Biotechnology Principles and Processes - Class 12 Biology - Chapter 9 - Notes, NCERT Solutions & Extra Questions
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Extra Questions - Biotechnology Principles and Processes | NCERT | Biology | Class 12
Which of the following steps is catalysed by Taq polymerase?
A. Denaturation of template DNA
B. Annealing of primers to template DNA
C. Extension of primer end on the template DNA
D. Maintenance of high temperature during PCR
Taq polymerase is extensively utilized in the polymerase chain reaction (PCR), which is a technique designed to amplify DNA sequences. PCR involves several critical steps:
Denaturation: This step involves the separation of the double-stranded DNA into two single strands, typically induced by high temperatures around $94^\circ C$.
Annealing: During annealing, short sequences known as primers bind, or anneal, to specific sections of the single-stranded DNA, providing a starting point for DNA synthesis.
Extension: Here, the crucial role of Taq polymerase is evident. This enzyme catalyzes the synthesis of a new DNA strand by extending the primer along the template DNA sequence.
Given its function, Taq polymerase is directly responsible for the extension of the primer end on the template DNA during PCR. This enzyme is highly valued in molecular biology for its ability to efficiently synthesize DNA at the high temperatures typical of PCR processes.
While sequencing the DNA fragment in a BAC, it is further cut into fragments containing 2000 base pairs. How are the DNA fragments from BACs separated from the human DNA?
A. BAC DNA is cut out early, and only human DNA is cut into smaller fragments.
B. The DNA sequence of BAC is known; therefore, during tallying the DNA sequence of all fragments, the BAC DNA is left out.
C. BAC DNA has specific markers that can be read and identified during sequencing.
D. None of the above.
The correct answer is Option B: "The DNA sequence of BAC is known; therefore, during tallying the DNA sequence of all fragments, the BAC DNA is left out."
Explanation: Before DNA sequencing begins, both human DNA and BAC DNA are fragmented into smaller pieces. All these fragments are then sequenced. Since the sequence of the BAC is already known, these specific sequences can be identified and excluded when analyzing the data. By doing this, only the sequences that belong to human DNA are considered for further analysis. This technique ensures that the final DNA sequence data represents only the human DNA, while the known BAC sequences are ignored.
In pBR322, a gene is inserted using the PvuI restriction enzyme site, which makes the ampicillin-resistant gene non-functional. However, the recombinant is resistant to tetracycline. This means:
A. pBR322 has a tetracycline-resistant gene as well as an ampicillin-resistant gene.
B. The insertion of the gene has given pBR322 tetracycline-resistant properties.
C. The tetracycline-resistant property of pBR322 is functional only in the absence of the ampicillin-resistant property.
D. The recombinant is resistant to both ampicillin and tetracycline.
The correct answer is A: pBR322 has a tetracycline-resistant gene as well as an ampicillin-resistant gene.
The plasmid pBR322 contains two key antibiotic resistance genes: one for ampicillin and another for tetracycline. The PvuI restriction enzyme site is located within the ampicillin resistance gene. When a foreign gene is inserted at this site, it disrupts the ampicillin resistance gene, rendering the recombinant bacteria sensitive to ampicillin. However, because the tetracycline resistance gene is not affected by this insertion, the recombinant plasmid confers resistance to tetracycline to the host bacteria. Thus, the bacteria harboring this recombinant plasmid are resistant to tetracycline but not to ampicillin.
Which of the following sequences can be recognized by a restriction enzyme?
A) 5' GAATTC 3' 3' CTTAAG 5'
B) 5' GATC 3' 3' CTAG 5'
C) 5' GTTA 3' 3' CAAT 5'
D) 5' CATGG 3' 3' GTACC 5'
The correct answer is option A:
$5'$ GAATTC $3'$
$3'$ CTTAAG $5'$
This DNA sequence displays the property of a palindromic sequence, meaning it reads the same from both $5'$ to $3'$ and $3'$ to $5'$ directions:
Forward read: $5' \rightarrow$ GAATTC $\rightarrow 3'$
Reverse complement: $3' \rightarrow$ CTTAAG $\rightarrow 5'$
Restriction enzymes specifically recognize such sequences. They typically cleave the DNA a short distance away from the center of these palindromic sites, but between the same two bases on oppoiste strands. This action results in leaving overhanging single-stranded portions at each end, facilitating further genetic manipulation or analysis.
DNA or RNA segment tagged with a radioactive molecule is called
A) vector
B) probe
C) clone
D) plasmid
The correct answer is B) probe.
Molecular probes are DNA or RNA segments used to identify the presence of complementary sequences within nucleic acid samples. Typically, these probes range from 200 to 500 nucleotides in length and can be labeled with either radioactive or nonradioactive substances. The primary function of probes is to assist in the identification and isolation of specific DNA and RNA sequences.
Which of the following is not a use of USG technique?
A. Detecting foetal heartbeat
B. Measuring Nuchal translucency
C. Genetic disorders in foetus
D. Extracting chorionic villi cells
The correct answer is D. Extracting chorionic villi cells
Ultrasound (USG) is a noninvasive imaging technique widely used in detecting various conditions and developments in a fetus.
It enables the detection of a fetal heartbeat, verifying the viability of the pregnancy.
USG is also employed to measure Nuchal translucency which is the fluid under the baby's skin at the back of the neck, aiding in assessments for certain genetic conditions.
Moreover, USG can detect signs that suggest genetic disorders in the fetus.
However, USG does not involve the extraction of chorionic villi cells. This process, known as chorionic villus sampling, is a separate procedure where cells from the placenta are taken for genetic testing, and it requires different methods not involving USG.
The process of making proteins from mRNA sequence is called:
A. translation
B. translocation
C. transcription
The correct answer is A. translation.
Translation is the biological process where the sequence of bases in mRNA provides the template for assembling a sequence of amino acids to form a protein. Each triplet codon in the mRNA corresponds to a specific amino acid or a stop signal for the translation process. This occurs in the ribosome, where tRNA molecules bring the appropriate amino acids that match the codons on the mRNA. The ribosome reads the mRNA, directing the addition of amino acids in the correct order to build the protein chain. When the ribosome encounters a stop codon, it ceases adding amino acids, resulting in the release of the completed protein. This entire mechanism of synthesizing proteins guided by the mRNA template is called translation.
"Can a plasmid integrate with a eukaryotic genome?"
Yes, plasmids can integrate with a eukaryotic genome, particularly through a process known as transfection. This method is commonly used to transfer genes and facilitate the creation of genetic clones in various organisms.
DNA Fingerprinting was invented by:
A) Dr. Verghese Kurien
B) Sir Alec Jeffreys
C) Dr. Norman Borlaug
The correct option is B) Sir Alec Jeffreys.
DNA fingerprinting was invented by Sir Alec Jeffreys. This technique involves identifying the specific patterns in the nucleotide sequences of DNA, which are unique to each individual.
Difference between eukaryotes and prokaryotes in transcription
Differences between Prokaryotic and Eukaryotic Transcription
Prokaryotic Transcription | Eukaryotic Transcription | |
---|---|---|
1 | Transcription and translation occur simultaneously in the cytoplasm. | These are separate processes: transcription in the nucleus; translation in the cytoplasm. |
2 | Simple initiation machinery; DNA is not associated with histones. | Complex initiation machinery due to DNA-histone association. |
3 | One RNA polymerase synthesizes all RNA types (mRNA, rRNA, tRNA). | Three RNA polymerases: I for rRNA, II for mRNA, III for tRNA and 5S rRNA. |
4 | RNA polymerase consists of 5 subunits; functional form is 2α1β1β'1ω. | Multiple subunits: RNA polymerase I (14), II (10-12), III (12). |
5 | σ factor present, essential for initiation. | σ factor not required; initiation factors are used instead. |
6 | RNA polymerase can recognize/bind the promoter with the help of σ factor. | Requires initiation factors to pre-occupy the promoter for binding. |
7 | Promoter always upstream of the start site. | Promoter usually upstream, but variations exist like in RNA polymerase III. |
8 | Promoter contains a Pribnow box; lacks TATA and CAT boxes. | Promoter includes TATA box (~35-25 upstream), CAT box (~70 upstream), GC box (~110 upstream); Pribnow box absent. |
9 | Termination via rho-dependent or rho-independent mechanisms. | Termination method unclear; possibly involves poly A signal or DNA termination sequence. |
10 | No post-transcriptional modifications of primary transcript. | Extensive post-transcriptional modifications (RNA editing). |
11 | No RNA capping; mRNA lacks 5' guanosine cap. | RNA capping present; occurs at mRNA's 5' position. |
12 | No Poly A tail on mRNA. | Poly A tail present at mRNA’s 3' end. |
13 | Introns absent in mRNA. | Introns present in primary transcripts. |
14 | No mRNA splicing; introns absent. | Splicing present; introns removed and exons rejoined. |
15 | Genes usually polycistronic; one transcript may encode several polypeptides. | Genes monocistronic; each transcript encodes for a single polypeptide. |
16 | Shine-Dalgarno sequence (SD sequence) present; acts as ribosome binding site. | No SD sequence in eukaryotes. |
This table highlights the main distinctions in transcription mechanisms between prokaryotic and eukaryotic cells, such as the simplicity versus complexity of the transcription machinery, gene structure, and post-transcriptional processing. These differences are crucial for understanding cellular processes and genetic expression in different organisms.
Biotechnology medical products include:
A) insulin
B) growth hormone
C) tPA (tissue plasminogen activator)
D) all of these
The correct answer is D) all of these.
Insulin, growth hormone, and tPA (tissue plasminogen activator) are all medical products developed through recombinant DNA technology. This is a common method in biotechnology used to produce various therapeutic substances.
"Bt" in Bt cotton stands for:
A) Biotechnology
B) Bollworm toxin
C) Bacterial tolerant
D) Bacillus thuringiensis
The correct answer is D) Bacillus thuringiensis.
Bt in Bt cotton refers to the bacterium Bacillus thuringiensis. This bacterium is known for its cry genes, which are utilized in genetic engineering to confer resistance to pests in crops like cotton. Thus, 'Bt' signifies Bacillus thuringiensis.
Enzyme that is central to the PCR technology is:
A) Taq polymerase
B) Polymerase
C) Helicase
D) Reverse transcriptase
The correct answer is A) Taq polymerase.
Polymerase Chain Reaction (PCR) is a method used to rapidly copy small segments of DNA. One key component in PCR technology is the enzyme that facilitates the synthesis of new strands of DNA. Taq polymerase is a thermostable enzyme derived from the bacterium Thermus aquaticus, which thrives in high-temperature environments, such as hot springs. This enzyme is vital because it remains active at the high temperatures necessary for denaturing double-stranded DNA during the PCR cycle, allowing for the efficient amplification of DNA.
If pepsin is lacking in gastric juice, then the event in the stomach will be affected:
A. Digestion of starch into sugars
B. Proteins break down into peptides
C. Breaking of fats into glycerol and fatty acids
D. Digestion of nucleic acids
Pepsin is an enzyme primarily involved in the breakdown of proteins into smaller peptides in the stomach. Here are how the options relate to the role of pepsin:
Option A: Digestion of starch into sugars – This process is facilitated by the enzyme amylase, not pepsin. Therefore, the absence of pepsin won't affect this process.
Option B: Proteins break down into peptides – This is the primary function of pepsin. If pepsin is lacking, this process will be disrupted, leading to impaired protein digestion.
Option C: Breaking of fats into glycerol and fatty acids – This process is done by lipase enzymes and is also assisted by bile, which is not related to pepsin. Thus, the absence of pepsin does not impact fat digestion.
Option D: Digestion of nucleic acids – This process involves other specific enzymes and does not depend on pepsin for its action.
Given these points, the most affected event in the stomach if pepsin is lacking is the breakdown of proteins into peptides. Therefore, the answer is:
B. Proteins break down into peptides
Plasma membrane is made up of
A Protein
B Lipid
C Carbohydrate
D Both (1) and (2)
Plasma Membrane Composition
The plasma membrane, often known as the cell membrane, is a critical structure of cells that helps in maintaining its integrity and facilitating various functions like transport and cellular communication. We understand more about its composition primarily through the Fluid Mosaic Model.
According to this model, the plasma membrane is made up of:
Lipids - Specifically, it consists of a bilipid layer. This bilayer structure is the fundamental framework providing the membrane's flexible yet sturdy nature.
Proteins - Proteins are embedded within the lipid bilayer. They perform a variety of functions, from forming channels and carriers for substance movement into and out of the cell to signaling and structural roles.
While carbohydrates are also associated with the plasma membrane, they are typically found attached to lipids or proteins and play a role in cell recognition and protection, rather than forming the core structural component.
Given the answer choices:
A) Protein
B) Lipids
C) Carbohydrate
D) Both Protein and Lipids
The correct answer is D) Both Protein and Lipids, as these two are the primary components of the plasma membrane structure, as highlighted by the Fluid Mosaic Model.
Indian scientist known for research on cosmic rays and nuclear energy:
Dr. Prafullachandra Roy
Chandra Shekhara Venkat Raman
Dr. Panchanan Maheshwari
Dr. Homi Jahangir Bhabha
The Indian scientist known for his substantial contributions to the research on cosmic rays and nuclear energy is Dr. Homi Jahangir Bhabha. Dr. Bhabha showcased significant findings in the field by demonstrating the scattering of electrons and protons in 1935. This foundation led to profound advancements in nuclear physics and also contributed to his global recognition, including prestigious awards like the Nobel Prize.
While others mentioned like Dr. Prafullachandra Roy, Sir Chandra Shekhar Venkat Raman, and Dr. Panchanan Maheshwari have made their significant contributions to science, they did not specifically focus on cosmic rays as Dr. Bhabha did. Hence, among the given options, Dr. Homi Jahangir Bhabha is the correct answer.
Pancreatic juice contains more than one enzyme. Which among the following combination is correct?
A. Pepsin and lipase
B. Amylase and pepsin
C. Pepsin and trypsin
D. Trypsin and lipase
Pancreatic juice plays a critical role in digestion by releasing several essential enzymes. The prime enzymes found in pancreatic juice are trypsin, amylase, and lipase.
Trypsin helps in the breakdown of proteins.
Amylase assists in the digestion of starch.
Lipase is vital in the breakdown of fats.
From the options given:
Option A, Pepsin and lipase is incorrect because pepsin is not found in pancreatic juice but is instead secreted in the stomach.
Option B, Amylase and pepsin, is incorrect for similar reasons as A; pepsin is not a component of pancreatic juice.
Option C, Pepsin and trypsin, again includes pepsin which is not present in pancreatic juice.
Option D, Trypsin and lipase, is indeed correct because both enzymes are critical components of pancreatic juice.
Thus, the correct answer is Option D - Trypsin and lipase.
Hydrogen can be prepared by mixing steam and water gas at $500^{\circ} \mathrm{C}$ in the presence of $\mathrm{Fe}_{2} \mathrm{O}_{3}$ and $\mathrm{Cr}_{2} \mathrm{O}_{3}$. This process is called:
A Nelson process
B Serpeck's process
C Bosch process
D Parke's process
The correct option is C. Bosch process
In the Bosch process, large quantities of hydrogen are produced from inexpensive raw materials such as water and coke. When steam is passed over red hot coke (carbon) at a temperature of about $1200^{\circ} \mathrm{C}$, a mixture of carbon dioxide and hydrogen is produced. In this process, $\mathrm{Fe}_{2} \mathrm{O}_{3}$ acts as a catalyst and $\mathrm{Cr}_{2} \mathrm{O}_{3}$ as a promoter.
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Can you list 10 recombinant proteins which are used in medical practice? Find out where they are used as therapeutics (use the internet).
A list of 10 recombinant proteins commonly used in medical practice along with their therapeutic applications:
Insulin: Used to treat diabetes mellitus.
Erythropoietin (EPO): Used to treat anemia, especially in chronic kidney disease.
Human Growth Hormone (HGH): Used to treat growth disorders and growth hormone deficiency.
Interferon-alpha: Used to treat various forms of cancer and viral infections like hepatitis C.
Interferon-beta: Used to treat multiple sclerosis.
Factor VIII: Used to treat Hemophilia A.
Factor IX: Used to treat Hemophilia B.
Tissue Plasminogen Activator (tPA): Used to dissolve blood clots in patients with myocardial infarction, pulmonary embolism, and stroke.
Granulocyte Colony-Stimulating Factor (G-CSF): Used to stimulate the production of white blood cells and reduce the risk of infection in patients undergoing chemotherapy.
Monoclonal antibodies (e.g., Rituximab, Trastuzumab): Used to treat various cancers and autoimmune diseases.
These recombinant proteins are critical in the management and treatment of numerous health conditions and have revolutionized modern therapeutics. Recombinant DNA technology has enabled the production of these proteins in large quantities, making them readily available for clinical use.
Make a chart (with diagrammatic representation) showing a restriction enzyme, the substrate DNA on which it acts, the site at which it cuts DNA and the product it produces.
Restriction Enzyme: EcoRI
Structure of EcoRI:
EcoRI is a restriction enzyme from the bacterium *Escherichia coli*. Its function is to cut DNA at specific recognition sequences. The enzyme recognizes the palindromic sequence 5'-GAATTC-3'.
Substrate DNA:
DNA sequence: $$ \begin{aligned} & 5'-GAATTC-3' \\ & 3'-CTTAAG-5' \end{aligned} $$
Site of Action:
EcoRI specifically recognizes and cuts the DNA sequence between the G and the A on both DNA strands:
$$ \begin{aligned} & 5'-G \downarrow AATTC-3' \\ & 3'-CTTAA \uparrow G-5' \end{aligned} $$
Product:
After the DNA is cut by EcoRI, it results in sticky ends, which are the overhangs exposed from the staggered cut: $$ \begin{aligned} & 5'-G & & AATTC-3' \\ & 3'-CTTAA & & G-5' \end{aligned} $$
Diagrammatic Representation:
Diagram Steps:
Recognition: EcoRI binds to the DNA at the recognition sequence (5'-GAATTC-3').
Cleavage: EcoRI cuts the DNA between G and A on both strands.
Product Formation: The DNA fragments produced have sticky ends (overhangs).
Chart Summary:
Enzyme | Substrate DNA | Recognition Site | Cleavage | Products |
---|---|---|---|---|
EcoRI | 5'-GAATTC-3' | 5'-GAATTC-3' | Between G and A | DNA fragments with sticky ends (5'-G AATTC-3' and 3'-CTTAA G-5') |
From what you have learnt, can you tell whether enzymes are bigger or DNA is bigger in molecular size? How did you know?
DNA molecules are generally bigger in molecular size compared to enzymes.
Here's how we know:
DNA Composition: DNA molecules consist of long chains of nucleotides, which can create very large structures. An example is the human genome, which contains about 3 billion base pairs.
Enzyme Composition: Enzymes are typically made up of shorter chains of amino acids (polypeptides). Even large enzymes rarely exceed the length of medium-sized DNA sequences.
Furthermore, the process of recombinant DNA technology involves manipulating relatively small sections of DNA to introduce into hosts, yet even these "small" segments of DNA are larger than typical enzymes used in various reactions. Thus, the typical size of an enzyme is much smaller than lengthier DNA molecules.
What would be the molar concentration of human DNA in a human cell? Consult your teacher.
The molar concentration of human DNA in a human cell is approximately 1.54 nanomolar (nM) or 1.54 × 10^-9 M.
This means that the molar concentration of DNA in a human cell is very low, considering the small amount of DNA and the tiny volume of a cell.
Do eukaryotic cells have restriction endonucleases? Justify your answer.
No, eukaryotic cells do not generally possess restriction endonucleases. These enzymes are primarily found in prokaryotic organisms, particularly bacteria, where they serve as a defense mechanism against viral DNA. The key points to understand here are:
Source: Restriction endonucleases are enzymes typically found in bacteria and archaea. They are absent in eukaryotic cells.
Function in Prokaryotes: In bacteria, restriction endonucleases protect against invading viral DNA by cutting it into smaller, non-functional pieces. Bacteria also have methylation systems to protect their own DNA from being cut by these enzymes.
Eukaryotic Defense Mechanism: Eukaryotic cells have different mechanisms for protecting their genomes and dealing with foreign DNA, such as the immune system and RNA interference (RNAi).
Here’s an important point from the provided text:
"In the year 1963, the two enzymes responsible for restricting the growth of bacteriophage in *Escherichia coli* were isolated. One of these added methyl groups to DNA, while the other cut DNA."
This indicates that restriction endonucleases are a part of bacterial defense mechanisms rather than eukaryotic defense systems.
Besides better aeration and mixing properties, what other advantages do stirred tank bioreactors have over shake flasks?
Stirred tank bioreactors have several advantages over shake flasks beyond better aeration and mixing properties:
Controlled Environment: Stirred tank bioreactors allow precise control over various parameters such as temperature, pH, and oxygen levels, which are essential for optimal growth and productivity of the microorganisms or cells.
Scalability: They can be scaled up to process large volumes (100-1000 liters) of culture, making them suitable for industrial-scale production.
Sterility: Stirred tank bioreactors have better mechanisms for maintaining a sterile environment, crucial for avoiding contamination and ensuring product quality.
Monitoring and Sampling: They are equipped with sampling ports for regular monitoring, allowing the extraction of small volumes of culture to check for growth conditions and product synthesis.
Foam Control: They include foam control systems, which help manage foam that is often produced during microbial growth and can interfere with aeration and mixing.
Automated Control Systems: They can be integrated with automated control systems that continually adjust the environmental parameters and thus optimize the production process.
By providing a controlled, scalable, and monitored environment, stirred tank bioreactors significantly enhance the efficiency and consistency of bioproduction processes.
Collect 5 examples of palindromic DNA sequences by consulting your teacher. Better try to create a palindromic sequence by following base-pair rules.
Five examples of palindromic DNA sequences:
GAATTC
$5' \rightarrow 3'$ : GAATTC
$3' \rightarrow 5'$ : CTTAAG
AGCT
$5' \rightarrow 3'$ : AGCT
$3' \rightarrow 5'$ : TCGA
AAGCTT
$5' \rightarrow 3'$ : AAGCTT
$3' \rightarrow 5'$ : TTCGAA
GGATCC
$5' \rightarrow 3'$ : GGATCC
$3' \rightarrow 5'$ : CCTAGG
TTAATTAA
$5' \rightarrow 3'$ : TTAATTAA
$3' \rightarrow 5'$ : AATTAATT
To create your own palindromic sequence, follow these base-pair rules:
Write a sequence of bases in the $5' \rightarrow 3'$ direction (e.g., "AGCT").
Reverse it and replace each base with its complement (A with T, G with C, etc.) for the $3' \rightarrow 5'$ sequence (e.g., "AGCT" becomes "TCGA").
Can you recall meiosis and indicate at what stage a recombinant DNA is made?
Recombinant DNA is typically not made during the natural process of meiosis, which is the cell division process that results in four genetically diverse haploid cells from one diploid cell. However, in the context of recombinant DNA technology, the creation of recombinant DNA involves several stages such as isolation of DNA, cutting of DNA at specific locations using restriction enzymes, and inserting the cut DNA into vectors which are then transferred to host cells.
To clarify in the context of your biology chapter, recombinant DNA is created artificially *before* any insertion into a host organism. It is not a product of meiosis but rather a product of laboratory techniques involving genetic engineering.
Key Stages in Creating Recombinant DNA:
Isolation of Genetic Material: Extraction of DNA from a cell.
Cutting of DNA: Use of restriction enzymes to cut DNA at specific sequences.
Ligation: Joining of DNA fragments using DNA ligase.
Insertion: Introduction of recombinant DNA into a host organism.
This process is separate from meiosis and is a part of genetic engineering to create organisms with modified traits.
Can you think and answer how a reporter enzyme can be used to monitor transformation of host cells by foreign DNA in addition to a selectable marker?
A reporter enzyme can be used to monitor the transformation of host cells by foreign DNA, in conjunction with a selectable marker, through the following process:
Insertion of Reporter Gene: A reporter gene that encodes the reporter enzyme (e.g., $\beta$-galactosidase, luciferase) is inserted into the vector alongside the foreign DNA.
Expression and Detection: After the transformation process, the host cells are cultured under conditions that allow for the expression of the reporter gene. The presence of the reporter enzyme is then detected using specific substrates that produce a detectable signal when acted upon by the enzyme.
Signal Observation: For example, if $\beta$-galactosidase is used as the reporter enzyme, the transformed cells can be plated on a medium containing X-gal. The enzyme cleaves X-gal, resulting in the formation of a blue color. Only cells that have taken up the vector containing the reporter gene and the foreign DNA will exhibit this blue color.
Thus, the **signal generated by the reporter enzyme provides a visual or measurable confirmation of successful transformation, complementing the use of a selectable marker, which ensures that only the transformed cells survive under selective conditions. This dual approach enhances the efficiency and accuracy of identifying transformed cells.
Describe briefly the following:
(a) Origin of replication
(b) Bioreactors
(c) Downstream processing
(a) Origin of Replication
The origin of replication (ori) is a specific sequence on a DNA molecule where replication begins. This sequence is crucial in genetic engineering because:
When a piece of DNA is linked to this sequence, it can replicate within host cells.
The copy number of the DNA sequence linked to the origin can be controlled.
For effective cloning, the vector should support a high copy number if many copies of the target DNA are needed.
(b) Bioreactors
Bioreactors are vessels used for the large-scale production of biotechnology products. They provide an optimal environment for the growth of microorganisms or cells to produce desired products. Key features include:
Stirrer system for even mixing and oxygen distribution.
Oxygen delivery system to maintain aerobic conditions.
Foam control system, temperature control, and pH control systems.
Sampling ports to withdraw small volumes for testing. The stirred-tank bioreactor is the most commonly used type.
(c) Downstream Processing
Downstream processing refers to the purification and formulation of biological products for marketing. This stage includes:
Separation of the desired product from the culture.
Purification to achieve the required standard.
Formulation with preservatives for stability and usability.
Quality control testing and clinical trials (if the product is medicinal). Downstream processing ensures that the final product meets all necessary standards and regulations before it is brought to market.
Explain briefly
(a) PCR
(b) Restriction enzymes and DNA
(c) Chitinase
(a) PCR (Polymerase Chain Reaction)
PCR is a technique used to amplify a specific segment of DNA. It involves the following steps:
Denaturation: The double-stranded DNA is heated to separate it into two single strands.
Primer Annealing: Short DNA primers bind to the complementary sequences on the single-stranded DNA.
Extension: DNA polymerase extends the primers, synthesizing new strands complementary to the original template.
This cycle is repeated multiple times to create billions of copies of the target DNA sequence.
Figure: Polymerase Chain Reaction Steps
(b) Restriction Enzymes and DNA
Restriction enzymes are proteins that cut DNA at specific sequences known as recognition sites. These enzymes are essential tools in genetic engineering for creating recombinant DNA. For example, EcoRI always cuts DNA at the sequence GAATTC.
Action: Restriction enzymes cut both DNA strands at specific points, leaving 'sticky ends' which can form hydrogen bonds with complementary sticky ends of other DNA fragments.
Use: They allow precise cutting and pasting of DNA fragments, making it possible to insert genes into plasmids.
Figure: Formation of Recombinant DNA
(c) Chitinase
Chitinase is an enzyme that breaks down chitin, a major component of the cell walls in fungi and the exoskeletons of insects.
Function in Biotechnology: In the context of biotechnology, chitinase is used to lyse fungal cells to release their DNA for further genetic analysis or manipulation.
Application: It aids in the isolation of DNA from fungal cells by breaking down the cell wall components to release genetic material.
Discuss with your teacher and find out how to distinguish between
(a) Plasmid DNA and Chromosomal DNA
(b) RNA and DNA
(c) Exonuclease and Endonuclease
(a) Plasmid DNA and Chromosomal DNA
Plasmid DNA:
Form: Usually circular and autonomously replicating.
Location: Exists independently within the cytoplasm of bacterial cells.
Function: Often carries genes that provide selective advantages, like antibiotic resistance.
Copy Number: Can have multiple copies per cell.
Use in Biotechnology: Commonly used as vectors for gene cloning due to their ability to replicate independently.
Chromosomal DNA:
Form: Linear in eukaryotes (and circular in prokaryotes), constituting the primary genetic material.
Location: Found within the nucleus (eukaryotes) or nucleoid region (prokaryotes).
Function: Contains essential genes necessary for the survival and normal function of the organism.
Copy Number: Typically exists as a single copy in prokaryotic cells, or as pairs of homologous chromosomes in eukaryotic cells.
Use in Biotechnology: Genetic material for studying gene functions and inheritance, can be manipulated but not used as vectors.
(b) RNA and DNA
RNA (Ribonucleic Acid):
Sugar: Contains ribose.
Structure: Usually single-stranded.
Bases: Has uracil (U) instead of thymine (T).
Function: Involved in protein synthesis (mRNA, tRNA, rRNA) and gene regulation.
Stability: More prone to enzymatic degradation due to the hydroxyl group on the 2' carbon of ribose.
DNA (Deoxyribonucleic Acid):
Sugar: Contains deoxyribose.
Structure: Double-stranded helix.
Bases: Contains thymine (T) instead of uracil (U).
Function: Stores genetic information that directs the development and functioning of living organisms.
Stability: More stable due to the lack of the hydroxyl group on the 2' carbon of deoxyribose, making it less prone to hydrolysis.
(c) Exonuclease and Endonuclease
Exonuclease:
Function: Removes nucleotides one at a time from the ends of a DNA molecule.
Action Site: Works on the terminal ends of DNA strands.
Use in Biotechnology: Used in DNA repair processes and for trimming ends of DNA fragments.
Endonuclease:
Function: Cuts DNA at specific positions within the molecule, not just at the ends.
Action Site: Works internally, making cuts at specific recognition sequences within the DNA strand.
Use in Biotechnology: Essential for genetic engineering to create recombinant DNA by cutting DNA at precise locations.
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Comprehensive Class 12 Notes on Biotechnology: Principles and Processes
Introduction to Biotechnology
Biotechnology: An Overview
Biotechnology deals with the techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans. Everyday examples of biotechnology include making curd, bread, or wine, all microbe-mediated processes. However, in contemporary use, biotechnology refers to processes that employ genetically modified organisms to achieve results on a larger scale.
Definition and Scope of Biotechnology
The European Federation of Biotechnology (EFB) defines biotechnology as "The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services." This definition encompasses both traditional views and modern molecular biotechnology.
Principles of Biotechnology
Core Principles of Biotechnology
Two core techniques enable the modern biotechnology landscape:
- Genetic Engineering: This involves altering the chemistry of genetic material (DNA and RNA) to introduce them into host organisms and change the organism's phenotype.
- Bioprocess Engineering: This involves maintaining sterile conditions in chemical engineering processes to ensure the growth of only desired microbes/eukaryotic cells in large quantities for manufacturing biotechnological products like antibiotics and vaccines.
Recombinant DNA Technology
Biotechnology utilises genetic engineering or recombinant DNA technology, which combines genetic material from different sources to create new genetic combinations.
Key Tools of Recombinant DNA Technology
To carry out genetic engineering, several key tools are used:
- Restriction Enzymes
- DNA Polymerases
- DNA Ligases
- Vectors
- Host Organisms
Steps in Recombinant DNA Technology
Key Steps of Genetic Modification
The process of genetically modifying an organism involves three basic steps:
- Identification of DNA with Desirable Genes
- Introduction of DNA into the Host
- Maintenance and Transfer of DNA in Host
Tools for Genetic Engineering
Restriction Enzymes
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Types and Functions Restriction enzymes are proteins used to cut DNA at specific sites. They are crucial in creating recombinant DNA by generating compatible DNA fragments that can be joined together.
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Mechanism of Action Restriction enzymes cut DNA at specific nucleotide sequences called recognition sites, creating sticky ends that facilitate the joining of DNA fragments.
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Naming Conventions and Examples The enzymes are named based on the genus and species of the bacteria from which they were isolated, e.g., EcoRI comes from Escherichia coli.
Gel Electrophoresis
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Process Description Gel electrophoresis is a technique used to separate DNA fragments by size using an electric field.
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Separation of DNA Fragments Smaller DNA fragments move faster through the gel matrix than larger ones, allowing for size-based separation.
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Visualisation Techniques The separated DNA fragments are stained with compounds like ethidium bromide and visualised under UV light.
graph LR
A[DNA Extraction]
B[Restriction Enzyme Digestion]
C[Gel Electrophoresis]
D[DNA Fragment Separation]
A --> B --> C --> D
Cloning Vectors
Vectors are DNA molecules that can carry foreign DNA into host cells where it can be replicated.
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Types of Cloning Vectors Common vectors include plasmids and bacteriophages, which can replicate independently of the chromosomal DNA in bacterial cells.
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Origin of Replication This sequence allows the vector to replicate within the host cells.
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Selectable Markers and Cloning Sites Markers help identify successful transformants, while cloning sites are specific sequences where foreign DNA can be inserted.
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Vectors for Plants and Animals Vectors like the Ti plasmid in plants and retroviruses in animals are used to introduce foreign genes into higher organisms.
Techniques and Processes
Insertion of Recombinant DNA
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Methods of Introducing DNA into Cells Various methods, such as heat shock, microinjection, and use of gene guns, are employed to introduce recombinant DNA into host cells.
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Competent Host Cells Preparation Host cells are made competent to take up DNA by treating them with calcium ions and applying heat shock.
Polymerase Chain Reaction (PCR)
PCR amplifies specific DNA sequences exponentially using primers and the enzyme DNA polymerase.
graph TD
A[DNA Denaturation]
B[Primer Annealing]
C[DNA Extension]
A --> B --> C --> A
Production and Expression of Foreign Genes
Scaling Up Production
Once recombinant DNA is introduced, the host organism can be cultured to produce the desired protein on a large scale.
- Use of Bioreactors Bioreactors facilitate the growth of large volumes of culture, optimising conditions like temperature and pH for maximum yield.
Downstream Processing
After production, the recombinant protein undergoes purification and quality control steps before being formulated into final products.
Conclusion
Biotechnology, through genetic engineering and bioprocess engineering, has revolutionised the production of medicinal and industrial products. The intricate processes of recombinant DNA technology and the tools employed ensure the efficient creation, multiplication, and utilisation of genetically modified organisms for human benefit.
By understanding these principles and processes, students can appreciate the depth and breadth of biotechnology and its applications in various fields.
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