Gene Cloning - Class 12 Biotechnology - Chapter 3 - Notes, NCERT Solutions & Extra Questions
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Describe the methods used for isolation of DNA.
Isolation of DNA involves several crucial steps to ensure purity and integrity of the extracted DNA, which is essential for various molecular biology experiments and applications. The first step is the cell lysis, where cellular membranes are disrupted. This can be achieved through physical methods such as grinding in liquid nitrogen, or using detergents and enzymes like lysozyme or SDS to dissolve the cell membrane.
In the second step, known as deproteinization, proteins must be removed to prevent contamination of the DNA sample. This often involves the use of protease enzymes and further purification using phenol-chloroform extraction. The phenol-chloroform mixture helps in separating the proteinaceous phase from the aqueous DNA solution.
The third and final step is the precipitation of DNA using alcohol, typically ethanol or isopropanol. During this process, the soluble DNA in the aqueous solution forms a precipitate upon addition of alcohol, especially under cold conditions. This precipitate is then washed and solubilized in a suitable buffer for further use.
Each of these steps must be carefully controlled to avoid degradation and loss of the DNA, ensuring that the final product is suitable for accurate and efficient downstream applications.
What is the role of biological detergent in the process of isolation of nucleic acid?
In the isolation of nucleic acids, biological detergents play a crucial role in lysing cell membranes or cell walls, which is pivotal for releasing nucleic acids into the extraction medium. These detergents, such as sodium dodecyl sulfate (SDS), disrupt the lipid bilayer of cell membranes due to their surfactant properties, effectively breaking down the protective barrier and ensuring cellular components are exposed.
For bacteria, an enzyme called lysozyme is used to digest peptidoglycan in the cell wall, followed by SDS to dissolve the cell membrane. In plant cells, which have a tougher structural makeup, detergents like cetyl trimethylammonium bromide (CTAB) are preferred for their effectiveness in breaking down the rigid cell walls. CTAB also aids in separating polysaccharides from nucleic acids during DNA extraction by exploiting differential solubility in varying ionic strength solutions. This property ensures that the nucleic acids can be purified effectively without contamination from other macromolecules.
How does DNA isolation from plant tissue differ from that of bacterial cell?
DNA isolation from plant tissues is more challenging than from bacterial cells due to the presence of cell walls and higher content of polysaccharides and polyphenolic compounds in plant cells. These substances can interfere with the DNA extraction process. To isolate DNA from plant tissues, cell walls are mechanically disrupted using methods such as grinding in liquid nitrogen. Special detergents like cetyl trimethyl ammonium bromide (CTAB) are used to separate DNA from polysaccharides; the ionic strength of the solution is adjusted to selectively precipitate these contaminants. Additionally, polyvinyl pyrrolidone (PVP) is often added to neutralize polyphenols which might otherwise degrade the DNA.
In contrast, bacterial cell DNA isolation is generally simpler. Bacterial cells lack complex structural components like those of plants. Here, an enzyme like lysozyme is used to digest the cell wall, typically made of peptidoglycan. Then, detergents like sodium dodecyl sulphate (SDS) aid in lysing the cell membrane to release DNA.
How many types of restriction enzymes (REs) are there? Can all REs be used in rDNA technology? Give justification.
There are three main types of restriction enzymes (REs), categorized as Type I, Type II, and Type III, based on their cofactor requirements and the position of their DNA cleavage site relative to the recognition sequence. Type I enzymes cleave DNA at random sites far from the recognition site and are not commonly used in rDNA technology due to their unpredictable cutting patterns. Type II enzymes, which cleave within or at specific short distances from their recognition sites, are the most useful in rDNA technology because they produce predictable and reproducible cuts, facilitating the creation of recombinant DNA. Type III enzymes also cleave at specific distances from their recognition sites but, like Type I, require ATP and are more complex, making them less favored than Type II enzymes. Therefore, not all REs are equally suitable for rDNA technology; Type II REs are preferred for their precision and simplicity in gene cloning and molecular biology projects.
What are the challenges faced during the process of nucleic acid extraction?
During the process of nucleic acid extraction, several challenges arise that must be addressed to ensure the integrity and purity of the extracted nucleic acids. Firstly, the limited availability of DNA and RNA in cells compared to other macromolecules like proteins creates a need for sensitive extraction techniques to maximize yield. Secondly, the molecular stability of nucleic acids is a concern as they are susceptible to degradation by nucleases; in the cell, these enzymes can rapidly degrade unprotected DNA and RNA following extraction.
Another challenge involves the separation of nucleic acids from cellular contaminants. The presence of proteins, lipids, and carbohydrates can interfere with downstream applications, necessitating rigorous purification steps. Additionally, the physical shearing forces during extraction can fragment the long strands of DNA or RNA, leading to potential loss of information or function. Addressing these challenges requires careful optimization of extraction protocols, protective measures against nucleases, and gentle handling to ensure high-quality, intact nucleic acids for research and diagnostic purposes.
Write the role of alkaline phosphatase, DNA ligase, terminal transferase in rDNA technology.
In recombinant DNA (rDNA) technology, several enzymes play pivotal roles:
- Alkaline phosphatase is crucial for preparing DNA for the ligation process. It removes the 5' phosphate groups from DNA fragments, preventing unwanted self-ligation and ensuring that these fragments can only join with other DNA segments that are introduced with complementary overhangs or blunt ends.
- DNA ligase is fundamental in the process of gene cloning. It catalyzes the formation of phosphodiester bonds between adjacent DNA fragments, effectively sealing nicks and joining DNA strands. This enzyme is especially important in the final steps of vector and insert combination, allowing for the stable insertion of a gene of interest into vector DNA.
- Terminal deoxynucleotidyl transferase (or terminal transferase) adds nucleotides to the 3' end of a DNA molecule. In rDNA technology, it is used to add poly(A) tails or other nucleotide sequences, facilitating cloning or increasing the stability of recombinant constructs.
Describe the role of chelating agent in the process of DNA extraction.
In the process of DNA extraction, the role of a chelating agent is crucial. One commonly used chelating agent is ethylene diamine tetraacetic acid (EDTA). The primary function of EDTA is to bind divalent metal ions, such as magnesium (Mg²⁺) and manganese (Mn²⁺). These metal ions are essential cofactors for enzymes like nucleases that can degrade nucleic acids. By binding to these ions, EDTA inhibits the activity of these enzymes, thereby protecting DNA from enzymatic degradation during the extraction process.
The presence of EDTA in the extraction buffer helps in maintaining the integrity of the DNA by preventing its breakdown. This is particularly important during the cell lysis stage, where cellular enzymes that can digest DNA are released into the mixture. By chelating the metal ions, EDTa ensures that these nucleases remain inactive, thus enhancing the yield and purity of the extracted DNA. This makes EDTA an indispensable component in the buffers used for DNA extraction, contributing significantly to the success of downstream applications such as cloning, PCR, and genomic analysis.
Briefly describe the modes of DNA transfer into the host.
Modes of DNA transfer into a host cell play a pivotal role in recombinant DNA technology. These methods are broadly categorized into natural and artificial techniques.
Natural methods include:
1. Transformation: Direct uptake of exogenous DNA from the environment into the cell.
2. Transduction: Transfer of DNA from one cell to another via bacteriophages.
3. Conjugation: Transfer of DNA between bacteria through direct cell-to-cell contact, mediated by a pilus.
Artificial techniques involve:
1. Electroporation: Creates temporary pores in cell membranes using an electric pulse to introduce DNA.
2. Microinjection: Direct injection of DNA into the cell using a fine needle.
3. Biolistic or Gene Gun: High-velocity microprojectiles coated with DNA are shot directly into cells.
4. Lipofection: Uses lipid-based particles to encapsulate the DNA and facilitate its introduction into cells.
Each of these methods ensures that the foreign DNA is efficiently delivered into the host's genome, enabling the expression of new genetic information vital for various applications in biotechnology and medicine.
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.
(b) It is based on the expression of lacZ gene.
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
Correct Match:
(c) Western blot: Detect specific proteins
Explanation:
Western blotting is indeed used to detect specific proteins in a sample of tissue homogenate or extract.
The other options are incorrectly matched as follows:
(a) Northern blot is used to detect specific RNA molecules, not DNA.
(b) Southern blot is used to detect specific sequences of DNA, not RNA.
(d) Eastern blot is used for detecting specific post-translational modifications of proteins, not transcriptional modifications in RNA.
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
The incorrect matched pair from the provided options is:
(d) PstI: Pyrococcus stuartii
Correct: PstI is derived from *Providencia stuartii*, not *Pyrococcus stuartii*.
How are recombinants screened? Describe the methods in detail.
Screening of recombinants is crucial in genetic engineering to identify cells that have successfully incorporated the gene of interest. The two main methods are:
1. Direct Selection: This involves using a selectable marker in the vector, such as an antibiotic resistance gene. Transformed cells are cultured on a medium containing the antibiotic; only those cells that have taken up the recombinant plasmid survive, allowing direct selection of recombinants.
2. Insertional Inactivation (Blue-White Selection): This method uses vectors containing a lacZ gene, which codes for β-galactosidase. When the gene of interest is inserted into the lacZ sequence, it disrupts its function, preventing the enzyme from being produced. Transformed cells are plated on media containing X-gal and IPTG. Cells with non-recombinant plasmids produce β-galactosidase, hydrolyzing X-gal and turning blue. Conversely, recombinant plasmids yield white colonies due to the inactivation of lacZ, allowing easy identification of successful recombinants.
Both methods provide efficient ways to distinguish recombinant cells from non-transformed ones, streamlining the process of genetic manipulation.
Differentiate between the Southern, Northern and Western blotting.
Parameter | Southern Blotting | Northern Blotting | Western Blotting |
---|---|---|---|
Purpose | Detects and analyzes specific DNA sequences in DNA samples. | Used for the detection of specific RNA sequences in a mixture of RNA. | Identifies specific proteins in a sample of tissue homogenate or extract. |
Sample Type | DNA fragments. | RNA molecules. | Proteins, separated by SDS-PAGE. |
Probe Type | DNA probes, which hybridize to complementary DNA sequences. | RNA or DNA probes that hybridize to RNA sequences of interest. | Antibodies specific to the protein of interest, used to detect the protein on the blot. |
Detection | The probe is labeled (radioactive, fluorescent, or chemiluminescent) and visualized to locate DNA. | Similar labeling and visualization to detect RNA. | Proteins are detected using antibodies coupled to a reporter enzyme or a fluorescent marker for visualization. |
Scientific Relevance | Useful for gene mapping, genotyping, and detecting gene rearrangements. | Essential for studying gene expression patterns, RNA processing, and RNA transcript sizes. | Critical for studying protein expression, modifications, and protein-protein interactions. |
What is PCR? Describe in detail.
Polymerase Chain Reaction (PCR) is a powerful method used to amplify small segments of DNA or RNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. It is highly efficient, rapid, and can amplify DNA from a single cell.
The technique relies on thermal cycling, consisting of cycles of repeated heating and cooling for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary in the region surrounding the desired DNA region, a DNA polymerase enzyme, and nucleotides are key components.
The steps include:
1. Denaturation: Heating the reaction strongly to separate the DNA strands.
2. Annealing: Cooling to allow primadoc to bind to the complementary DNA template.
3. Extension: DNA polymerase synthesizes a new DNA strand complementary to the DNA template.
PCR is pivotal in various applications including research, medical diagnostics, forensic analysis, and more. Its ability to detect and quantify DNA or RNA in samples has revolutionized scientific studies.
Write a comparative account of the genomic and cDNA libraries.
Genomic DNA Library entails a comprehensive collection of DNA fragments from an organism's genome. These fragments, cloned into vectors, represent the entirety of the genome. It includes both coding and non-coding sequences, providing a full genomic overview. This is especially useful for studying organismal genetics in its entirety, including evolutionary aspects and comparisons between healthy and diseased tissues.
cDNA Library, on the other hand, contains only the expressed sequences of a genome. These libraries are constructed from mRNA, reflecting the actively expressed genes under specific conditions or tissues. This is achieved by reverse-transcribing mRNA into cDNA. Hence, cDNA libraries are highly specific and useful for studying gene expression profiles and identifying functional genes relevant to particular physiological states.
In summary, while genetic libraries provide a snapshot of all possible genes, cDNA libraries offer a targeted view of active genetic expressions, making them invaluable for functional genomics and gene expression studies.
Diploid human genome contains:
(a) 3.2 × 109 base pairs
(b) 6.4 × 108 base pairs
(c) 3.2 × 108 base pairs
(d) 6.4 × 109 base pairs
The correct answer is: (a) $3.2 × 10^9$ base pairs
This represents the approximate number of base pairs in a haploid human genome; for a diploid genome, this number would double, but since each chromosome pair comes from different parental origins, the $3.2 × 10^9$ bp figure is commonly referenced as the size of the human genome in each set of chromosomes.
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
The incorrectly matched pair is:
(d) EcoRI: Type I Restriction Enzyme
EcoRI is a Type II restriction enzyme, not a Type I. Type II restriction enzymes cleave DNA within or at short specific distances from their recognition sites and do not require ATP, in contrast to Type I enzymes which cleave at sites remote from their recognition sequence and require ATP.
Assertion: PCR can be used to amplify very small amount 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.
(a) Both assertion and reason are true and the reason is the correct explanation of the assertion.
Assertion: PCR can be used to amplify very small amount of DNA using DNA modifying enzymes. This assertion is true because PCR (Polymerase Chain Reaction) is specifically designed to exponentially amplify small quantities of DNA, utilizing the enzymatic activity of DNA polymerases to replicate DNA sequences.
Reason: PCR uses Taq Polymerase. This is a true statement. Taq Polymerase is a thermostable DNA polymerase enzyme derived from the bacterium *Thermus aquaticus*. It is capable of withstanding high temperatures used in PCR to denature DNA, making it ideal for the cycles of heating and cooling in the PCR process.
The reason also correctly explains the assertion because the use of Taq Polymerase is essential for PCR to function across multiple cycles involving high temperatures. Its thermostability allows it to synthesize new DNA strands from the DNA template repeatedly, which is fundamental for the amplification of DNA in PCR.
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.
(b) Both assertion and reason are true but the reason is not the correct explanation of the assertion.
Explanation:
Assertion: True. Techniques like electroporation are indeed used to introduce foreign DNA into a host bacterium.
Reason: True. Bacteria do possess cell walls/membranes. However, this is not why electroporation can be used. Electroporation involves applying an electric field to increase the permeability of the cell membrane, allowing DNA to enter the cell. The presence of cell walls/membranes merely identifies a characteristic of bacteria but isn't the reason why electroporation is effective.
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Gene Cloning Class 12 Notes: Comprehensive Guide for Students
Introduction to Gene Cloning
Gene cloning is a fundamental technique in biotechnology that allows scientists to produce multiple copies of a specific gene. This technology has revolutionary applications in fields ranging from agriculture to medicine. Gene cloning typically involves transferring a DNA fragment containing the gene of interest into a host cell, where it can be replicated and analysed.
Steps Involved in Gene Cloning
Identification of the Candidate Gene
The first step in gene cloning is identifying a candidate gene, which is chosen based on its significance in medical, economic, or evolutionary contexts. Scientists often rely on biochemical and physiological studies to locate genes that, for instance, cause diseases or confer desirable traits like pest resistance in crops.
Isolation of Nucleic Acids
Once a candidate gene is identified, DNA or RNA must be isolated from the organism. This process involves several critical steps to ensure the purity and integrity of the nucleic acids:
- Cell Disruption: Breaking the cell wall or membrane to release nucleic acids.
- Protecting Nucleic Acids: Using chemicals to guard against degradation by enzymes.
- Separating Nucleic Acids: Removing other cellular molecules.
- Precipitation: Concentrating the nucleic acids by adding ethanol or isopropanol.
Here is a basic flowchart summarising these steps:
graph TD;
A[Identification of Candidate Gene] --> B[Isolation of Nucleic Acids]
B --> C[Cell Disruption]
C --> D[Protecting Nucleic Acids]
D --> E[Separating Nucleic Acids]
E --> F[Precipitation]
Enzymes Used in Recombinant DNA Technology
Numerous enzymes facilitate the manipulation of DNA in gene cloning:
-
Nucleases: Cut DNA strands by hydrolysing phosphodiester bonds.
- Exonucleases: Remove nucleotides from the end of DNA molecules.
- Endonucleases: Break internal phosphodiester bonds.
- Restriction Endonucleases: Cut DNA at specific sequences.
- DNA Ligase: Joins DNA strands by forming phosphodiester bonds.
- DNA Polymerases: Synthesize new DNA strands from nucleotides.
- Reverse Transcriptase: Converts RNA into complementary DNA (cDNA).
Methods of DNA Transfer
There are several ways to insert recombinant DNA into host cells:
- Transformation: Uptake of naked DNA by cells.
- Transduction: Viruses introduce DNA into bacterial cells.
- Conjugation: DNA transfer between bacterial cells through direct contact.
In addition to these natural methods, scientists use techniques like electroporation, microinjection, and lipofection to introduce DNA into cells.
Screening and Selection of Recombinant Cells
Once the host cells have taken up the recombinant DNA, it’s crucial to identify and isolate the transformed cells.
Direct Selection: Transformed cells express a trait (e.g., antibiotic resistance) that untransformed cells do not.
Insertional Inactivation: Disrupting a marker gene in the vector to differentiate between recombinant and non-recombinant cells.
A common method for selection is the blue-white screening technique, which involves inserting the gene of interest into the LacZ gene. Transformed cells that have the recombinant plasmid produce white colonies, while those that do not produce blue colonies.
Blotting Techniques
Blotting techniques help to identify specific DNA, RNA, or proteins from a mixture.
- Southern Blotting: Detects specific sequences in DNA.
- Northern Blotting: Identifies RNA molecules in a sample.
- Western Blotting: Detects specific proteins in a tissue extract.
Polymerase Chain Reaction (PCR)
PCR is a technique that amplifies small amounts of DNA into millions of copies through thermal cycling. The steps involved are:
- Denaturation: Heating DNA to break hydrogen bonds.
- Annealing: Binding of primers to target sequences.
- Extension: DNA polymerase synthesizes new DNA strands.
Real-time PCR (qPCR) and Reverse Transcription PCR (RT-PCR) are advanced PCR methods used for various applications, including gene expression analysis.
DNA Libraries
Genomic DNA Library
A genomic library contains cloned DNA fragments that represent the entire genome of an organism. This library is useful for sequencing genomes and identifying unknown genes.
cDNA Library
A cDNA library consists of cloned cDNA fragments synthesized from mRNA. This library represents the genes expressed in a specific tissue or under certain physiological conditions.
Applications and Ethical Considerations
Gene cloning has broad applications in agriculture and medicine:
- Agriculture: Creating pest-resistant crops.
- Medicine: Producing insulin and human growth hormones.
However, ethical concerns, such as the impact on natural ecosystems and genetic privacy, must be considered.
Conclusion
Understanding the steps and components involved in gene cloning enables students to grasp its vast applications and importance. As technology advances, gene cloning continues to be an invaluable tool in scientific discovery and innovation.
By grasping these fundamental concepts, you’re well on your way to mastering gene cloning and appreciating its real-world impacts.
Feel free to revisit this guide whenever you need a clear and concise understanding of gene cloning for your exams and beyond.
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