Questions - Answers
Very Short Answer Type Questions
1. Define Biotechnology.
A: Biotechnology is the science which utilises properties and uses of microorganisms or exploit cells and cell constituents at industrial level for generating useful products essential to life and human welfare.
2. What are molecular scissors? Where are they obtained from?
A: The restriction enzymes which cut the DNA at specified sites are called molecular scissors. They are obtained from bacteria.
3. Name any two artificially restructured plasmids.
A: pBR322, pUC19, 101.
4. What is EcoRI? How does it function?
A: EcoRI is a restriction endonuclease enzyme obtained from the bacteria Escherichia coli. It cuts the DNA between G and A (Guanine and Adenine)
5. What are cloning vectors? Give an example.
A: Vectors used for multiplying the foreign DNA sequences are called cloning vectors.
e.g.: Plasmids and Bacteriophages
6. What is recombinant DNA?
A: The DNA formed by ligation of a cut gene of interest from a source and the cut vector is called rDNA or chimeric DNA.
7. What is palindromic sequence?
A: A palindromic sequence is a sequence of nitrogen bases that reads the same on the two strands in opposite direction.
e.g.: 3’GAATTC 5’
5’CTTAAG 3’
8. What is the full form of PCR? How is it useful in biotechnology?
A: PCR = Polymerase Chain Reaction
It is used to synthesise multiple copies of DNA or gene (amplification of gene). This method is employed to diagnose some diseases.
9. What is downstream processing?
A: The process of separation and purification of a gene product to make it a finished product for marketing is called downstream processing.
10. How does one visualize DNA on an agar gel?
A: We can visualize DNA fragments on agar gel by staining with ethidium bromide and then exposing to UV radiation.
11. How can you differentiate between exonucleases and endonucleases?
A: Exonucleases remove nucleotides from the ends of DNA.
Endonucleases make cuts at specific positions within the DNA.
Short Answer Type Questions
1. Write short notes on restriction enzymes.
A: * Restriction enzymes belong to the class of nucleases.
* They were first isolated from Escherichia coli.
* More than 900 restriction enzymes have been isolated so far from 230 strains of bacteria.
* The restriction enzymes are named after the genus and species from which they are isolated.
e.g.: EcoRI = ‘E’ the first letter in the genus Escherichia
‘co’ the first two letters of the species coli.
‘R’ is the name of the strain
‘I’ the roman number indicating the order of the Strain
* Restriction enzymes are of two kinds: Exonucleases and Endonucleases
* Exonucleases remove nucleotides from the ends of the DNA.
* Endonucleases cut DNA at specific positions.
* The restriction endonucleases recognize specific palindromic nucleotide sequences and cut them to form staggered ends.
e.g.: Hind II always cuts DNA at specific sequence of six base pairs.
2. What is a bio-reactor? Describe briefly the stirring type of bio-reactor.
A: A bio-reactor is a large vessel which is used for biological conversion of raw material into specific products like enzymes, hormones antibiotics etc., It provides optimal conditions of temperature, pH, substrate, salts, vitamins, oxygen etc., to achieve the desired product.
Stirring type of bio-reactor:
It is a vessel which is cylindrical and has a curved base to facilitate mixing of contents. It has an agitator system, an oxygen delivery system, a foam control system, a temperature control system, pH control system and sampling ports. A stirrer facilitates even mixing and oxygen supply.
3. What are the different methods of insertion of recombinant DNA into the host cell?
A: Recombinant DNA can be inserted into host cells through the following methods:
1) Ice Incubation and heat shock
2) Micro injection
3) Gene gun method or biolistic method
4) Disarmed pathogen vectors
Heat shock: rDNA can be injected into cells by incubating on ice and briefly subjecting it to heat shock at 42º and again putting back on ice. This method is used for bacteria.
Micro injection: The rDNA is directly injected into the nucleus of a cell. This method is used for animal cells.
Gene-gun method or biolistic method: The cells are bombarded with high velocity micro particles of gold or tungsten coated with rDNA. This method is used for plant cells.
Disarmed pathogens: The bacteriophage vectors are disarmed and allowed to infect the bacterial cells. They transfer the rDNA into the host.
Long Answer Type Questions
1. Explain briefly the various processes of recombinant DNA technology.
A: Recombinant DNA technology involves the following steps:
1) Isolation of DNA
2) Fragmentation and isolation of DNA by restriction endonucleases
3) Ligation of DNA with vector and its multiplication
4) Insertion of rDNA into the host
5) Selection of transformed host cells
6) Culturing the host cells in a medium and extraction of the desired product.
Isolation of DNA
* To obtain pure form of DNA the cells should be treated with various substances.
* The cell wall is digested with lysozymes (bacteria), cellulase (Plant), chitinase (fungi) etc.,
* The cell membranes are digested by using powered detergents (detergent lysis).
* Ribonucleases are used to remove RNA and proteases to remove proteins.
* Other molecules are removed by suitable treatments.
* Finally purified DNA is obtained by precipitating it with chilled ethanol.
* The fine threads of DNA can be separated by spooling.
Fragmentation of DNA by restriction endonucleases and its isolation:
* The purified DNA is incubated with a suitable restriction endonuclease enzyme at optimal conditions.
* The enzyme cuts the DNA at regular sites (digestion).
* Agarose gel electrophoresis is used to separate the DNA fragments.
* The separated DNA fragments are visualized by staining with ethidium bromide and exposing to UV radiation.
* Then the separate bands of DNA are cut (elution).
* The process is repeated with vector DNA using the same restriction enzyme.
Ligation of DNA with vector and its amplification:
* The cut gene of interest and the cut vector which have sticky ends are mixed with ligase enzyme.
* Ligation of gene of interest with cut vector results in formation of a recombinant DNA or chimaeric DNA.
* To obtain multiple copies of gene of interest PCR (Polymerase Chain Reaction) is employed.
* Taq polymerase from Thermus aquaticus bacterium is used in PCR.
Insertion of rDNA into the host cell or organism:
Recombinant DNA is inserted into host cells through the following methods:
* Ice Incubation and heat shock (42º) in bacterial cells.
* Micro injection in animal cells.
* Gene gun method or biolistic method using gold or tungsten in plant cells.
* Disarmed pathogen vectors are also used for this purpose.
Selection of transformed host cells:
The transformed host cells may be selected by using selectable markers, insertional activation or colony hybridization using probes.
Culturing the host cells in a medium and extraction of the desired product:
* The host cells are grown in a culture medium on a small scale in the laboratory.
* The cells are then multiplied in a continuous culture system in which the used medium is removed and fresh medium is added continuously.
* This culture produces large biomass of the host cells from which the desired protein is extracted.
* To convert the raw materials into specific products biologically bioreactors (stirring type) are used.
* After separation and purification of the product (downstream processing) the product is ready for marketing.
* Clinical trials or quality control tests are performed before releasing the product for use.
2. Give a brief account of the tools of recombinant DNA technology.
A: Tools of recombinant DNA technology:
1) Enzymes
2) Vectors
3) Competent hosts
1) Enzymes: Enzymes are the real tools of rDNA technology without which this science would not have evolved.
Three kinds of enzymes are necessary for rDNA technology. They are
i. Restriction enzymes endonucleases and exonucleases
ii. Polymerases
iii. Ligases
* The restriction endonucleases are the enzymes that cut the DNA at specific recognition sequences called palindromic sequences.
e.g.: EcoRI is a restriction endonuclease enzyme obtained from Escherichia coli bacterium. It cuts the DNA between G and A recognizing a palindrome with 6 nucleotides (GAATTC).
* The exonucleases help to remove nucleotides from the ends of DNA.
* Polymerases like DNA polymerase and RNA polymerase help to synthesize DNA and RNA strands from a template.
* Taq polymerase is a thermostable enzyme used for amplification of rDNA in PCR.
* Ligase enzymes help in joining the staggered ends of cut genes of interest and the vectors.
2) Vectors
The DNA used as a carrier for transferring a segment of foreign DNA into a host is called vector.
Vectors used for multiplying the foreign DNA sequences are called cloning vectors.
e.g.: Plasmids, bacteriophages, cosmids.
* T1 plasmid from Agrobacterium tumifaciens in plants.
* Retroviruses (disarmed) in animal cells.
* Plasmids are extra chromosomal circular DNA molecules found in bacteria.
* As they are easy to isolate and reintroduce into host cells they are considered to be most suitable as vectors.
* pBR 322 and pUC 19, 101 are the popularly used artificial plasmids.
* A cloning vector should have the following properties:
* Ori (origin of replication) with high copy number.
* Should have selectable markers e.g.: antibiotic resistance markers.
* One or few recognition sites for cloning.
* Low molecular weight.
3) Competent hosts:
* DNA cannot pass through the cell walls and membranes easily as it is hydrophilic.
* The host cell can take the plasmid (rDNA) only when it is made competent.
* The host cells are made competent by treating with divalent cation like calcium which allows the DNA to pass through the pores in the cell walls.
