NEET COACHIG PROGRAMME, 2016-17
II PUC- BIOLOGY
UNIT-BIOTECHNOLOGY
Biotechnology may be defined the use of microorganism, plant or animal cells or their products for the welfare of mankind.”
According to EFB (European Federation of Biotechnology):Biotechnology is the
integration of natural science and organisms, cells, parts thereof and molecular analogues forproducts and services.
Recombinant DNA technology or genetic engineering: The techniques to alter the chemistry of genetic material (DNA and RNA) and introduction of it into host organisms to change its phenotype.
Tools of recombinant DNA technology:
Restriction enzymes polymerase enzymes, ligases vectors and host organisms.
Restriction enzymes: belong to a larger class of enzymes called nucleases and cut DNA at specific sites called “Restriction site”.
These are of two types :exonucleases and endonucleases.
Palindromic Sequence: Complementary DNA sequences that are the same when each strandis read in the same direction (5'— 3'). These sequences act as recognition sites for restriction endonucleases.
5'—GAATTC - 3'
3'— CTTAAG - 5'
Sticky ends: Single stranded portions of DNA which can form hydrogen bonds with their complementary cut DNA segments.
Taq polymerase is a thermostableDNA polymerase isolated from bacterium Thermusaquaticusand is frequently used in polymerase chain reaction (PCR), a method for greatly amplifying short segments of DNA.
Ligases :An enzyme used by a genetic engineer to join the cut ends of the double stranded DNA.
Plasmid : Extra chromosomal, self replicating circular DNA molecule found in bacteria. Plasmids are used as cloning vector in recombinant DNA technology.
Features of cloning vector :
(a) Origin of Replication (Ori) :This is a sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells.
(b) Selectable Marker: It is a gene which helps in identifying and eliminating nontransformants from transformants (having recombinant DNA) by selectively permitting the growth of transformants. The process through which a piece of DNA is introduced in a host bacterium is called transformation. Eg: normally genes encoding resistance to anitibiotics such as ampicillin, chloramphenicol, tetracycline etc. are useful selectable markers for E.coli.
(c) Cloning Sites: A location on a cloning vector into where a foreign gene can be introduced iscalled a cloning site. The vector must have very few (preferably single) recognition sites.Eg: E.coli cloning vector pBR 322.
Vectors for cloning genes in plants and animalsTi Plasmid:An extra chromosomal, double stranded and self replicating DNA molecule found in Agrobacterium tumefaciensthat causes tumor in plants modified into a cloning vector.
Retrovirus is used as vector to transfer desired gene in animals.
Host organisms: It is the cell /organisms that accept rDNA and produce essential substance in large quantity. Eg: plant cell, animal cell and most commonly used host cell in genetic engineering is E.Coli and Yeast.
Bioreactor : A large vessel in which raw materials are biologically converted into specific products under optimal conditions such as temperature, pH, substrate, salts, vitamins, oxygen. Most commonly used is Stirred tank bioreactor.
Process of Recombinant DNA Technology:
(i)Isolation of Genetic Material (DNA):DNA can be obtained from the cell by treating with enzymes like, Lysozyme(bacteria)Cellulase(plant cell)Chitinase(fungus). Histoneprotein and RNA can be removed by treating with proteases and ribonuclease. Purified DNAultimately precipitated by the addition of chilled ethanol. Fine threads of DNA are obtained inthe suspension.
(ii) Cutting of DNA at specific location: The purified DNA is cut by use of restriction
enzymes. Agarose gel electrophoresis is used to check the progression of restriction enzymesdigestion.
(iii) Amplificationof gene of interest using PCR:Amplification is the process of makingmultiple copies of desired DNA segment in vitro.
(iv) Ligation:The cut out gene of interest from the source of DNA and cut vector withappropriate space, are mixed and ligase enzyme is added. This results in recombinant DNA(r-DNA).
(v) Transfer of recombinant DNA into the host: The ligated DNA is introduced into therecipient cell. The recipient cell makes itself competent to receive and take up DNA present inthe surrounding.
(vi) Obtaining the foreign gene product: The cell containing the foreign gene is cultured onsuitable medium and the product can be extracted from the medium. Bioreactors are used forprocessing large volume of culture for obtaining products of interest in sufficient quantities.
(vii) Downstream Processing: The products so obtained undergo a series of processesbefore putting them in market as a finished product. The processes include separation andpurification. The products are formulated with suitable preservation and subjected to qualitycontrol testing and clinical trials.
Gel Electrophoresis:DNA fragments are negatively charged molecules. They can beseparated by forcing them to move towards anode under anelectric field through a medium. Agarose gel is used as medium. Ethidium bromide is used as stain for DNA, which on exposure to UV-light appear as orange coloured bands. Separated bands of DNA are cut out from agarose gel called elution. These DNA fragments are used in recombinant DNA by joining them with cloning vectors.
Green Revolution:Substantial increase in crop yields due to use of high yielding varieties, use of fertilizers and pesticides, improved agricultural practices etc.
Genetically Modified Organisms (GMO):plants,bacteria, fungi and animals whose genes have been altered by manipulation. These are called transgenic organisms.
GM Plants: Tolerant to Abiotic stress, reduced dependence on chemical pesticides,
less post harvest-loss, efficient use of minerals, enhanced nutritional value.
RNA Interference (RNAi): Process used to develop pest resistant plants. It involves silencing of a specific mRNA due to complementary double stranded RNA.
Gene Therapy:It is a technique of inserting genes into the cells and tissue of an individual totreat a hereditary disease.
The first clinical gene therapy was given in 1990 to a four year old girl with adenosinedeaminase (ADA) deficiency. ADA enzyme is required for proper functioning at immunesystem. This disorder is caused due to the deletion of the gene for adenosine deaminaseenzyme.
Bt. Cotton:The soil bacterium Bacillus thuringiensisproduced crystal protein calledcryprotein that kills certain insect‘s larvae such as tobacco budworm,armyworm, beetles andflies.The proteins encoded by the genes: crylAcand cryllAbcontrol the cotton bollworms andcrylAbcontrol corn borer.
Biopesticides :Biological agents that are used to control weeds, insects and other pests.
Cry Gene: The Bt toxins are coded by a gene named Cry.
Cry Protein: The insecticidal protein which is produced by Bacillus thuringiensis.
Transgenic Animals: used to study normal physiology and development,to study diseases, to get biological products, to test vaccine and chemical safety testing.
1.Biotechnology is present being used to do which of the following?
- Produce vaccines
- Produce human gene products
- Correct defects in human germ cells
- Only (a) and (b) are correct
2.Restriction endonucleases
- Are synthesized by bacteria as a part of their defence mechanism
- Are present in mammals cell for degeneration of DNA when the cell dies
- Are used in Genetic engineering for ligating two DNA molecules
- Are used for invitro DNA synthesis
3.Which of the following is a palindrome
1. CCGTA/GGCAT 2. GAATTC/CTTAAG
3. CCTTG/GTAAC 4. AAAA/TTTT
4. The polymerase chain reaction is a technique that
- Is used for in vivo replication of DNA
- Is used for in vivo synthesis of mRNA
- Is sued for in vitro synthesis of mRNA
- Is used for in vitro replication of specific DNA sequence using thermostable DNA polymerase.
5.A genetically engineered bacteria used for clearing oil spills is
- Escherischia coli
- Bacillus subtilis
- Agrobacterium tumifaciens
- Pseudomonas putida
6.The process of reverse transcriptase was brought to light by the work of
- George Beadle and Edward Tatum 3) H.W. Temin and D. Baltimore
- Garrod 4) R.W.Holley
7.The linking of antibiotic resistance gene with plasmid vector became possible with
- DNA polymerase 3.Exonuclease
- DNA 4.Endonuclease
8.Plasmids present in bacterial cells are
a)Circular double helical RNA molecules
b) Circular double helical DNA molecules
c) Linear double helical RNA molecules
d) Linear double helical DNA molecules
9. Gel electrophoresis is used for
- Construction of recombinant DNA by joining with cloning vector
- Isolation of DNA molecules
- Cutting of DNA into fragments
- Separation of DNA fragments according to their size
10.Polyethylene glycol method is used for
- Biodiesel production 3. Energy production from sewage
- Seedless fruit production 4. gene transfer without a vector
- second generation vaccines are prepared by recombinant DNA technology , which of the following is an example for such Vaccines?
a) Hepatitis B Vaccines b) Herpes virus vaccine
c)Salk’s Polio Vaccines d) Both (a) and (c)
- Which one of the following is used as vector for cloning genes into higher organisms?
a.Baculovirus b.Salmonella typhimurium
c.Rhizopus nigricans d.Retrovirus
13.EnzymeTaq polymerase is obtained from
a.Thermus aquaticus b.Trichoderma aquatic
c.Tremetes aquaticus d.All of the above
14.Stirred tank bioreactors have been designed for
- Availability of oxygen throughout the process
- Addition of preservative to the product
- Purification of the product
- Ensuring anaerobic condition in the culture level