate enzymes like DNases, which degrade DNA. By chelating these ions, the chelating agent inhibits DNase activity, protecting the extracted DNA from enzymatic degradation.
2. Stabilizing Protein-DNA Interactions:
*Metal ions can also stabilize interactions between proteins and DNA, making them difficult to remove during purification steps. Chelating agents can compete with proteins for binding to these metal ions, weakening the protein-DNA interactions and facilitating easier separation.
3. Preventing Oxidation:
*Certain chelating agents can also scavenge free radicals, preventing oxidative damage to DNA. This is particularly important for samples susceptible to oxidation, such as those rich in lipids or exposed to sunlight.
4. Maintaining Buffer Stability:
*Chelating agents can complex with metal ions that might precipitate or interfere with the function of other components in the extraction buffer. This helps maintain the stability and effectiveness of the buffer throughout the extraction process.
Choosing the Right Chelating Agent:
The specific chelating agent used depends on the extraction method, sample type, and desired outcomes. Some factors to consider include:
*Binding Affinity: The strength of the chelating agent's binding to target metal ions.
*Specificity: Whether the agent primarily binds desired metal ions or can also chelate other ions potentially impacting downstream applications.
*Solubility and Stability: The ability of the chelating agent to remain soluble and active in the extraction buffer and throughout the process.
Overall, chelating agents are valuable tools in DNA extraction, protecting DNA from degradation, facilitating protein removal, and ensuring the successful isolation of high-quality DNA for various research and diagnostic applications.
8. Briefly describe the modes of DNA transfer into the host.
Ans: There are three main modes of DNA transfer into a host cell:
1. Transformation:
*Recipient cell directly takes up free DNA molecules from its environment.
*Requires the recipient cell to be in a "competent" state, meaning its membrane is temporarily permeable to DNA.
*Common in bacteria, can also occur in some fungi and protists.
*Examples: Bacterial natural transformation, plasmid uptake.
2. Transduction:
*DNA is transferred by a virus acting as a vector.
*The virus accidentally packages host cell DNA during its own replication cycle.
*This viral particle then infects another host cell, injecting the transferred DNA.
*Common in bacteria, can also occur in some archaea and eukaryotes.
*Examples: Bacterial transduction by bacteriophages.
3. Conjugation:
*Direct cell-to-cell contact transfers DNA through a physical structure called a pilus.
*Donor cell actively replicates and transfers a specific piece of DNA (plasmid) to the recipient cell.
*Primarily occurs in bacteria.
*Examples: Bacterial conjugation for plasmid exchange.
Each mode has its own advantages and limitations, influencing the frequency and types of DNA transfer that can occur in different organisms and environments. Understanding these modes is crucial for studying gene flow, bacterial evolution, and developing genetic engineering techniques.
9. Identify the correct statement for blue-white selection method.
(a) A specific dye is used to stain bacterial colony.
(b) It is based on the expression of lacZ gene.
(c) The recombinant bacterial colony remains blue.
(d) lacZ gene is inserted in an antibiotic resistant gene.
Ans: (b) It is based on the expression of lacZ gene.
10. Identify the correctly matched pair from the following options.
(a) Northern blot: Detect specific sequence of DNA
(b) Southern blot: Detect specific sequence of RNA
(c) Western blot: Detect specific proteins
(d) Eastern blot: Detect transcriptional modifications in RNA
Ans: (c) Western blot: Detect specific proteins.
11. Identify the incorrect matched pair from the following options.
(a) Taq polymerase: Thermus aquaticus
(b) Pfu polymerase: Pyrococcus furiosus
(c) HindIII: Haemophilus influenzae
(d) PstI: Pyrococcus stuartii
And: (d) PstI: Pyrococcus stuartii.
12. How are recombinants screened? Describe the methods in detail.
Ans: Recombinant screening involves identifying cells that successfully took up foreign DNA and incorporated it into their genome. Several methods are employed, each with its own advantages and limitations:
1. Selection Markers:
*Most vectors carry antibiotic resistance genes or other selectable markers. Transformed cells grow on selective media containing the antibiotic, while untransformed cells die.
*Simple and efficient: Ideal for large-scale screening.
*Limitations: Not all genes of interest confer selectable phenotypes, and some unwanted mutations can lead to false positives.
2. Reporter Genes:
*Certain vectors express reporter genes like β-galactosidase or GFP upon successful insertion of the target DNA. These genes produce observable products like color change or fluorescence.
*Directly identify recombinants: Provides immediate visual confirmation.
*Limitations: Reporter expression levels can vary, may require additional substrates or equipment, and not all vectors offer suitable reporter genes.
3. Southern Blotting:
*This technique uses labeled probes to hybridize with specific sequences present in the recombinant DNA. Only clones harboring the insert will show a positive signal.
*Highly specific: Can detect even single copy insertions.
*Time-consuming and labor-intensive: Requires gel electrophoresis and hybridization steps.
4. PCR (Polymerase Chain Reaction):
*PCR amplifies specific DNA sequences using primers flanking the insertion site. Only recombinant DNA will generate the expected PCR product.
*Sensitive and specific: Detects low levels of target DNA.
*Requires primer design and optimization: Time-consuming for large libraries.
5. DNA Sequencing:
*Direct sequencing of the inserted DNA fragment confirms its identity and orientation within the vector.
*Definitive verification: Provides detailed information about the insert.
*Expensive and time-consuming: Not suitable for high-throughput screening.
Choosing the optimal screening method depends on several factors:
*Size and complexity of the insert: Small inserts might be easier to detect with PCR, while larger fragments might require Southern blotting.
*Desired level of sensitivity and specificity: High-throughput screens might utilize antibiotic resistance, while confirming specific modifications might require sequencing.
*Available resources and expertise: PCR may be readily accessible in most labs, while Southern blotting might require specialized equipment and technical skills.
13. Differentiate between the Southern, Northern and Western blotting.
Ans: All three blotting techniques - Southern, Northern, and Western - share the principle of transferring target molecules from a gel to a membrane for subsequent detection. However, they differ in the type of molecule they target and the detection methods used.
Target Molecule:
*Southern blotting: Detects specific DNA sequences
*Northern blotting: Detects specific RNA sequences
*Western blotting: Detects specific proteins
Detection Method:
*Southern and Northern blotting: Hybridization with labeled probes (radioactive, fluorescent, or enzyme-linked) specific to the target sequence.
*Western blotting: Use of primary antibodies that bind to the protein of interest, followed by secondary antibodies linked to a reporter molecule for visualization.
Applications:
*Southern blotting: Identifying genetic mutations, analyzing gene rearrangements, detecting transgenic organisms.
*Northern blotting: Studying gene expression levels, identifying differentially expressed genes.
*Western blotting: Protein expression analysis, post-translational modifications, protein-protein interactions.
Here's a table summarizing the key differences:
14. What is PCR? Describe in detail.
Ans: PCR: Revolutionizing Molecular Biology
PCR, or Polymerase Chain Reaction, is a fundamental technique in molecular biology that allows for the specific amplification of DNA fragments. Developed in 1983 by Kary Mullis, PCR has become a ubiquitous tool for various research disciplines, revolutionizing how we study genes and genomes.
Here's how it works:
1. Target selection: You define the specific DNA fragment you want to amplify. This is done by designing short DNA sequences called primers that flank the target region.
2. Reaction setup: The reaction mixture includes the DNA template, primers, DNA polymerase enzyme, nucleotides (building blocks for DNA synthesis), and buffer.
3. Thermal cycling: The mixture undergoes repeated cycles of temperature changes:
*Denaturation: High temperature (95°C) separates the double-stranded DNA into single strands, making the target region accessible.
*Annealing: Lower temperature (around 50-60°C) allows the primers to bind to their complementary sequences on the single-stranded target DNA.
*Extension: Optimal temperature (around 72°C) for the DNA polymerase to synthesize new DNA strands using the primers as starting points, copying the target sequence.
4. Exponential amplification: With each cycle, the amount of target DNA doubles. After 30-40 cycles, millions or even billions of copies of the target DNA fragment are generated.
Applications of PCR are vast and include:
*Gene cloning: Amplifying specific genes for subsequent characterization or manipulation.
Diagnosis of genetic diseases: Detecting mutations or gene deletions associated with specific diseases.
DNA fingerprinting: Analyzing individual variations in DNA for forensic applications or paternity testing.
*Gene expression analysis: Measuring the levels of mRNA transcripts to understand gene activity.
*Ancient DNA analysis: Amplifying and studying DNA from ancient remains like fossils or museum specimens.
Advantages of PCR:
*High sensitivity: Can detect even minute amounts of target DNA.
*Specificity: Targets only the desired DNA sequence defined by the primers.
*Versatility: Applicable to various research areas and sample types.
*Speed and efficiency: Generates millions of copies in a short time compared to traditional cloning methods.
Limitations of PCR:
*Primer design: Requires careful selection of primers to avoid non-specific amplification.
*Contamination: Sensitive to contamination with extraneous DNA, leading to false positives.
*Error rate: DNA polymerase can introduce errors during the copying process, affecting downstream applications.
15. Write a comparative account of the genomic and cDNA libraries.
Ans: Genomic vs. cDNA Libraries: Capturing Different Worlds of DNA
Genomic and cDNA libraries represent two fundamental tools in molecular biology, offering distinct snapshots of an organism's genetic information. Here's a comparative account of their key features:
Starting Material:
*Genomic Library: Constructed from an organism's genomic DNA, which includes both coding and non-coding regions, encompassing the entire genome.
*cDNA Library: Derived from an organism's mature mRNA molecules, representing only transcribed and potentially translated regions of the genome.
Content:
*Genomic Library: Represents the complete genetic blueprint, including introns, exons, regulatory sequences, and even repetitive elements. Offers a comprehensive view of an organism's genetic information.
*cDNA Library: Focuses on expressed genes, containing only exons spliced together after RNA processing. Ignores non-coding regions and rarely includes alternative splicing variants.
Applications:
*Genomic Library: Useful for identifying and isolating entire genes, studying regulatory elements, mapping mutations, and analyzing genome organization.
*cDNA Library: Ideal for studying gene expression patterns, identifying differentially expressed genes, isolating expressed genes for functional analysis, and cloning protein-coding sequences.
Advantages:
*Genomic Library: Provides a complete picture of the genome, potentially revealing regulatory elements and unannotated genes. Offers a broader perspective for genetic studies.
*cDNA Library: Easier to work with as it eliminates introns and simplifies gene identification and manipulation. Useful for studying gene expression and protein function.
Disadvantages:
*Genomic Library: Large size and complexity make downstream applications like screening and manipulation more challenging. May contain non-functional or irrelevant sequences.
*cDNA Library: Misses non-coding regions and lacks information about gene regulation and alternative splicing. Only reflects genes actively expressed under specific conditions.
Choosing the Right Library:
The choice between a genomic and cDNA library depends on the specific research question:
*Study gene structure and regulation: Opt for a genomic library.
*Analyze gene expression and protein function: Choose a cDNA library.
*Comprehensive understanding of a gene: Combine both library types for in-depth analysis.
In conclusion, both genomic and cDNA libraries serve unique purposes in exploring an organism's genetic landscape. Understanding their distinct features and applications empowers researchers to choose the appropriate tool for unraveling the intricate secrets of genes and genomes.
16. Diploid human genome contains:
17. Select the incorrectly matched pair from the following.
(a) Nucleases : Hydrolyse phosphodiester bond
(b) Restriction enzymes: Cleave DNA at specific sequence
(c) Palindromic sequence: Read same backwards and forward
(d) EcoRI: Type I Restriction Enzyme
Ans: (d) EcoRI: Type I Restriction Enzyme.
18. Assertion: PCR can be used to amplify very smallamount of DNA using DNA modifying enzymes.
Reason: PCR uses Taq Polymerase.
(a) Both assertion and reason are true and the reason is the correct explanation of the assertion.
(b) Both assertion and reason are true but the reason is not the correct explanation of the assertion.
(c) Assertion is true but reason is false.
(d) Both assertion and reason are false.
Ans: (a) Both assertion and reason are true and the reason is the correct explanation of the assertion.
19. Assertion: Foreign gene can be introduced into host bacterium by transformation techniques like electroporation.
Reason: Bacteria have cell wall/membrane.
(a) Both assertion and reason are true and the reason is the correct explanation of the assertion.
(b) Both assertion and reason are true but the reason is not the correct explanation of the assertion.
(c) Assertion is true but reason is false.
(d) Both assertion and reason are false.
Ans: (c) Assertion is true but reason is false.
Questions and Answer Type By: Himashree Bora.
Post ID: DABP007075
