A. Restriction Enzymes (Molecular Scissors)

In 1963, two enzymes responsible for restricting the growth of bacteriophage in E. coli were isolated. One added methyl groups to DNA (methylase), while the other cut DNA. The latter was called restriction endonuclease.

Key Points:

• First Type II Endonuclease: Hind II. It cuts DNA molecules at a particular point by recognizing a specific sequence of six base pairs (Recognition Sequence).
• Current Status: More than 900 restriction enzymes have been isolated from over 230 strains of bacteria.
• Naming Convention: First letter from Genus, second two letters from Species, fourth letter from Strain. Roman number indicates the order of isolation (e.g., EcoRI comes from Escherichia coli RY 13).

Mechanism of Action

Restriction enzymes belong to the class of enzymes called Nucleases.

• Exonucleases: Remove nucleotides from the ends of the DNA.
• Endonucleases: Make cuts at specific positions within the DNA.

Each restriction endonuclease functions by inspecting the length of a DNA sequence. Once it finds its specific recognition sequence, it will bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar-phosphate backbones.

Sticky Ends vs. Blunt Ends: Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands. This leaves single stranded portions at the ends called sticky ends. They form hydrogen bonds with their complementary cut counterparts. This stickiness facilitates the action of the enzyme DNA ligase.

B. Cloning Vectors

Vectors are DNA molecules used as a vehicle to carry foreign genetic material into another cell. Plasmids and Bacteriophages are commonly used vectors.

Features required to facilitate cloning into a vector:

1. Origin of Replication (ori): Sequence from where replication starts. Any piece of DNA linked to this sequence can be made to replicate within the host cells. It also controls the copy number of the linked DNA.
2. Selectable Marker: Helps in identifying and eliminating non-transformants and selectively permitting the growth of transformants. Usually, genes encoding resistance to antibiotics (ampicillin, chloramphenicol, tetracycline, kanamycin) are useful selectable markers for E. coli.
3. Cloning Sites (Recognition Sites): The vector needs to have very few, preferably single, recognition sites for the commonly used restriction enzymes. Presence of more than one recognition site generates several fragments which hampers gene cloning.

Alternative Selectable Markers: Selection based on antibiotic resistance is cumbersome. Alternative markers differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate (e.g., Blue-White Screening using Beta-galactosidase gene. Recombinants are white, non-recombinants are blue).

Vectors for Plants and Animals:

• Plants: Agrobacterium tumefaciens (pathogen of dicots) delivers a piece of DNA known as 'T-DNA' to transform normal plant cells into tumor. The Ti plasmid is now modified into a cloning vector which is no more pathogenic but still delivers genes.
• Animals: Retroviruses transform normal cells into cancerous cells. Disarmed retroviruses are used to deliver desirable genes into animal cells.

C. Competent Host (Transformation)

DNA is a hydrophilic molecule; it cannot pass through cell membranes. Bacterial cells must be made 'competent' to take up DNA.

Methods to introduce Alien DNA:

1. Chemical Treatment: Treating bacteria with a specific concentration of a divalent cation like Calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall.
2. Heat Shock: Incubation of cells with recombinant DNA on ice, followed by placing them briefly at 42°C (Heat Shock), and then putting them back on ice.
3. Micro-injection: Recombinant DNA is directly injected into the nucleus of an animal cell.
4. Biolistics / Gene Gun: Cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA. Suitable for plants.

A. Isolation of Genetic Material (DNA)

Nucleic acid is the genetic material. To cut DNA with restriction enzymes, it needs to be in pure form, free from other macromolecules.

• Cell Lysis: Bacterial cells/plant or animal tissue are treated with enzymes like Lysozyme (bacteria), Cellulase (plant cells), Chitinase (fungus) to break the cell wall.
• Removal of Impurities: RNA is removed by treatment with Ribonuclease. Proteins are removed by Protease.
• Precipitation: Purified DNA precipitates out after the addition of Chilled Ethanol. It can be seen as collection of fine threads in the suspension (Spooling).

B. Separation and Isolation of DNA Fragments

The cutting of DNA by restriction endonucleases results in fragments of DNA.

Gel Electrophoresis:

• DNA fragments are negatively charged molecules. They can be separated by forcing them to move towards the anode under an electric field through a medium/matrix.
• Matrix: Most commonly used is Agarose (natural polymer extracted from sea weeds).
• Principle: DNA fragments separate according to their size (sieve effect). Smaller fragments move farther.
• Visualization: DNA fragments cannot be seen in visible light. They are stained with Ethidium Bromide and exposed to UV radiation. Bright orange coloured bands are seen.
• Elution: The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece.

C. Amplification of Gene of Interest using PCR

PCR (Polymerase Chain Reaction): In this reaction, multiple copies of the gene (or DNA) of interest is synthesized in vitro using two sets of primers and the enzyme DNA polymerase.

Steps of PCR:

1. Denaturation: Heating the DNA (approx 94°C) to separate the two strands.
2. Annealing: Two sets of primers (small chemically synthesized oligonucleotides that are complementary of the regions of DNA) bind to the complementary regions on the DNA strands (approx 50-60°C).
3. Extension: The enzyme DNA polymerase extends the primers using the nucleotides provided in the reaction.

If the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times (1 billion copies are made).

D. Ligation and Transformation

• The amplified fragment is ligated with a vector using DNA ligase.
• The recombinant DNA is introduced into the host cell (Transformation).

E. Obtaining the Foreign Gene Product

The ultimate aim of recombinant DNA technology is to produce a desirable protein. The host cells expressing the gene are grown on a large scale.

Bioreactors: Large vessels (100-1000 litres) in which raw materials are biologically converted into specific products, individual enzymes, etc. using microbial plant, animal or human cells. A bioreactor provides optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen).

• Stirred-tank bioreactor: Usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen availability.
• Sparged stirred-tank bioreactor: Sterile air is sparged through the reactor.

F. Downstream Processing

After the biosynthetic stage, the product has to be subjected through a series of processes before it is ready for marketing as a finished product.

• Processes: Separation and Purification.
• Formulation: The product has to be formulated with suitable preservatives.
• Clinical Trials: In case of drugs, strict clinical trials are required.
• Quality Control: Strict quality control testing for each product is also required.

A. Both A and R are true and R is the correct explanation of A.

B. Both A and R are true but R is NOT the correct explanation of A.

C. A is true but R is false.

D. A is false but R is true.

A. Circular structure

B. Transferable

C. Single-stranded

D. Independent replication

A. Both A and R are true and R is the correct explanation of A.

B. Both A and R are true but R is NOT the correct explanation of A.

C. A is true but R is false.

D. A is false but R is true.

A. Template

B. Carrier

C. Transformer

D. Vector

A. 5'-CGTATG-3'

B. 5'-CGAATG-3'

C. 5'-GAATTC-3'

D. 5'-GATACC-3'

A. Both A and R are true and R is the correct explanation of A.

B. Both A and R are true but R is NOT the correct explanation of A.

C. A is true but R is false.

D. Both A and R are false.

A. E. coli

B. Salmonella typhimurium

C. Bacillus thuringiensis

D. Yeast

A. Disarming pathogen vectors

B. Transformation of plant cells

C. Constructing recombinant DNA by joining with vectors

D. DNA fingerprinting

A. Both A and R are true and R is the correct explanation of A.

B. Both A and R are true but R is NOT the correct explanation of A.

C. A is true but R is false.

D. A is false but R is true.

A. Cloning site

B. Origin of replication

C. Antibiotic resistance gene

D. All of the above

A. Polymerase chain reaction

B. Electrophoresis

C. Restriction mapping

D. Centrifugation

A. Denaturation (94°C)

B. Annealing (50-60°C)

C. Extension (72°C)

D. All of these

A. Both A and R are true and R is the correct explanation of A.

B. Both A and R are true but R is NOT the correct explanation of A.

C. A is true but R is false.

D. A is false but R is true.

A. Upstream processing

B. Downstream processing

C. Bioprocessing

D. Postproduction processing

A. Purification of product

B. Addition of preservatives to the product

C. Availability of oxygen throughout the process

D. Ensuring anaerobic conditions in the culture vessel

A. Both A and R are true and R is the correct explanation of A.

B. Both A and R are true but R is NOT the correct explanation of A.

C. A is true but R is false.

D. A is false but R is true.

A. Restriction enzyme

B. Vector

C. Polymerase enzyme

D. Introns

A. Spectrophotometry

B. Tissue culture

C. PCR

D. Gel electrophoresis

A. Binding of DNA to the cell wall

B. Uptake of DNA through membrane pores

C. Uptake of DNA through cell wall pores

D. Expression of antibiotic resistance gene

A. Both A and R are true and R is the correct explanation of A.

B. Both A and R are true but R is NOT the correct explanation of A.

C. A is true but R is false.

D. A is false but R is true.

Biotechnology is the use of living systems, organisms, or parts of this to develop or make products. The European Federation of Biotechnology (EFB) has given a definition that encompasses both traditional view and modern molecular biotechnology: 'The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services'.

Genetic engineering refers to the techniques to alter the chemistry of genetic material (DNA and RNA), to introduce these into host organisms and thus change the phenotype of the host organism. It allows the isolation and introduction of only one or a set of desirable genes without introducing undesirable genes into the target organism.

The construction of the first recombinant DNA emerged from the possibility of linking a gene encoding antibiotic resistance with a native plasmid of Salmonella typhimurium. This was accomplished by Stanley Cohen and Herbert Boyer in 1972. They cut the piece of DNA using restriction enzymes and ligated it into the plasmid vector.

Restriction enzymes are called 'molecular scissors'. They belong to a larger class of enzymes called nucleases. They are of two kinds: exonucleases and endonucleases. Exonucleases remove nucleotides from the ends of the DNA whereas, endonucleases make cuts at specific positions within the DNA. They are critical tools for rDNA technology.

Each restriction endonuclease inspects the length of a DNA sequence. Once it finds its specific recognition sequence, it binds to the DNA and cuts the two strands of the double helix at specific points in their sugar-phosphate backbones. Each restriction endonuclease recognizes a specific palindromic nucleotide sequence in the DNA.

The palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same. For example, the following sequences reads the same on the two strands:
5' G A A T T C 3'
3' C T T A A G 5'
This creates a mirror-like structure recognized by enzymes which then cleave correctly.

Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands. This leaves single stranded portions at the ends. There are overhanging stretches called sticky ends on each strand. These are named so because they form hydrogen bonds with their complementary cut counterparts.

The convention for naming restriction enzymes: the first letter of the name comes from the genes and the second two letters come from the species of the prokaryotic cell from which they were isolated. E.g., EcoRI comes from Escherichia coli RY 13. The letter 'R' is derived from the name of the strain. Roman numbers indicate the order in which the enzymes were isolated.

DNA fragments are negatively charged molecules. They can be separated by forcing them to move towards the anode under an electric field through a medium/matrix. Nowadays the most commonly used matrix is agarose which is a natural polymer extracted from sea weeds. The DNA fragments separate according to their size through sieving effect.

The separated DNA fragments can be visualized only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation. You cannot see pure DNA fragments in visible light and without staining. You can see bright orange coloured bands of DNA in an ethidium bromide stained gel exposed to UV light.

The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution. The DNA fragments purified in this way are used in constructing recombinant DNA by joining them with cloning vectors. This ensures only the desired fragment is used for cloning.

A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted for cloning purposes. Plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA, making them ideal vectors. Plasmids are autonomously replicating circular extra-chromosomal DNA.

This is a sequence from where replication starts and any piece of DNA linked to this sequence can be made to replicate within the host cells. This sequence is also responsible for controlling the copy number of the linked DNA. If one wants to recover many copies of the target DNA it should be cloned in a vector whose origin support high copy number.

In addition to 'ori', the vector requires a selectable marker, which helps in identifying and eliminating non-transformants and selectively permitting the growth of transformants. Transformation is a procedure through which a piece of DNA is introduced in a host bacterium. Markers are usually antibiotic resistance genes (like ampR, tetR).

Selection of recombinants due to inactivation of antibiotics is a cumbersome procedure. Therefore, alternative selectable markers have been developed which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. This is called insertional inactivation (e.g. blue-white screening).

In this method, a recombinant DNA is inserted within the coding sequence of an enzyme, beta-galactosidase. This results in inactivation of the enzyme. The presence of a chromogenic substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results in white colonies (recombinants).

Agrobacterium tumefaciens, a pathogen of several dicot plants is able to deliver a piece of DNA known as 'T-DNA' to transform normal plant cells into a tumor and direct these tumor cells to produce the chemicals required by the pathogen. The tumor inducing (Ti) plasmid of Agrobacterium tumefaciens has now been modified into a cloning vector.

Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. In order to force bacteria to take up the plasmid, the bacterial cells must first be made 'competent' to take up DNA. This is done by treating them with a specific concentration of a divalent cation, such as calcium, which increases the efficiency of entry.

Recombinant DNA can be forced into calcium-treated cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42°C (heat shock), and then putting them back on ice. This enables the bacteria to take up the recombinant DNA. It creates transient pores in the bacterial cell wall allowing DNA entry.

For plants, cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA in a method known as biolistics or gene gun. This is a direct method of gene transfer suitable for plant cells which have a rigid cell wall. Another method is micro-injection, where DNA is directly injected into the nucleus of an animal cell.

In order to cut the DNA with restriction enzymes, it needs to be in pure form, free from other macromolecules. Since the DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids.

To release DNA, the cell walls are digested by specific enzymes. Bacteria are treated with lysozyme, plant cells with cellulase, and fungus with chitinase. RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease. This step ensures that pure DNA is obtained for downstream applications.

Purified DNA ultimately precipitates out after the addition of chilled ethanol. This can be seen as collection of fine threads in the suspension. The process of removing this DNA from the suspension using a glass rod is called spooling. If the DNA is not pure, it will not precipitate cleanly.

PCR stands for Polymerase Chain Reaction. In this reaction, multiple copies of the gene (or DNA) of interest is synthesized in vitro using two sets of primers (small chemically synthesized oligonucleotides that are complementary to the regions of DNA) and the enzyme DNA polymerase. This revolutionized molecular biology.

The enzyme used in PCR is thermostable DNA polymerase (Taq polymerase) isolated from a bacterium, Thermus aquaticus. It remains active during the high temperature induced denaturation of double stranded DNA. Normal DNA polymerase would be denatured at such temperatures (above 90°C).

The three steps of PCR are:
1. Denaturation: Heating DNA to separate strands (94°C).
2. Annealing: Primers bind to complementary sequences on ssDNA (54°C).
3. Extension: Taq polymerase extends primers by adding nucleotides (72°C).
This cycle is repeated 30-40 times.

After completion of the biosynthetic stage, the product has to be subjected through a series of processes before it is ready for marketing as a finished product. The processes include separation and purification, which are collectively referred to as downstream processing. The product is then formulated with suitable preservatives.

Small volume cultures cannot yield appreciable quantities of products. To produce in large quantities, the development of bioreactors, where large volumes (100-1000 litres) of culture can be processed, was required. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions.

A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. Alternatively air can be bubbled through the reactor. This ensures that all cells in the culture get equal access to nutrients and oxygen.

In the context of the pUC18 vector, the lacZ gene codes for beta-galactosidase. If an insert is placed within this gene, the gene is disrupted (insertional inactivation). The bacteria can no longer produce beta-galactosidase. When grown on X-gal, these colonies remain white, while those without the insert turn blue.

The 'rop' gene in pBR322 codes for the proteins involved in the replication of the plasmid. It helps regulate the copy number of the plasmid. Understanding the function of each part of a vector is crucial for designing cloning experiments. Mutations in 'rop' can lead to uncontrolled replication (runaway plasmid).

The Ti (tumor inducing) plasmid of Agrobacterium tumefaciens naturally transfers a DNA segment (T-DNA) into the plant genome. Scientists have disarmed this plasmid by removing the tumor-causing genes but keeping the transfer machinery intact, making it a powerful vector for delivering desirable genes into plants.

Retroviruses in animals have the ability to transform normal cells into cancerous cells. A disarmed retrovirus, which has been modified to remove its ability to cause disease/cancer, helps to deliver desirable genes into animal cells. This is a common method used in gene therapy trials.

Micro-injection is a physical method of introducing foreign DNA into host cells. In this method, recombinant DNA is directly injected into the nucleus of an animal cell using a very fine glass micropipette. This technique requires high precision and specialized equipment (micromanipulator).

In the DNA isolation process, the addition of chilled ethanol is the final step that causes the purified DNA to precipitate out of the solution. DNA is insoluble in alcohol, whereas other cell components might remain in solution. This allows for the visible spooling of DNA threads.

Agarose is a natural polymer extracted from sea weeds. It forms a gel matrix with pores. The size of these pores depends on the concentration of agarose. This sieving property is exploited in gel electrophoresis to separate DNA fragments based on their size (length).

DNA fragments are negatively charged molecules due to the phosphate groups in their backbone. In gel electrophoresis, they are forced to move towards the positive electrode (anode) under an electric field. The rate of migration is inversely proportional to the size of the DNA fragment.

In this system, the used medium is drained out from one side while fresh medium is added from the other to maintain the cells in their physiologically most active log/exponential phase. This type of culturing method produces a larger biomass leading to higher yields of desired protein compared to batch culture.

Hind II always cuts DNA molecules at a particular point by recognizing a specific sequence of six base pairs. This was the first restriction endonuclease to be characterized found to act not only on phage DNA but also on chromosomal DNA. It produces blunt ends (cuts in the middle of recognition site).

Transformants are cells that have taken up the plasmid (vector), whether it has the insert or not. Recombinants are transformants that have the vector WITH the new DNA insert. Non-recombinants are transformants with the empty vector. Selectable markers help distinguish transformants from non-transformants, and recombinants from non-recombinants.

A sparged stirred-tank bioreactor is a variation of the stirred-tank reactor where sterile air is sparged through the reactor. Sparging increases the surface area for oxygen transfer. The bubbles dramatically increase the oxygen transfer area, ensuring aerobic microbes have sufficient oxygen for growth.

The ampicillin resistance gene (ampR) in pBR322 behaves as a selectable marker. If bacteria are grown on medium containing ampicillin, only those containing the plasmid (transformants) will survive. This is a positive selection method. It is often used in conjunction with tetR for insertional inactivation.

Primers used in PCR are small chemically synthesized oligonucleotides that are complementary of the regions of DNA. Two sets of primers are needed, one for each strand of the DNA double helix. They provide the free 3'-OH group required by DNA polymerase to initiate synthesis of the new DNA strand.

The annealing step in PCR involves cooling the reaction mixture to 50-60°C. This temperature allows the primers to hydrogen bond with their complementary sequences on the single-stranded template DNA. If the temperature is too high, primers won't bind. If too low, they may bind non-specifically.

Quality control testing is a critical part of downstream processing. Strict quality control testing for each product is required before it is released for marketing. The downstream processing and quality control testing vary from product to product. For pharmaceuticals, this is regulated by agencies like FDA.

During DNA isolation, enzymes like protease are used to digest proteins. Proteins are the major contaminants associated with DNA in the chromosomes (histones). Their removal is essential because they can interfere with the action of restriction enzymes and other downstream applications.

DNA ligase is the enzyme that joins DNA fragments together. It acts by forming phosphodiester bonds between the 3'-hydroxyl end of one nucleotide and the 5'-phosphate end of another. In rDNA technology, it is used to seal the nicks between the vector and the insert, creating a continuous circle of recombinant DNA.

Cohen and Boyer are credited with the birth of recombinant DNA technology. In 1972, they isolated an antibiotic resistance gene from a plasmid of Salmonella typhimurium and linked it with a plasmid vector. This demonstrated the feasibility of moving genes between organisms, laying the foundation for modern biotechnology.

Calcium ions (Ca2+) are commonly used divalent cations to make bacterial cells competent. They interact with the negatively charged DNA and the negatively charged lipopolysaccharides in the cell wall, neutralizing the charge repulsion and facilitating DNA binding to the cell surface, crucial for heat-shock transformation.

Bioprocess engineering is the maintenance of sterile (microbial contamination-free) ambience in chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc. Sterility is paramount.