(CLASS XII) TUITION - BIOLOGY

CHAPTER - 04
BIOTECHNOLOGY : PRINCIPLES AND PROCESSES

BIOTECHNOLOGY
Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce
products and processes useful to humans.
Two core techniques that enabled birth of modern biotechnology:
 Genetic engineering : Techniques to alter the chemistry of genetic material (DNA and RNA) to
introduce into host organisms and thus change the phenotype of the host organism.
 Maintenance of sterile (microbial contamination-free) ambient chemical engineering processes
to enable growth of only the desired microbe/eukaryotic cell in large quantities.
Three basic steps in genetically modifying an organism :
 Identification of DNA with desirable genes.
 Introduction of the identified DNA into the host.
 Maintenance of introduced DNA in the host and transfer of the DNA to its progeny..
Conceptual development of the principle of genetic engineering
 Asexual reproduction preserves the genetic identity of species.
 Sexual reproduction creates variation and creates unique combinations of genetic makeup.
 Traditional hybridization procedures used in plant and animal breeding lead to inclusion of
undesirable genes along with desired genes.
 The techniques of genetic engineering which includes creation of recombinant DNA, use of gene
cloning and gene transfer, over come this limitation and allows us to isolate and introduce only
one or a set of desirable genes without introducing undesirable genes into target organism.
Tools of Recombinant DNA Technology
Restriction Enzymes
 In the year 1963 two enzymes discovered from Escherichia coli which restrict the growth of
bacteriophage in it.
 One of these added methyl groups of DNA
 Other cut the phage DNA (restriction endonuclease)
 The first restriction endonuclease discovered is Hind II
 Hind II always cut DNA molecules at particular point of recognizing a specific sequence of six base
pairs. This is called recognition sequence for Hind II.
 Restriction enzyme belongs to nucleases
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 There are two kind of nucleases :

 Exonuclease
 Endonuclease
 Exonuclease removes nucleotides from the free ends of the DNA.
 Endonuclease make cuts at specific positions within the DNA.
 Each restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence. Each restriction
endonuclease recognizes a specific palindromic nucleotide sequences in the DNA.

eg., 5  GAATTC  3
3  CTTAAG  5

 Palindromes are the group of letters that read same both forward and backward, eg; “MALAYALAM”.

5  GAATTC  3 5  G AATTC  3
3  CTTAAG  5 3  CTTAA G  5

 The restriction enzyme cut the strand of DNA little away from the centre of the palindrome sites, but
between the same two bases on the opposite strand. This leaves single stranded portions at the
ends. There are overhanging stretches called Sticky ends on each strand.
 The foreign DNA and the host DNA cut by the same restriction endonuclease, the resultant DNA
fragments have the same kind of ‘sticky-ends’ and these can be joined together using DNA ligases.
Convention for naming restriction endonuclease :
 The first letter of the name comes from the genus.
 Second two letters come from the species of the prokaryotic cell from which the enzyme isolated
 The fourth letter is in capital form derived from the strain of microbes.
 The Roman letter followed is the order of discovery..
 Best example : EcoRI comes from Escherichia coli RY 13
Separation and isolation of DNA fragments :
 The cutting of DNA by restriction endonucleases results in the fragments of DNA.
 These fragments are separated by a technique called gel electrophoresis.
 Since the DNA fragments are negatively charged, they can be separated by forcing them to
move towards anode under an electric field through a medium/matrix.
 Most commonly used matrix is agarose, a natural polymer extracted from sea weed.
 DNA fragments separate according to their size through sieving effect provided by the agarose
gel. Hence the smaller the fragment size, farther it moves.
 The separated fragments are visualized by staining them with Ethidium bromide followed by
exposure of UV radiation.
 The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece.
This step is called elution.

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Cloning Vectors
Plasmid is an autonomously replicating circular extrachromosomal DNA that contains only few genes
and is generally found in bacterial cells. Plasmids and bacteriophages have the ability to replicate
within the bacterial cell independent of chromosomal DNA, so they are used as cloning vectors.
Features of Cloning vector:
Origin of replication
 This is the sequence where the replication starts called ori gene.
 The alien DNA linked with vector also replicates
 Controls the copy number of the linked DNA.
Selectable marker
 It is required to identify recombinant from the non-recombinant
 Helps in identifying and eliminating non-transformants and selectively permitting the growth of the
transformants.
 Transformation is a procedure through which a piece of foreign DNA is introduced in a host
bacterium.
 Normally, the gene coding resistance to antibiotics such as ampicillin. Tetracycline, chloramphenicol
or kanamycins etc are considered as useful selectable markers for E.coli
Cloning sites
 In order to link the alien DNA, the vector needs to have very few, preferably single, recognition
sites (palindromic site) for the commonly used restriction endonuclease.
 Commonly used vector is pBR322 for E.coli
 If a foreign DNA ligated or inserted at the Bam HI site of tetracycline resistance gene in the vector
pBR322, the recombinant plasmid will lose tetracycline resistance. (insertional inactivation)

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Insertional Inactivation

 The foreign DNA is introduced within the coding sequence of an enzyme   galactosidase, which
convert X-Gal (chromatogenic substrate) into Galactose and 5-bromo+4-chloro indigo ( blue colour)
 The non-recombinant produce enzyme and give blue coloured colonies.
 The recombinant unable to produce   galactosidase and does not produce blue coloured colonies
after addition of chromatogenic substrate ie., X-Gal.
 This inactivation of insertion of foreign DNA called insertional inactivation.
Vectors for cloning genes in plants and animals
Agrobacterium tumifaciens transfer its T-DNA to normal plant cells and causes tumor. Similarly
retroviruses in animals have the ability to transform normal cells into cancerous cells. Now retroviruses
have disarmed and used to deliver desirable genes into animal cells.
Competent Host (for transformation with recombinant DNA)
 DNA is a hydrophilic molecule; it cannot pass through cell membranes.
 In order to force bacteria to take-up the plasmid, the bacterial cells must first be made ‘competent’ to
take up DNA.
 The bacterial cell is treated with divalent cations such as calcium, which increases the efficiency of
DNA up take by the bacteria.
 Recombinant DNA and the bacterial cells are incubated in ice, followed by placing them briefly at
420C (heat shock) and then putting them back in ice.
 Micro-injection : Recombinant DNA is directly injected into the nucleus of an animal cells.
 Biolistics (gene gun) : Cells are bombarded with high velocity micro particles of gold or tungsten
coated with DNA. It is suitable for plants. This method uses ‘disarmed pathogen’ vectors, which when
allowed to infect the cell, transfer the recombinant DNA into the host.
Process of Recombinant Technology
 Isolation of DNA
 Fragmentation of DNA by restriction endonuclease.
 Isolation of desired DNA fragment by gel electrophoresis
 Ligation of DNA fragment with a vector by DNA ligase
 Transferring the recombinant DNA into the host
 Culturing the host cells in a medium at large scale in a bioreactor
 Extraction of desired product by downstream processing.
 Isolation of the Genetic material (DNA)
 Bacterial cell wall digested by Lysozyme.
 Plant cell wall is digested by cellulase and pectinase.
 Fungal cell wall is digested by chitinase.
 RNA of the cellular content is digested by ribonuclease.
 Proteins are removed by Proteases.

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 Purified DNA ultimately precipitated out after addition of chilled ethanol.

 The precipitated DNA is separated and removed by spooling.
 Amplification of Gene of Interest using PCR
 PCR stands for, Polymerase Chain Reaction
 Multiple copy of gene of interest can be synthesized in-vitro.

 PCR includes following steps :

Denaturation
 Double stranded DNA made single stranded
 It is done by heating the DNA at 940C
 Each single stranded DNA is called Template strand

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Annealing

 Two sets of primer (small oligonucleotide chain that are complementary to the DNA at 3 end of
the DNA template) added to the medium.
 This is done at around 500C.
Extension
 Deoxyribonucleotides triphosphates are added in the medium.
 Taq polymerase catalyses the polymerisation reaction using nucleotides extending from the
primer towards 5 end of the template.

 Taq polymerase is a thermostable polymerase isolated from a bacterium called Thermus

aquaticus.
 It catalyses polymerisation reaction at 740C.
 Obtaining the Foreign Gene product or, Recombinant product
 If is necessary to produce target protein ie., recombinant protein on a small scale, rDNA transferred
into the host and cloned genes of interest must be grown in laboratory. Then the protein is extracted
and purified.
Bioreactor
 A bioreactor provides optimum growth conditions (temperature, pH, substrate, salts, vitamins,
oxygen)
 Bioreactor convert the raw materials into specific product, specific enzyme.
 Bioreactors are large vessels (100-1000L) in which raw materials are biologically converted into
specific products, individual enzymes etc, using microbial, plant, animal or human cells.
Downstream Processing
After the desired product is formed, it is subjected to a series of processes.
It includes :
 Separation and purification
 Formulation with suitable preservatives
 Clinical trials as in case of drugs
 Quality control testing

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Descriptive Questions
1. DNA cannot pass through cell membrane. Give reason.
2. Mention the significance of sticky ends.
3. Write a note on bioreactor and its working.
4. Explain taq polymerase
5. Give reason for the movement of DNA towards the anode.
6. Write a note on naturally occurring plasmids in E.coli, Agrobacterium
7. Describe downstream processing.
8. Explain the naming of Hind II.
9. Write 2 selectable marker genes in pBR322.
10. PCR is just like a photocopying document. Justify
Objective Questions
1. The first restriction endonuclease isolated was
A) EcoRI B) Hind II C) Bam HI D) Sal I
2. The letter ‘R’ in EcoRI stands for
A) name of genus B) name of strain C) name of species D) the term restriction
3. The sticky ends of a fragmented DNA molecules are made of
A) Ca salts B) Endonuclease enzyme
C) Unpaired bases D) Methyl groups
4. Identify the palindromic sequence :

GAATTC GGATCC CCTGG CGATA

A) B) C) D)
CTTUUG CCTAGG GGACC GCTAA
5. _______ a crown gall bacterium, called as natural genetic engineer of plants.
A) E.coli B) Streptomyces albus
C) Agrobacterium tumifaciens D) Azotobacter
6. Which of the following is required for microinjection method of gene transfer ?
A) Microparticles B) Micropipettes C) Divalent cations D) UV radiations
7. Which among the following is not used to transfer the recombinant DNA into the host ?
A) Microinjection B) Gene gun
C) Bioreactor D) Disarmed pathogen vector
8. Select the incorrect pair.
A) Plant cells - Cellulose B) Bacteria - Methylase
C) Fungi - Chitinase D) None of these
9. A device in which large volume of living cells are cultured in order to get a specific product ;
A) PCR B) Agitator C) Bioreactor D) Assimilator
10. Select the one which is not a component of downstream processing.
A) Separation B) Purification C) Preservation D) Expression
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Answer Key
1. B
2. B
3. C
4. B
5. C
6. B
7. C
8. B
9. C
10. D