12. BIOTECHNOLOGY

-Created By-

Deshmukh A. B.

Asst. Teacher,

Agasti Arts, Commerce and Dadasaheb Rupwate

Science Junior College, Akole

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A. B. Deshmukh

XII Science BIOLOGY NOTES

12. BIOTECHNOLOGY

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• Biotechnology is the product of interaction between the biological science and technology.

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• It is infact, an applied branch of biology.

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• The term biotechnology was first used by Karl Ereky in 1919 to describe a process for large scale production of pigs.

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A. B. Deshmukh

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12.1 Biotechnology

• It is defined by different organizations in different ways.
• It has been broadly defined as ‘the development and utilization of biological forms, products or processes for obtaining maximum benefits to man and other forms of life’.
• According to OECD (Organization for Economic Cooperation and Development, 1981)- ‘It is the application of scientific and engineering principles to the processing of materials by biological agents to provide goods and services to the human welfare’.

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A. B. Deshmukh

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• It uses scientific principles of
• microbiology,
• genetics,
• biochemistry,
• chemical engineering,
• mathematics,
• statistics,
• computers,
• industrial processes, etc.
• Biological agent means plants and animal cells, microorganisms, enzymes or their products.

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• History of origin of biotechnology is as old as human civilization.
• Development of biotechnology in terms of its growth, occurred in two phases viz,
• Traditional biotechnology
• Modern biotechnology.

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A. B. Deshmukh

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Traditional biotechnology (old biotechnology)

• It was primarily based on fermentation technology using microorganisms as in the preparation of curd, ghee, soma, vinegar, yogurt, cheese making, wine making, etc.
• It became an art of kitchen in Indian houses.
• It was more an art than science.

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• Till that time people did not know as to how exactly the process occurs and the organisms causing this process.

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• The contributions made by several chemists, biochemists and microbiologist, over the time, could explain the mechanism of process and also the nature of microorganisms causing the process.

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A. B. Deshmukh

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Modern biotechnology:

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• During 1970 a new technique of ‘recombinant DNA technology was developed and then established by Stanley Cohen and Herbert Boyer in 1973.
• This technique has changed the overall outlook, then.
• The technique permits to change/ modify genetic (heritable) material for getting new specific products.
• The combination of biology and production technology based on genetic engineering evolved into modern biotechnology (new biotechnology).

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A. B. Deshmukh

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• There are two major features of technology that differentiate modern biotechnology from classical or old biotechnology viz,
• i. Capability of science to change the genetic material for getting new specific products through
• rDNA technology,
• polymerase chain reaction (PCR),
• microarrays,
• cell culture and fusion, and
• bio-processing.
• ii. Ownership of technology and its sociopolitical impact.
• Now the conventional industries, pharmaceutical industries, agro industries, food industries, etc. are also focusing attention to produce biotechnology- based products.

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A. B. Deshmukh

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12.2 Principles and Processes of Biotechnology :

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• Modern biotechnology is based on two core techniques viz.
• Genetic engineering and
• Chemical engineering.

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• Genetic engineering deals with alteration of genetic material (DNA and RNA)

• Chemical engineering deals with maintaining sterile environment for manufacturing variety of useful products including vaccines, antibodies, enzymes, organic acids, vitamins, therapeutics, etc.

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A. B. Deshmukh

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• Genetic engineering is defined as the manipulation of genetic material towards a desired end and in a directed and predetermined way, using in vitro process.
• Manipulation of genes involve
• repairing of the defective genes or replacing of defective genes by healthy genes or normal genes;
• artificially synthesizing of a totally new gene;
• transfer of genes into a new location or into a new organism;
• introducing an all together new gene;
• manipulation of genes for improvement of living organisms;
• combining of genes from two organisms, altering the genotype;
• gene cloning etc.
• Therefore, the genetic engineering is alternatively called recombinant DNA technology or gene cloning.

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A. B. Deshmukh

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Extra information :

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• John E. Smith (1996) gave definition of genetic engineering as
• ‘the formation of new combination of heritable material by the insertion of nucleic acid molecule produced by whatever means outside the cells, into any virus, bacterial plasmid or other vector system so as to allow their incorporation into a host organism in which they do not occur naturally but in which they are capable of continued propagation’.

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Technique of gene cloning and rDNA technology :

• In gene cloning, a gene of known function can be transferred from its normal location into a cell (that of course does not contain it) via a suitable vector.
• The transferred gene is replicated normally and is handed over to the next progeny.

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A. B. Deshmukh

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A. Tools and techniques for gene cloning/ rDNA technology:

• Basic requirements for the technique –
• I. Different instruments (devices)
• II. Biological tools

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A. B. Deshmukh

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I. Different instruments (devices):

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• Macromolecule such as DNA, RNA, proteins, etc. are synthesized in the living cells.
• They vary in their
• molecular weight,
• solubility,
• presence of charges,
• absorbance of light, etc.
• Several techniques are used to isolate and characterize the macromolecules.
• The size of different types of molecules varies.
• Therefore, their molecular weights also vary.

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A. B. Deshmukh

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• The techniques used based on molecular weight, are
• gel permeation chromatography,
• osmotic pressure,
• ion exchange chromatography,
• spectroscopy,
• mass spectrometry,
• electrophoresis, etc.

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Gel Permeation:

A. B. Deshmukh

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Osmotic Pressure:

A. B. Deshmukh

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Ion Exchange Chromatography:

A. B. Deshmukh

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Spectroscopy:

Spectroscopy is the study of the interaction between matter and electromagnetic radiation as a function of the wavelength or frequency of the radiation.

A. B. Deshmukh

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Mass Spectrometry:

Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. 

A. B. Deshmukh

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Electrophoresis:

A. B. Deshmukh

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• Electrophoresis is the separation of charged molecules, applying an electric field.
• It is applied for the separation of DNA, RNA and proteins.
• DNA being negatively charged, migrates to anode.
• Small fragments of DNA molecules, move faster and thus separate faster.
• Different methods of electrophoresis are –
• Agarose gel electrophoresis,
• PAGE (Polyacrylamide gel electrophoresis),
• SDA PAGE (sodium dodecyl sulphate– polyacrylamide gel electrophoresis)

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A. B. Deshmukh

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Polymerase chain reaction (PCR) :

• Polymerase chain reaction (PCR) is another device used for gene cloning or gene multiplication in vitro.
• It is the amplification of gene of interest, through PCR.
• In 1985, Kary Mullis made an important discovery (contribution) in the form of an extremely powerful technique called polymerase chain reaction (PCR).
• PCR can generate a billion copies of the desired segment of DNA or RNA, with high accuracy and specificity, in a matter of few hours.
• The process of PCR is completely automated and involves automatic thermal cycles for denaturation and renaturation of double stranded DNA.

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A. B. Deshmukh

XII Science BIOLOGY NOTES

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March 23

Q. 29 (a) What is polymerase chain reaction (PCR)?

(b) Describe three steps involved in

mechanism of PCR.

March 24

Q. 19 Explain different steps involved in PCR

technique.

A. B. Deshmukh

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12. BIOTECHNOLOGY

• The device required for PCR is called thermal cycler.
• PCR is in vitro amplification of a desired DNA segment, which requires :
• DNA containing the desired segment to be amplified,
• Several molecules of four deoxyribonucleoside triphosphates (dNTPs),
• excess of two primer molecules,
• heat stable DNA polymerase and
• appropriate quantities of Mg⁺⁺ ions.

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A. B. Deshmukh

XII Science BIOLOGY NOTES

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Mechanism of PCR:

• At the start of PCR, following components are mixed in the ‘eppendorf tube’.
• the DNA segment,
• excess of two primer molecules,
• four deoxyribonucleoside triphosphates
• the thermostable DNA polymerase
• The following operations are performed sequentially.
• Step i: Denaturation
• Step ii: Annealling
• Step iii: Renaturation

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A. B. Deshmukh

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Step i : Denaturation -

• The reaction mixture is heated to a temperature (90–98 ^oC)
• to separate two strands of desired DNA.
• This is called denaturation.

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Step ii : Annealing -

• The mixture is allowed to cool at 40–60 ^oC.
• it permits pairing of the primer to the complementary sequences in DNA.
• This step is called annealing.

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March 22

Q. 1(x) Annealing step of PCR, operates at

.............. ⁰C

(a) 90 – 98 (b) 40 – 60

(c) 70 – 75 (d) 100 -120

A. B. Deshmukh

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Step iii : Renaturation -

• The temperature is now increased up to 70–75 ^oC.
• It allows thermostable Taq DNA polymerase to use single-stranded DNA as template and adds nucleotides.
• This is called primer extension.
• It takes around two minutes duration.

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Taq Polymerase

A. B. Deshmukh

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• One cycle takes around 3 to 4 minutes.
• To begin second cycle, DNA is again heated to convert double stranded DNA into single strands.
• In an automatic thermal cycler, the above three steps are automatically repeated 20-30 times.
• Thus, at the end of ‘n’ cycles 2ⁿ copies of DNA segments, are produced.
• The machine performs the entire operations automatically and precisely.

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July 23

Q. 1 (iii) If only one DNA molecule is subjected to PCR and the time required for each cycle is three minutes, then after five cycles, how many DNA molecules are obtained?

(a) 10 (b) 15 (c) 32 (d) 64

A. B. Deshmukh

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• Once the desired number of cycles is completed, the amplified DNA segment is purified by gel electrophoresis.
• After its sequencing, the amplified DNA segment can be inserted into a cloning vector.
• Desired gene can also be obtained from gene library.

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A. B. Deshmukh

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II. Biological tools :

• There are three types of biological tools used viz,
• Enzymes,
• Cloning vectors (vehicle DNA) and
• Competent host (cloning organisms) for transformation with recombinant DNA.

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A. Enzymes :

• Different enzymes include
• Lysozymes,
• Nucleases such as
• exonucleases
• endonucleases,
• restriction endonucleases,
• DNA ligases,
• DNA polymerases,
• alkaline phosphatases,
• reverse transcriptases, etc.

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i. Restriction enzymes :

• Enzymes that cut the phosphodiester bonds of polynucleotide chains are called nuclease.
• There of two types –
• Exonuclease - cut nucleotides from the ends of DNA strands
• Endonuclease - cut DNA from within
• During the 1970s, it was found that bacteria contain nucleases that would recognize short nucleotide sequence with duplex DNA and cut.

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• The phosphodiester back bone at highly specific sites on both strands of duplex, is cut by these enzymes, called restriction endonucleases or simply restriction enzymes.

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• They were given this name because they are used by the bacteria to destroy various viral DNAs that might enter the cell, thereby restricting the potential growth of the virus.
• Thus, restriction enzymes serve as defense mechanism.
• The bacteria protect its own DNA from nucleolytic attack by methylating the bases at susceptible sites,
• a chemical modification that blocks the action of the enzyme.

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• The restriction enzymes are thus the molecular scissors that are used to recognize and cut DNA at specific sequences.
• The sites recognized by them, are called recognition sequences or recognition sites.
• Different restriction enzymes found in different organisms recognize different nucleotide sequences and therefore cut DNA at different sites.
• Table encloses list of some restriction endonucleases and the site at which they cleave DNA.

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ii. Recognition sequences :

• The sequences recognized by restriction enzymes are 4 to 8 nucleotides long.
• It is characterized by a particular type of internal symmetry.

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• Consider the particular sequence recognized by the enzyme EcoRI .

3’------- C T T A A G-----5’

5’------- G A A T T C-----3’

• When one reads the sequence in opposite direction (3’ to 5’ or 5’ to 3’) it is identical/ same.
• A sequence with this type of symmetry is called a palindrome.

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March 24 Q. 1 (vi)

A. B. Deshmukh

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• When the enzyme EcoRI attacks this palindrome,
• it breaks each strand at the same site in the sequence, which is indicated by the arrow between the A and G residues.

3’ ---- C T T A A G -------5’

5’ ---- G A A T T C -------3’

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July 23 Q. 30 (b) Write the appropriate palindrome for

Eco RI and indicate by an arrow its

recognition sequence.

A. B. Deshmukh

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• Restriction enzymes either
• cut straight across the DNA in the region of palindrome to give blunt ends or
• cuts producing short, single stranded projections at each end of DNA to produce, cohesive or sticky ends or staggered ends.

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A. B. Deshmukh

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B. Cloning vectors (vehicle DNA) -

• Vectors are DNA molecules that carry a foreign DNA segment and replicate inside the host cell.
• Vectors may be
• plasmids,
• bacteriophages (M13, lambda virus),
• cosmid,
• phagemids,
• BAC (bacterial artificial chromosome),
• YAC (yeast artificial chromosome),
• transposons,
• baculoviruses and
• mammalian artificial chromosomes (MACs),.

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July 23 Q. 30 (a) Give full form of the cloning vectors BAC and YAC.

A. B. Deshmukh

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• Most used vectors are
• plasmid vectors
• pBR 322,
• pUC,
• Ti plasmid
• Bacteriophages
• lambda phase,
• M13 phage.

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A. B. Deshmukh

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Do you know ?

• There are three types of restriction enzyme viz,
• Type I - Which function simultaneously as endonuclease and methylase
• e.g., Eco K.
• Type II - Which have separate activities for cleaving and methylation;
• they are more stable and are used in rDNA technology
• e.g., Eco RI, BgII;
• these enzymes cut DNA at specific sites within the palindrome.
• Type III - Which cut DNA at specific non palindromic sequences

e.g., HpaI, MboII.

• There are thousands of type II R. E. enzymes are recognized/ discovered.

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• A good vector should have the ability of independent replication so that as the vector replicates (through ori gene) and large number of copies of the DNA insert will be formed.
• Moreover, vector should be able to easily introduce into host cells.
• A vector should have marker genes for antibiotic resistance;
• It must contain unique cleavage site in one of the marker genes for restriction enzyme;
• It should have at least suitable control elements like promoter, operator, ribosomal binding sites, etc.

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Characteristics of Vector:

A. B. Deshmukh

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• The plasmids obtained naturally do not posses all the characteristics.
• Hence, they are constructed by inserting gene for antibiotic resistance.
• e.g., pBR 322, pBR 320, pACYC 177 are the constructed plasmids.
• pBR 322 is mostly used in rDNA technology in plants.

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i. Plasmid :

• Used in recombinant DNA technology
• Must replicate in E. coli.

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• Investigators have engineered these plasmids to optimize their use as vectors in DNA cloning.

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A. B. Deshmukh

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ii. Plasmid vectors for plants :

• An important vector for carrying new DNA into many types of plants is a plasmid that is found in Agrobacterium tumefaciens.
• This bacterium lives in the soil
• It causes a plant disease called crown gall.
• characterized by the presence of over- growths, or tumors, in the plant.
• A. tumefaciens contains a plasmid called Ti (for tumor-inducing).
• Ti plasmid contains a transposon, called T DNA,
• It inserts copies of itself into the chromosomes of infected plant cells.
• The transposon, with the new DNA, can still be inserted into the host cell’s chromosomes.
• A plant cell containing this DNA, can then be grown in culture or induced to form a new, transgenic plant.

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March 22 Q. 2 (v) Name the plant disease caused by Agrobacterium tumefaciens.

A. B. Deshmukh

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C. Competent host (cloning organism)

• Competent host used are usually the bacteria like
• Bacillus haemophilus,
• Helicobacter pyroli and
• E. coli.
• Mostly E. coli is used for the transformation with recombinant DNA

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12.3 Methodology for rDNA technology :

• The steps involved in gene cloning are as follows :

a. Isolation of DNA (gene) from the donor organism

b. Insertion of desired foreign gene into a cloning vector

(vehicle DNA)

c. Transfer of rDNA into suitable competent host or

cloning organism

d. Selection of the transformed host cell

e. Multiplication of transformed host cell

f. Expression of the gene to obtain the desired product

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a. Isolation of DNA (gene) from the donor organism :

i. Isolation of desired DNA:

• Desire gene is isolated obtained from the source organism (donor).
• Initially, the cells of the donor organism are
• sheared with the blender and
• treated with suitable detergent.
• Genetic material from the donor is removed, isolated and purified by using several techniques.
• Isolated DNA can be spooled on to a glass rod.

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ii. Cleaving of the isolated DNA:

• Isolated purified DNA is then cleaved by using restriction enzymes particularly Restriction Endonucleases (RE).
• R. E. cleave DNA at specific sites, called restriction sites and break the DNA into fragments.
• There are several types of restriction endonucleases.
• Cleaved DNA fragments have cohesive, sticky, staggered ends or blunt ends.
• From cleaved DNA fragments, a fragment containing desired gene is isolated and selected for cloning.
• This is now called foreign DNA or passenger DNA.
• A desired gene can also be obtained directly from genomic library or cDNA library.

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Do you know ?

• Gene library is a collection of different DNA sequences from an organism where each sequence has been cloned into a vector for
• ease of purification,
• storage and
• analysis.
• There are two types of gene libraries on the basis of the source of DNA used.
• Genomic Library
• cDNA Library

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Genomic library :

• It is a collection of clones that represent the complete genome of an organism.
• The genomic library of prokaryotes can be constructed by using plasmid vector.
• It is because prokaryotic genome does not contain repetitive DNA.

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cDNA library :

• It represents the library of eukaryotic organisms only.
• DNA is produced from isolated mRNA by reverse transcription.
• The DNA so made is called complementary DNA (cDNA).
• The library is called cDNA library.
• Eukaryotic DNA genome contains introns, regulatory genes and repetitive DNA.
• Hence, the establishment of genomic library in eukaryotes is not meaningful.

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b. Insertion of desired foreign gene into a cloning vector � (vehicle DNA) :

• The foreign DNA or passenger DNA is now inserted into a cloning vector or vehicle DNA.
• The most used cloning vectors are
• plasmids of bacteria and
• The most used plasmid is pBR 322.
• the bacteriophage viruses like
• lambda phage and M13.

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Lambda phage

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• Plasmids are isolated from the vector organism i.e., bacterium.
• By using same RE (which is used in the isolation of the desired gene from the donor), plasmid i.e., vector DNA is cleaved.
• Now by using enzyme DNA ligase, foreign DNA is inserted/ integrated into the vector DNA.
• The combination of vector DNA and foreign DNA is now called Recombinant DNA or Chimeric DNA, and the technology is referred to as rDNA technology.

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c. Transfer of rDNA into suitable competent host or cloning organism :

• rDNA is now introduced (i.e., transferred for expression) into a competent host cell of the suitable cloning organism which is usually a bacterium.
• Host cell takes up naked rDNA by process of ‘transformation’ and incorporates into its own chromosomal DNA which finally expresses the trait controlled by passenger DNA.
• The transfer of rDNA into a bacterial cell is assisted by divalent Ca⁺⁺.
• The cloning organisms used in plant biotechnology are E.coli and Agrobacterium tumefaciens.
• The host/ competent cell which has taken up rDNA is now called transformed cell.

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July 22 Q. 1 (ix)

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• Foreign DNA can also be transferred directly into the naked cell or protoplast of the competent host cell, without using vector.
• This is done by using techniques like
• electroporation,
• microinjection,
• lipofection,
• shot gun,
• ultrasonification,
• biolistic method, etc.
• In plant biotechnology the transformation is done through Ti plasmids of A. tumefaciens.

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d. Selection of the transformed host cell :

• The transformation process generates a mixed population of transformed (recombinant) and non-transformed (non-recombinant) host cells.
• For isolation of recombinant cell from non-recombinant cell, marker gene of plasmid vector is employed.
• For example,
• pBR322 plasmid vector contains different marker gene (Ampicillin resistant gene and Tetracycline resistant gene).
• When pst1 RE is used, it knocks out Ampicillin resistant gene from the plasmid, so that the recombinant cell become sensitive to Ampicillin.

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e. Multiplication of transformed host cell:

• Once transformed, host cells are separated by the screening process.
• Transformed host cells are introduced into fresh culture media.
• Host cells divide and redivide along with the replication of the recombinant DNA carried by them.

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f. Expression of the gene to obtain the desired product:

• Involves the production of desired products like alcohol, enzymes, antibiotics, etc.
• Desired product is separated and purified through downstream processing using suitable bioreactor.

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12.4 Applications of Biotechnology:

• It is used in many sectors, such as
• Health industry,
• agriculture industry,
• Environment and
• Genomics

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a. Healthcare Biotechnology :

• It refers to a medicinal or diagnostic product or a vaccine.
• Biotechnology offers patients a variety of new solutions such as:
• unique, targeted and personalized therapeutic and diagnostic solutions for organ transplant,
• Stem cell technology,
• Genetic counseling,
• Forensic medicine,
• Gene probes,
• Genetic fingerprinting and karyotyping are outcomes of biotechnology.

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Human proteins produced by rDNA technology to treat human diseases

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Human insulin :

• Insulin - a peptide hormone - produced by β-cells of islets of Langerhans of pancreas.
• Discovered by Sir Edward Sharpey Schafer (1916) while studying Islets of Langerhans.
• Essential for the control of blood sugar levels.
• Diabetes mellitus - disease - some people cannot make insulin themselves.
• Hakura et al (1977), chemically synthesized DNA sequence of insulin for two chains A and B and separately inserted into two pBR322 plasmid vector.

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March 24 Q. 2 (vi)

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• Insulin production by recombinant DNA technology is designed by Gilbert and Villokomaroff in 1978.

​

• The genes are inserted by the side of β-galactosidase gene of the plasmid.

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• The recombinant plasmids were then separately transformed into E. coli host.

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• The host produced penicillinase + pre-pro insulin.

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• Insulin is later separated by trypsin treatment.

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Vaccine production:

• A vaccine is a biological preparation
• provides active acquired immunity against a certain disease.
• Usually, a vaccine consists of a biological agent
• represents the disease-causing microorganism.
• It is often made from
• a weakened or killed form of the microorganism,
• its toxins or
• one of its surface protein antigens.

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• Biotechnology has offered modern diagnostic test kits such as
• rickettsial, bacterial and viral vaccines

along with

• radiolabelled biological therapeutics for imaging and analysis.

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• Vaccines have eliminated small-pox, polio and other deadly diseases for the last several decades.

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• Biotechnology has made advancements in vaccination by making recombinant vaccines that have the potential to eradicate non-communicable diseases like cancer.
• [Russia claims to develop first cancer vaccine - mRNA vaccine _{19 Dec 24}]

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• Naked DNA vaccines, viral vector vaccines and plant-derived vaccines are found to be most effective against several bacterial and viral disorders.

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Oral vaccines: a novel approach :

• The latest hot spot in the field of vaccine.
• Immunogenic protein of certain pathogens is found to be active when administered orally.

​

• The gene corresponding to such proteins is isolated and a gene construct is produced.
• This is introduced and expressed in a plant genome,
• It results in production of such immunogenic proteins in the parts of the plant where it is expressed.
• These when fed into animals or mainly humans, the person becomes vaccinated against certain pathogen.
• Such vaccines are also known as edible vaccines.

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• An exciting invention is production of ‘melt in the mouth’ vaccines
• can be administered by placing them under your tongue - delivers it into the blood stream.
• The most important example is the production of flu vaccine by Bacillus which melts in the mouth.
• The tremendous benefit of such vaccines, is
• the comfort of administration,
• low cost
• ease of storage.

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Sept 21 Q. 31 (a) Vaccines have eliminated small-pox, polio and other deadly diseases.

Describe the production of edible vaccine.

(b) What are ‘melt-in-mouth’ vaccines?

(c) Give the benefits of oral vaccines.

July 22 Q. 20 How ‘melt in mouth’ vaccines are administered? Mention any two benefits of the same.

July 23 Q. 9 What are oral vaccines? Enlist the benefits of oral vaccines.

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b. Agriculture :

• Applications of biotechnology in agriculture involves
• the use of genetically modified crops through the gene manipulation.
• GM crops developed are pest resistant, stress tolerant etc.
• Used in improving productivity.
• G. modification helps in modifying plants, animals, and microorganisms and improve their agricultural productivity.

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• Tissue Culture is used in Micropropagation
• i.e., large-scale propagation of plants in very short durations.
• Tissue culture technique is also the best method for
• storing germplasm and
• maintaining a specific genetic type (Clone).
• This technique is used in those plants, which produce recalcitrant seeds or produce highly variable seeds.

​

• [Recalcitrant seeds are seeds that are difficult to germinate or store, often due to their unique physiological characteristics. These seeds are typically sensitive to desiccation, temperature fluctuations, and other environmental factors, making them challenging to work with.]

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c. Gene therapy:

• A gene is a stretch of DNA required to make a functional product such as
• part or entire protein.

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• During gene therapy, DNA that codes for specific genes is delivered to individual cells in the body.

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• Gene therapy is the treatment of disease by replacing, altering or supplementing a gene that is absent or abnormal and whose absence or abnormality is responsible for the disease.

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• Most, if not all, diseases have a genetic factor.

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• The genetic factor can be wholly or partially responsible for the disease.
• For example, in disorders such as cystic fibrosis, haemophilia, and muscular dystrophy, changes in a gene directly result in the condition.

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• In other conditions, such as high cholesterol and high blood pressure, genetic and environmental factors interact to cause disease.
• There are more than 5000 different human genetic diseases known to be caused by single gene defects
• e.g.
• sickle cell anaemia,
• thalassemia,
• Tay-sach’s disease, [a rare, inherited genetic disorder caused by a deficiency of the enzyme hexosaminidase A (Hex-A). This enzyme is necessary for the breakdown of certain fatty substances (gangliosides) in the brain and nerve cells]
• Cystic fibrosis, [genetic disorder that affects the respiratory, digestive, and reproductive systems]
• Huntington’s chorea, [is a rare, inherited disorder that causes progressive damage to the brain]
• haemophilia,
• alkaptonuria, [a rare, inherited genetic disorder caused by a deficiency of the enzyme homogentisate 1,2-dioxygenase (HGD)]
• albinism, [rare genetic disorders that affect the production of melanin] etc.

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Gene therapy is being used in many ways.

• For example, to:
• Replace missing or defective genes;
• Deliver genes that speed the destruction of cancer cells;
• Supply genes that cause cancer cells to revert back to normal cells;
• Deliver bacterial or viral genes as a form of vaccination;
• Deliver DNA to antigen expression and generation of immune response;
• Supply of gene for impairing viral replication;
• Provide genes that promote or impede the growth of new tissue; and
• Deliver genes that stimulate the healing of damaged tissue.

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Forms of gene therapy :

a. Germ line gene therapy :

• Healthy genes - introduced into germ cells like sperms, eggs, early embryos.

​

• It allows transmission of the modified genetic information to the next generation.

​

• It is highly effective in counteracting the genetic disorders,
• But it is not encouraged for application in human beings due to a variety of technical and ethical reasons.

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• b. Somatic cell gene therapy :
• In this type the gene is introduced only in somatic cells like
• bone marrow cells,
• hepatic cells,
• fibroblasts endothelium and
• pulmonary epithelial cells,
• central nervous system,
• endocrine cells and
• smooth muscle cells of blood vessel walls.
• Modification of somatic cells only affects the person being treated and the modified chromosomes cannot be passed on the future generations.

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• Somatic cell gene therapy is the only feasible option,
• the clinical trials have already employed for the treatment of acquired disorders such as
• cancer and
• rheumatoid arthritis and
• blood disorders including
• SCID, (Severe Combines Immunodeficiency)
• Gaucher’s disease,
• familial hypercholesterolemia,
• haemophilia,
• phenylketonuria,
• cystic fibrosis,
• sickle cell anaemia,
• Duchene muscular dystrophy,
• emphysema,
• thalassemia etc.

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d. Genetically Modified Organisms (GMOs):

• Genetic material of organism have been artificially manipulated in a laboratory through genetic engineering.

​

• This creates combinations of plant, animal, bacteria, and virus genes that do not occur in nature or through traditional crossbreeding methods.

​

• Most GMOs have been engineered to
• withstand the direct application of herbicide and/or to produce an insecticide.
• to artificially develop other traits in plants, such as
• a resistance to browning in apples, and
• to create new organisms using synthetic biology.

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I. Transgenic Plants :

• Traditional breeding procedures were used
• To improve the quality and productivity of agriculturally important plants.
• But traditional breeding procedures were painstakingly slow and difficult.
• It involve sexual crosses, which resulted in the high quality of present-day food plants such as wheat, rice, corn, potato, etc.
• Biotechnological approaches have been applied to these plants to create genetic variations that are beneficial for mankind.

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• First transgenic plant produced was tobacco.
• More than 60 transgenic dicot plants and several monocot plant like maize, oat, rice, wheat are known.
• Tomato, soybean, potato, sugar beet, grapes, brinjal, cotton are other transgenic plants.
• Transgenic plants are being looked up as bioreactors for molecular farming i.e., for production of novel drugs like,
• interferons,
• edible vaccines,
• antibodies,
• amino acids,
• immunotherapeutic drugs, etc

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July 24 Q. 13 Give any two examples of commercially exploited products of transgenic plants.

March 23 Q. 2 (iv) Give the name of first transgenic plant.

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Advantages of GM food-plants :

• The ways in which one thinks that genetically modified plants can help, are listed as follows:

a. Insect pest resistance:

• It help to reduce use of chemical pesticides,
• which in turn can reduce the cost of producing food.

​

• However, an alternative has been available for more than 30 years which is a biological insecticide from the bacterium, Bacillus thuringiensis (Bt).

​

• However, the use of B. thuringiensis sprays is limited because of low stability of the protein in the field.

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​

​

• Insect resistant plants contain either a gene from
• B. thuringiensis or
• the cowpea trypsin inhibitor gene.
• The gene called cry gene present in B. thuringiensis.
• It produces a protein that forms crystalline inclusions in bacterial spores.
• When ingested by a susceptible insect, a combination of high pH and the enzyme proteinase of the insect’s midgut, processes them hydrolytically to release the core toxic fragments.

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• Bt cotton is one of the best transgenic plants known for its insect resistance property.

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• Effect of these fragments is seen within minutes of ingestion,
• begins with midgut paralysis and
• ends with disruption of midgut cells of insect.
• Bt toxin activity has been against many species of insects within the orders of Lepidoptera, Diptera, and Coleoptera.

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• Golden rice - a transgenic food crop used to reduce vitamin A deficiency disease.

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• Similarly, the gene of α-amylase inhibitor (αAl-Pv) has been isolated from adzuki bean (Phaseolus vulgaris) and transferred to tobacco.
• This gene works against pests like Zabrotes subfasciatus and Callosobruchus chinensis.

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July 24 Q. 2 (iii) Which gene is introduced in tobacco against pest like Zabrotes subfasciatus?

लाल चवळी

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b. Improved nutritional qualities (biofortification):

• Transgenic plants have also been produced to provide functional food and neutraceuticals.

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• For millions of people in developing countries, rice is the main item in their diet.

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• Because rice does not contain many essential nutrients, malnutrition is very common in these countries.

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• Especially terrible is the blindness that results from a lack of vitamin A.
• Vit.A is abundant in milk and in vegetables such as carrots,
• most of the poor people of the world cannot afford.
• To solve this problem, Swiss researchers created
• transgenic rice (golden rice) and
• transgenic mustard (golden mustard)
• high in vitamin A.
• The golden colour is due to vitamin A.

​

• They hoped that this rice, if grown and eaten in developing countries, would reduce the diseases associated with vitamin A deficiency (VAD).

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Antioxidants are the substances that may protect body cells against free radicals which may play a role in heart disease, cancer and other diseases.

Free radicals are molecules produced when your body breaks down food or when you are exposed to tobacco smoke or radiation.

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• Improvement in oil content and oil quality
• Oil crops like soybean, oil palm, rapeseed and sunflower, have been achieved by transfer of ‘Arabidopsis genes ’ .
• Iron deficiency is also a serious nutritional problem,
• affecting an estimated 30% of the world population.
• For production of transgenic crops that will produce food rich in iron, an iron storage protein (ferritin) is targeted.
• Ferritin is found in many animals, plants and bacteria.
• Genes for ferritin protein isolated from soyabean and Phaseolus have been transferred to rice.

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c. Modification in Post-harvest characteristics:

• Diseases and pests,
• Bruising on soft fruits and vegetables,
• Heat and cold storage,
• Over ripeness,
• Loss of flavours and odours, etc.
• lead to great deal of losses during storage and transport of crops.
• Most of these physiological changes are due to endogenous enzyme activity.
• Genetic engineering has made it possible to slow down these activities.

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• In tomato the enzyme polygalacturonase breaks down the cell wall constituent- pectin, leading to softening of fruit during ripening.
• Thus, the fruits are easily bruised and damaged on shipment.
• By inhibiting the polygalacturonase by antisense genes,
• the tomato can remain on the vine until mature and
• be transported in a firm solid state.
• Genetically modified tomatoes are called Flavr savr tomatoes.

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d. Plants as factories :

• To produce novel biochemicals and vaccines (Biopharmaceuticals), plants are potential factories or bioreactors for high value biochemicals like
• starch, sugar,
• lipids,
• proteins, and
• products like fine chemicals,
• perfumes and
• adhesive compounds as well as
• industrial lubricants,
• biodegradable plastic and even
• ‘renewable’ energy crops to replace fossil fuels.

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• Biopharmaceuticals are proteins, hormones, antibodies, vaccines or enzymes isolated from transgenic plants.
• Some of the proteins that are being produced by transgenic crop plants:
• Human growth hormone with the gene inserted into the chloroplast DNA of tobacco plants.
• Humanized antibodies against such infectious agents as
• HIV, Respiratory syncytial virus (RSV), Herpes simplex virus (HSV), the cause of "cold sores"
• Protein antigens to be used in vaccines for
• e.g.,
• Patient-specific antilymphoma (a cancer) vaccines.
• B-cell lymphomas are clones of malignant B cells expressing on their surface a unique antibody molecule.

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• Currently many novel products have been commercially exploited for their products such as:

a. A ‘superglue’ produced by tobacco plants with genes encoding for powerful adhesive proteins, enables marine mussels to stick to rocks.

• It will be especially valuable as a biochemical glue for body repairs during surgery.

​

b. Transgenic plants, containing oil encoding gene from marine algae, produce oil that has nutritional value similar to cod-liver oil.

​

c. Plant that will produce the anti malarial drug, Artemisinin.

​

d. Genetically engineered opium (poppy) to produce more powerful painkillers.

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e. Transgenic plants producing edible vaccines:

• Genetically altered plants can provide protection to infectious diseases.
• Plant products acting as vaccines
• would be inexpensive to produce
• can easily be made available in developing countries.
• most common foods proposed for edible vaccine delivery -
• Potatoes, tomatoes, bananas, soybeans, alfalfa and cereals.

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II. Transgenic animals:

• Many transgenic animals such as mice, rats, rabbits, pig, sheep, cows, fowls, fish have been produced through rDNA technology.
• The term transgenic animal refers to an animal in which there has been a deliberate modification of the genome - the material responsible for inherited characteristics - in contrast to spontaneous mutation.
• Foreign DNA is introduced into the animal, using recombinant DNA technology,
• must be transmitted through the germ line so that every cell, including germ cells, of the animal contain the same modified genetic material.

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• A representative, but non-inclusive, list of purposes for which transgenic animals have been used, indicates the wide-ranging application of this biotechnology:
• In medical research, transgenic animals are used to identify the functions of specific factors in complex homeostatic systems through over- or under-expression of a modified gene (the inserted transgene);
• In toxicology: as responsive test animals (detection of toxicants);
• In mammalian developmental genetics;
• In molecular biology, the analysis of the regulation of gene expression makes use of the evaluation of a specific genetic change at the level of the whole animal;
• In the pharmaceutical industry, targeted production of pharmaceutical proteins, drug production and product efficacy testing;

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• In biotechnology: as producers of specific proteins;
• Genetically engineered hormones to increase milk yield, meat production;
• Genetic engineering of livestock and in aquaculture affecting modification of animal physiology and/or anatomy;
• Cloning procedures to reproduce specific blood lines; and
• Developing animals specially created for use in xenografting.
• [Tissue or organs from an individual of one species transplanted into or grafted onto an organism of another species, genus, or family. A common example is the use of pig heart valves in humans.]

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a. Transgenic mice and cancer research :

• Transgenic mice are genetically engineered mice.
• Used in laboratory investigations.
• Modified by adding particular oncogenes to develop cancer.
• This helps to study relationship between oncogenes and cancer development.
• They can also be used for research into cancer treatment and prevention of malignancy.
• In the laboratory of Philip Leder in Harvard (USA) the transgenic mouse model for the investigation of the breast cancer was developed.
• The oncogenes myc and ras were analysed to find out if they lead to breast cancer in mice transformed with these genes.

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b. Transgenic farm animals :

• Transgenic farm animals are produced to derive greater benefits.
• Many of the farm animals are improved for
• their meat production ability
• milk yields and quality
• disease-free status.
• At the beginning of the century, a dairy cow provided 2,000 to 3,000 liters of milk a year.
• Today, Holstein cow provides 6,000 liters on average and up to 8,000 – 10,000 for the best ones.

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• A century ago, a hen laid about 70 eggs a year.
• Today the best races lay up to 250 eggs per year.
• This could be possible because of the advent of biotechnology.
• The main objectives for improved animal breeding programmes coupled with this new technology of gene transfer are given below.
• Efficiency of meat production
• Improved quality of meat
• Milk quality and quantity
• Egg production
• Wool quality and quantity
• Disease resistance in animals
• Production of low-cost pharmaceuticals and biologicals

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c. Transgenic cattle for food production :

• Researchers introduced additional copies of bovine beta or kappa casein into dairy cattle
• Cattles are evaluated the effect on milk production and composition.
• Transgenic offspring had an 8 to 20% increase in beta casein and a two-fold increase in kappa casein.

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July 24 Q. 30 How transgenic pigs and cattle are commercially beneficial?

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d. Transgenic cattle for human therapeutic production :

• A second application for genetically modified cattle is the production of human therapeutic proteins.
• Human proteins that have been expressed in milk include
• human lactoferrin,
• human alpha lactalbumin,
• human serum albumin and
• human bile salt stimulated lipase.
• The mammary gland in dairy cows is an excellent protein production factory.
• On the other hand,
• one transgenic cow would be more than sufficient for production of annual world supply of factor IX (plasma thromboplastin component) that is used in the treatment of haemophilia.

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• In 1990 Tracy, the transgenic cow was born in Scotland and could produce a human protein in her milk for human therapeutics.
• Antibodies are currently used for many different human clinical applications;
• including treatment of infectious disease,
• cancer,
• transplanted organ rejection,
• autoimmune diseases and
• for use as antitoxins.
• To make a human antibody product, the genetically modified cows are immunized with a vaccine containing the disease agent.

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e. Transgenic Sheep :

• Transgenic sheep are
• able to achieve better growth and
• meat production as well as
• to serve as bioreactors.
• Human growth hormone gene
• is introduced in sheep for promoting the growth and meat production.
• Bacterial genes, cys E and cys M,
• concerned with biosynthesis of cysteine amino acids involved in formation of keratin protein found in wool.
• Both these genes are identified, cloned and introduced in sheep to increase wool production and to improve the quality of wool.

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f. Transgenic pigs :

• The objective of gene transfer in pigs is
• to increase growth,
• meat production,
• to act as bioreactors.
• Pigs are regarded as the most suitable animals to be bred for heart transplant because
• a pig’s heart is about the same size as a human heart, and
• pig heart valves have been used in human heart surgery for over a decade.
• The pig clone is the first step towards
• providing animal organs and
• tissues for human transplants (xenotransplantation).

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July 24 Q. 30 How transgenic pigs and cattle are commercially beneficial?

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g. Transgenic fish :

• The commercially important fishes are transfected with
• growth hormone,
• chicken crystalline protein and
• E.coli hygromycin resistance gene
• e.g., Atlantic salmon, catfish, goldfish, Tilapia, zebra-fish, common carp, rainbow trout, etc..
• Transgenic fish showed
• increased cold tolerance and
• improved growth,
• improved quantity and quality of fish proteins
• Improvement in preservation,
• Above factors are affecting the economic value of fish.

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h. Transgenic chicken :

• Carry and express foreign genes.
• They could be used
• to improve the genetic make-up of existing strains with respect to
• built-in (in vivo) resistance to viral and coccidial diseases,
• better feed efficiency,
• lower fat and cholesterol levels,
• high protein containing eggs,
• better meat quality.

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12.5 Bioethics:

• Ethics usually deals with the matters related to socially acceptable moral duty, conduct and judgment.
• It helps to regulate the behaviour of community by some set of standards.
• However, the concepts differ according to culture and traditions.
• Moreover, concepts change with the time due to shifting of perception of values which are affected by progress in science and technology.
• Bioethics helps to study moral vision, decision and policies of human behaviour in relation to biological phenomena or events.
• Ethics deals with ‘Life’
• e.g., in vitro fertilization, sperm bank, gene therapy, cloning, gene manipulations, euthanasia, death, maintaining those who are in comatose state, prenatal genetic selection, etc.

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• The era of biotechnology has brought wide spectrum on new topics like
• cloning,
• transgenic,
• gene therapy,
• eugenics,
• rDNA technology, etc.
• The use of all these has drawn a wide range of reactions in the society.
• The reactions are based on individual’s own perception and moral.
• Ethical aspects pertaining to the use of biotechnology seems to be more controversial and frightening.
• These concerns are broadly summarized below :

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• Use of animals causes great sufferings to them;
• violation of integration of species caused due to
• transgenosis;
• transfer of human genes into animals and vice versa;
• indiscriminate use of biotechnology pose risk to the environment, health and biodiversity.

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• The introduction of Genetically Modified Organisms (GMOs) has led to a wider debate on bioethical concerns affecting social, economic and environmental spheres.
• These include the effects
• on non-target organisms,
• insect resistance crops,
• gene flow and the
• loss of diversity as well as
• the issue on interfering with nature in which the modification process itself is disrupting the natural process of biological entities.
• Ethics in biotechnology also includes the general subject of what should and should not be done in using recombinant DNA techniques

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12.6 Effects of Biotechnology on the Environment:

a. Herbicide Use and Resistance :

• Effects on the environment are a particular concern with regard to GMO crops and food production.
• One area of development involves adding
• the ability to produce pesticides and
• resistance to specific herbicides.
• These traits are helpful in food production, allowing farmers
• to use fewer chemicals, and
• to grow crops in less than ideal conditions.
• However, herbicide use could be increased.
• This will have a larger negative effect on the surrounding environment.

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• Also unintended hybrid strains of weeds and other plants can develop resistance to these herbicides through cross-pollination.
• Thus, negating the potential benefit of the herbicide.
• One such herbicide that has already been added is Round Up.
• Crops of Round Up ready soybeans have already been implemented into agricultural practices, possibly conferring Round Up resistance to neighboring plants.

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b. Effects on Untargeted Species :

• Bt corn, which produces its own pesticide, is also in use today.

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• It has adverse effects on Monarch butterfly populations, which are not the original target of the pesticide.

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• It can also have unintentional effects on neutral or even beneficial species.

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Pest on Corn

Monarch Butterfly

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12.7 Effects of Biotechnology on Human Health :

a. Allergies :

• GM crops could potentially have negative effects on human health as well.
• Consumers have developed unexpected allergic reactions.
• e.g., Researchers used a gene from the Brazil nut to increase the production of Methionine in soya beans.
• The insertion of this gene inadvertently caused allergic reactions to the soya bean in those with known nut allergies (“Biotech Soybeans”).

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b. Long-Term Effects :

• Because GMO technology has been available for such a short amount of time,
• There is relatively little research which has been conducted on the long-term effects on health which we cannot anticipate at this point.

c. New Proteins :

• Proteins that have never been ingested before by humans are now part of the foods that people consume every day.
• Their potential effects on the human body are yet unknown.

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March 22 Q. 13 What are the effects of biotechnology with relation to human health?

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d. Food Additives :

• GMOs also present us with possibilities of
• introducing additional nutrients into foods, as well as
• antibiotics and vaccines.
• This availability of technology can provide nutrition and disease resistance to those countries that don’t have the means to provide these, otherwise.
• However, there is possibility of the creation of antibiotic and vaccine-resistant strains of diseases.

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• This shows that the vast advances in life sciences and our multicultural and pluralistic modern societies create numerous bioethical problems requiring some stringent regulation.

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• In terms of GMOs, the Indian Government has set up the Genetic Engineering Approval Committee (GEAC).

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• This organization makes decisions regarding
• the validity of research involving GMOs and
• addresses the safety of GMOs introduced for public use.

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12.8 Bio-patent and Biopiracy:

1. Bio-patent :

Patent:-

• Special right granted to the inventor by the government.
• A personal property of inventor.
• It can be sold like any other property.
• A patent consists of three parts –
• grant (agreement with the inventor),
• specification (subject matter of invention) and
• claims (scope of invention to be protected).

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• Bio-patent: -
• Biopatent is a biological patent.
• Biopatents are awarded for
• strains of microorganisms,
• cell lines,
• genetically modified strains,
• DNA sequences,
• biotechnological processes,
• product processes,
• product and product applications.
• Awarded to recognize real innovative contributions made by the inventor to the cause of human welfare.
• The awards are given
• to inculcate encouragement and values in developing scientific culture and
• in emphasizing the role of biology in shaping human society.

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• Indian Patent: -
• Indian patent allows ‘process patent’ and not the ‘product patent’.
• Biopatent allows the patent holder
• to exclude others from making, using, selling or
• importing protected invention for a limited period.
• Duration of bio-patent is
• five years from the date of the grant or
• seven years from the date of filling the patent application, which ever is less.

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• First biopatent was patented pertaining to genetically engineered bacterium ‘Pseudomonas’ used for clearing oils spills.

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• Patent under the title ‘control of plant gene expression’
• Issued jointly to Delta and Pineland company and U. S. department of agriculture.
• Patent is based on a gene that produces a protein toxic to plant thus, do not allow seeds to germinate.
• However, this patent was not granted by Indian government.
• Such a patent is considered morally unacceptable and fundamentally unequitable.
• This is because financially powerful corporations would acquire monopoly over biotechnological process.
• This in turn would pose a threat to global food security.

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b. Biopiracy :

• Pirates in general terms are those who steal and kill others to enrich themselves.

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• Bio pirates are those who do not kill but steal the patent (misuse the patent).

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• Biopiracy is defined as ‘theft of various natural products and then selling them by getting patent without giving any benefits or compensation back to the host country’.

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• In short, it is unauthorized misappropriation of any biological resource and indigenous knowledge.

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• Why Biopiracy occurs?
• Most developed, industrialized and financially rich nations are poor in biodiversity or traditional knowledge.
• Developing and underdeveloped nations
• have ample of biodiversity, and
• they traditionally better know the use of their bio-resources.
• Traditional Knowledge naturally includes
• a deep understanding of ecological processes and
• the ability to sustainably extract useful products from the local habitat.
• Most Traditional Knowledge is handed down through generations.
• This helps them to develop modern, commercial applications that save the makers time, money and effort.

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• Components of Traditional Knowledge that are especially relevant to our global survival include knowledge of:
• Food, crop varieties and agricultural/ farming practice
• Sustainable management of natural resources and conservation of biological diversity
• Biologically important medicines

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• The conservation of species, habitat, and biodiversity are essential to the continued survival of indigenous and rural people.

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• By conserving the customs and habitat of indigenous persons, we concurrently reduce emissions from deforestation and ecosystem degradation.

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• Furthermore, the opportunity for cultural survival is a basic human right.

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• The traditional knowledge is facing a problem of bio-piracy.

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• The act of Piracy is unauthorized publication or reproduction of another person’s work or material.

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• When someone indulges in piracy,
• the accused is using someone’s work illegally or without taking any permission.

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• The innovations and discovery of the pharmaceutical and agricultural researches are not new as to qualify as invention as they are based on centuries of knowledge of the traditional societies.

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Examples of Biopiracy :

1. Patenting of Neem (Azadirachta indica) :

• The people of India in a variety of ways have used neem, since time immemorial.
• Indians have shared the knowledge of the properties of the neem with the entire world.
• Pirating this knowledge, the USDA and an American MNC W.R. Grace in the early 90s sought a patent from the European Patent Office (EPO) on the “method for controlling on plants by the aid of hydrophobic extracted neem oil.”
• The patenting of the fungicidal properties of Neem, was an example of biopiracy.

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March 22 Q. 26 Explain any three examples of biopiracy.

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2. Patenting of Basmati :

• Basmati is a long-grained, aromatic variety of rice indigenous to the Indian subcontinent.
• In 1997 the US Patent and Trademark Office (USPTO) granted a patent to a Texas based American company Rice Tec Inc for “Basmati rice line and grains” having trade name Texmati.
• The patent application was based on 20 very broad claims on having “invented” the said rice.
• Due to people's movement against Rice Tec in March 2001, the UPSTO has rejected all the claims.

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3. Haldi (Turmeric) Biopiracy:

• Two American researchers of Indian origin of the University of Mississippi Medical Center, put a claim to the US Patent and Trademark Office, maintaining that they had discovered haldi's healing properties.
• Surprisingly, they were granted a patent in March 1995 for something you had known for years and our ayurvedas for centuries.
• It meant they had exclusive rights over any such haldi drug and were reexamination, to make millions of dollars.
• The Council of Scientific and Industrial Research (CSIR) applied to the US Patent Office for a reexamination, and they realized the mistake and cancelled the patent.
• This was after Indian scientists shouted from rooftops about how we are losing our traditional knowledge to marauding foreign companies who have started poaching on our ancient healing techniques.

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• It is the need of hour to launch genetic literacy movement in Indian school and colleges for
• better understanding of opportunities and risks related to biotechnology and
• also to promote the safe and meaningful use of technologies of modern life sciences.

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