Biotechnology and Tissue culture
1.Northern blotting.
Northern blotting is based on the hybridisation of specific DNA probes to complementary strands of RNA and identifying
the RNA transcript by identifying the bound probe. For this, first the RNA is isolated from the sample, resolved in an
agarose or polyacrylamide gel by electrophoresis. The resolved RNA is blotted onto a suitable membrane by
denaturation of the RNA followed by its transfer to the membrane pressed over it, enabled by capillary action of suitable
buffers made to move through the gel. The RNA gets adsorbed on the membrane while the buffer keeps moving into
the absorbent material stacked over it. The RNA which is now attached but exposed on the membrane is hybridised
with complementary DNA probes. Such probes will have markers such as fluorescent dyes, radioactive isotopes etc by
which they (and indirectly the RNA) can be identified. The identification of RNA transcripts gives an idea of the activity
of the cell at the molecular level.
2. rDNA technology and its applications in biotechnology
Isolation of the gene or its mRNA transcript; Amplification of the gene or cDNA by biological (using plasmids) or invitro
methods (by PCR); Purification of the gene of interest; Insertion of the isolated gene/cDNA into a suitable cell and its
transformation (role of restrict enzymes, ligases, selectable markers etc to be mentioned). Identification and
confirmation of the recombinant DNA; Culture or multiplication of transformed cells carrying the recombinant DNA
(rDNA) ; Induction of the modified organism to express the gene product; Harvesting of the product.
3.Site directed mutagenesis
A technique to make a protein that differs slightly from the protein normally produced by an organism or cell. A
primer with a single mutation is used for a PCR and the amplified product is used to express the mutated protein.
This results in the production of a different amino acid in the protein, using which differences between the normal
protein can be studied. The technique can also be used to create modified (engineered) proteins that have
desirable properties not currently available in the proteins produced by existing organisms. Also to compare two
same-species organisms possessing two different genes at the same site on the genome.
4.Bt toxin
The toxin produced by the bacteria Bacillus thuringiensis,is a crystalline protein (Cry protein) which breaks down to
delta endotoxin in the gut of lepidopteran larvae that feeds and destroys the plant. The toxin binds to the intestinal
lining of the larvae and generates holes which kill them. In genetically modified plants, the Bt gene is engineered
into it, so that the gene product enters the larvae that feed on the plant, killing it and thus work as a pesticide
resistant plant.
5.Recombinant vaccine and Attenuated vaccine
The recombinant vaccine produced is selected for its efficacy in evoking a robust immune response. It can be
selected so that it will not have any component that is toxic to humans. It can be produced easily by culturing the
engineered organism. It avoids the risks of using the attenuated vaccine such as infection from a revived
pathogen.
6.Homology modelling
In homology or comparative modelling, the unknown sequence is compared against the structure of an already
elucidated protein structure. This is done by first aligning the DNA, RNA, or protein sequence against the target
sequence and the degree of similarity is compared. The nearest model is taken as the starting point and the
structure is modified to get the best model.
7. Practical applications of DNA fingerprinting
Resolution of paternity and maternity disputes, identification of dead bodies, victims of crime, crime perpetuators
(in forensic science), identification adulteration of meat etc, identification of organisms or wild life by analysing
available material such as hair, blood samples etc.
8.Biostimulation and Bioaugumentation
Biostimulation is the release of nutrients, oxidants or electron donors into the environment to stimulate naturally
occurring microorganisms to clean up a contaminant.
Bioauguamentation is the addition of special microorganisms and their requirements to clean up the environment.
9.Biotechnology and food industry
In fermentation technology for the production of dairy and bakery industries, in the production of food
supplements such as vitamins, in brewing, in the fermentation of tea, preparation of pickles, production of single
cell proteins such as yeasts, spirulina, production of amino acids, enzymes, the use of enzymes for food processing,
enhancing flavours of natural food such as vegetables, meat etc.
10. Desirable traits of cloning vectors
Small size, ease of transfer from one host to the next, ability to isolate it by selective processes, easiness in
identification, induction of multiple copies in one cell, easy to detect presence in cell by the use of suitable
markers, easy to insert foreign DNA etc.
11. Anther culture
Used for the production of haploid or for the production of offspring which are multiples of the haploid. These are
useful in studying recessive mutation, development of homozygous recessive plants, enhancement of productivity
and desirable characteristics in progeny, in the creation of rare characters in ornamental plants etc.
12.Uses of a cDNA library
Clones of the DNA fragments contained in the bacteria get replicated each time the bacteria divide so that multiple
copies of the strands are produced. These can be transferred between laboratories for research and analysis.
13. DNA chip or DNA microarray
A glass slide that carries thousands of unique single stranded sequences of DNA to study gene expression or for
genome analysis by probing with complementary unique, labelled, single stranded oligonucleotides.
14.Ribozymes
RNA that works like an enzyme, usually helping in the synthesis of RNA strands.
15. Potential hazards of genetic engineering
Induction of unwanted genes resulting in unexpected outcome, implications of the antibiotic selectable markers
causing resistant microbes, evolution of undesirable organisms, transfer of the introduced gene to other organisms
etc.
16. HAT medium
Hypoxanthine, Aminopterin and Thymidine. They are blockers of alternative modes of nucleotide biosynthesis.
They are used to make the medium selective for hybridised (hybridoma) cells in Monoclonal antibody production.
17.Restriction enzymes
Restriction endonucleases: aka molecular scissors are enzymes that digest DNA at specific sites other than the 5’ or
3’ ends.
18.Biopharming
Inducing higher organisms to produce molecules of pharmaceutical utility in tissues from which it can be
harvested.
19.Electroporation
Technique that compels cells to take up DNA by applying swift, high voltage discharges.
20.Insitu Bioremediation
Treating environmental pollution by using microbial population that naturally occurs at the site of pollution and
treatment.
21.Hardening
The process of preparing a tissue culture plant for planting in the natural environment.
22.Episome.
A self replicating strand of DNA capable of integrating into the chromosome of bacteria.
23. Southern blotting
Devised by E. M. Southern, Southern blotting is based on the hybridisation of specific oligonucleotide DNA probes to
complementary strands of single stranded DNA and identifying the DNA by identifying the hybridised probe. For this,
first the DNA is isolated from the sample and resolved in an agarose or polyacrylamide gel by electrophoresis. The
resolved DNA is blotted onto a suitable membrane by denaturation of the DNA followed by its transfer to the membrane
pressed over it. This is enabled by capillary action of suitable buffers made to move through the gel and on to
hygroscopic material kept over it. The DNA strands get adsorbed on the membrane while the buffer keeps moving into
the absorbent material stacked over it. The DNA which is now attached and also exposed on the membrane, is
hybridised with complementary DNA probes carrying markers such as fluorescent dyes, radioactive isotopes etc. These
markers are identified by appropriate methods. The identification of DNA strands helps in similarity searches, presence
of abnormal sequences, mutations etc.
24.Organogenesis
Organogenesis is the induction of different organs from an undifferentiated callus by the appropriate use of
specific stimulants such as auxins, cytokinins etc. It can be controlled chemically by controlling the amounts and
types of stimulants used. Roots, shoots etc can be induced by adjusting the levels of plant hormones like IAA, IBA,
NAA etc. Different plants have different requirements which are identified by empirical methods.
25.Embryo culture.
It is used for the recovery of plants from distinct crosses, especially where it fails to develop due to degeneration of
the embryos. It is extensively used in plants such as orchids where natural propagation is slow and for the
production of different varieties to increase aesthetic values. Natural or artificially inseminated embryos are
harvested and cultured in suitable medium to produce callus. The callus is further processed through conventional
methods to produce plants
26. Northern blotting
It is the process by which RNA transcripts are resolved in agarose or polyacrylamide gels and are transferred to
suitable membranes for further analyses. Blotting RNA onto membranes leaves the sequence of nucleotide
exposed, to which complementary DNA can be hybridised. Such probes will have suitable markers such as
fluorescent dyes, radioactive isotopes etc by which they and indirectly the RNA can be identified. The
identification of RNA transcripts gives an idea of the activity of the cell at the molecular level.
27. Composting
Composting is a method of bioremediation by treating biodegradable solid waste with naturally occurring
organisms or by augmentation with cultured organisms to degrade matter into harmless, environment friendly
material. They can be done on site or at sites prepared for such operations. Larger quantities require human
intervention for efficient and speedy bioremediation. It is inexpensive, needs little care and least amount of
infrastructural inputs.
28.Monocolonal antibodies
They are used as diagnostic reagents in ELISA, as markers and probes in diagnostic procedures, as probes to
identify specific proteins and epitopes in research, in microarrays, as labelled markers for identification of specific
proteins, as vehicles for drug delivery etc.
29. Factors influencing Micropropagation
Selection of explants, presence or absence of contaminants, sterilisation protocols, selection of media,
identification of appropriate hormones and inducers, nature of the callus formed, amenability of the plant to the
standard processes, skill of the technician, precautions during cultures processes, hardiness of the plant to
downstream processes etc.
30. Liposome mediated gene delivery
A technique by which foreign DNA is introduced into a cell by enveloping them in lipid vesicles, which facilitates
entry by fusion of the vesicle to the outer plasma membrane as well as that of the nucleus.
31.Bioremediation
Bioremediation is the process of using live organisms [GMOs or natural], to remove contaminants, pollutants or
unwanted substances from soil or water.
32.Use of Gene therapy in medicine
Correction of inborn errors in genes, correction of mutations, introduction of genes or engineered pancreatic cells
to treat diseases such as diabetes, repair of damaged tissues such as cardiac cells, neurons, correction of genetic
diseases such as SCID etc.
33. Totipotent Callus
The term totipotent means that it posses the potency or capacity to develop in to any other type of tissue. Each
cell of the callus can give rise to daughter cells, each of which can be induced to differentiate into any other tissue
or numerous individual plants.
34. Viral vectors
They are agents which are capable of integrating into the host genome, used to transfer and insert genetic material
into the genome of specific hosts. Any viral vector such as adeno virus, adeno-associated virus, TMV,
Caulimoviruses, Geminiviruses, Tobamoviruses T4 etc.
35.Applications of micro-injection in reproductive biology
In Intracytoplasmic sperm injection, germ cell therapy, germ and stem cell cloning and therapy etc .
36. Advantages of Recombinant vaccine
It is an antigen produced by genetically engineering an organism so that the antigen produced can be used without
the side effects of a naturally occurring antigen or pathogen. In the event of an infection, the antibodies produced
give protection.
37. cDNA library
It is a genetically engineered micro organism that carries several cDNA strands produced by the reverse
transcription of mRNA.
38.Heterokaryon
Protoplast fusion, sometimes the nuclei do not fuse, but remain as two different nuclei with in the same cell.
These are called heterokaryons
39. Antifungal agents used in plant tissue culture
Mercuric chloride, merthiolate, AgNO3,
40.Clone
Organisms (or cells) produced from an individual cell through asexual processes, so that they have identical genetic
compositions.