Biotechnology and Tissue culture

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Oct 23, 2013 (4 years and 17 days ago)

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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 buff
er 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

it
s applications in biotechnology


Isolation of the gene or its mRNA transcr
ipt; 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, sel
ectable 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 o
f the product.

3.S
ite directed mut
agenesis


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 proper
ties 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 mo
dified 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.R
ecombinant va
ccine and Attenuated vaccine


The recombinant vaccine produced i
s 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 cul
turing the
engineered organism.

I t av oi d s t h e r i s k s of u s i n g t h e at t e n u at
e d v ac c i n e s u c h as i n f e c t i on f r om a r e v i v e d
p at h og e n
.


6.Homol og y mode l l i ng


I n h omo l og y or c omp ar at i v e mo d e l l i n g, t h e u n k n own s e qu e n c e i s c omp ar e d ag ai n s t t h e s t r u c t u r e of an al r e ad y
e l u c i d at e d p r ot e i n s t r u c t u r e. Th i s i s d on e b y f i r s t al i g n i n g t h e DNA,
RNA, or p r ot e i n s e qu e n c e ag ai n s t t h e t ar g e t
s e qu e n c e an d t h e d e g r e e of s i mi l ar i t y i s c omp ar e d. Th e n e ar e s t mo d e l i s t ak e n as t h e s t ar t i n g p oi n t an d t h e
structure is modified to get the best model.



7. P
ractical 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 suc
h 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.


Bioauguamentat
ion is the addition of special microorganisms and their requirements to clean up the environment.


9.B
iotechnology
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 ac
ids, enzymes, the use of enzymes for food processing,
enhancing flavours of natural food such as vegetables, meat etc.


10. D
esi
rable 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 forei
gn DNA etc.


11. Anther culture


Used f
or 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 produ
ctivity
and desirable characteristics in progeny, in the creation of rare characters in ornamental plants etc.


12.U
ses of

a cDNA library


Clones of the DNA fragments contained in the bacteria get replicated each time the bacteria divide so that multipl
e
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. P
otential

hazards of genetic engineering


Induction of unwanted genes resulting in unexpected outcome, implications of the antibiotic selectable markers
causing resistant microbes, evolution of undes
irable organisms, transfer of the introduced gene to other organisms
etc.







16. HAT medium


Hypoxanthine, Aminopterin and T
hymidine.

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 a
re 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.E
pisome.


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 isola
ted 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 an
d 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 se
arches, presence
of abnormal sequences, mutations etc.

24.O
rg
anogenesis


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 iden
tified by empirical methods.


25.E
mbryo 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 propaga
tion 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
met
hods 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, ra
dioactive 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 qua
ntities 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 dr
ug 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, amenabil
ity of the plant to the
standard processes, skill of the technician, precautions during cultures processes, hardiness of the plant to
downstream processes etc.


30. L
iposome
mediated gene delivery


A technique by which foreign DNA is introduced into a ce
ll 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 d
iabetes, 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. Vira
l 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, Geminiviruse
s, Tobamoviruses T4 etc.



35.A
pplications of micro
-
in
jection in reproductive biology


In I
ntracytoplasmic sperm injection, germ cell therapy, germ and stem cell cloning and therapy etc
.


36. Advantages of R
ecombinant va
ccine


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. A
ntifungal agen
ts 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.