MCB 311 & MCB 309 New Version

2019
Department of Biosciences and
Biotechnology, Kwara State
University, Malete.
DR. A.T. AJAO
LECTURE NOTES ON MICROBIAL GENETICS AND
MOLECULAR BIOLOGY & PRINCIPLES OF BIOTECHNOLOGY
(MCB 311 & MCB 309)

Biotechnology and its Applications
Describe biotechnological techniques for designing Bt cotton and RNAi strategy in
preventing infestation of Meloidegyne incognitia in Tobacco plants.
Answer
Bt Cotton
 Some strains of Bacillus thuringiensis have proteins that kill insects like coleopterans
(beetles) lepidopterans (tobaccobudworm, armyworm) & dipterans (flies, mosquitoes).
 B. thuringiensis forms a toxic insecticidal protein (Bt toxin) crystal during a particular
phase of their growth. It does not kill the Bacillus as it exists as inactive protoxins.
 When an insect ingest the inactive toxin, it is converted into active toxin due to the
alkaline pH of the gut which solubilise the crystals.
 The toxin binds to the surface of midgut epithelial cells and creates pores. It causes cell
swelling and lysis and death of the insect.
 Bt toxin genes were isolated from B. thuringiensis and incorporated into crop plants such
as cotton.
 Most Bt toxins are insect-group specific. The toxin is coded by a gene named cry. E.g.
the proteins encoded by the genes cryIAc and cryIIAb control the cotton bollworms that
of cryIAb controls corn borer.
Nematode resistance in tobacco plants:
 A nematode Meloidegyne incognitia infects the roots of tobacco plants and causes a great
reduction in yield.
 RNA interference (RNAi) strategy is used to prevent this infestation.
Lecture notes on Microbial genetics & Molecular biology/ Principles of Biotechnology by Dr. A.T
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 RNAi is a method of cellular defense in all eukaryotic organisms. It prevents translation
of a specific mRNA (silencing) due to a complementary dsRNA molecule.
 The source of this complementary RNA is from an infection by RNA viruses or mobile
genetic elements (transposons) that replicate via an RNA intermediate.
 Using Agrobacterium vectors, nematode-specific genes (DNA) were introduced into the
host plant.
 It produced both sense & anti-sense RNA in host cells. These two RNA‘s being
complementary to each other formed a double stranded (dsRNA) that initiated RNAi and
thus, silenced the specific mRNA of nematode.
 Thus the parasite cannot survive in a transgenic host expressing specific interfering
RNA.
The major different types of applications of Environmental Biotechnology are:
1. Biomarker or chemical that helps to measure the level of damage caused or the exposure of the
toxic or the pollution effect caused.
2. Bioenergy like Biogas, biomass, fuels, and hydrogen
3. Bioremediation for cleaning up the hazardous substances into non-toxic compounds…
4. Biotransformation to change of the complex/toxic to simple non-toxic compounds.
Biopharmaceuticals
By using the techniques of biotechnology, the drugs biopharmaceuticals were developed. There
are no chemicals involved in the synthesis of these drugs, but microorganisms have made it
possible to develop them. Large molecules of proteins are usually the source of
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biopharmaceuticals. They when targeted in the body attack the hidden mechanisms of the disease
and destroy them. Now scientists are trying to develop such biopharmaceutical drugs which can
be treated against the diseases like hepatitis, cancer and heart diseases.
These drugs are made by many ways and one method of developing such drugs is bioreactor.
Bioreactor is a container which is used to grow microorganisms under the specific temperature
and other required conditions. These microorganisms then make biopharmaceuticals. Though
genetically modified plants and animals can also be used to make biopharmaceuticals but then
there are various ethical and legal issues regarding these animals and plants.
Gene Therapy
Gene therapy is another technique of biotechnology which is used to treat and diagnose diseases
like cancer and Parkinson's disease. The mechanism of this technique is that the healthy genes
are targeted in the body which either destroy the damaged cells or replace them. In some cases,
the healthy genes make corrections in the genetic information and that is how the genes start
functioning in the favor of the body.
Pharmacogenomics
 Pharmacogenomics is another genetically modified technique which is used to study the
genetic information of an individual.
 It analyzes the body's response to certain drugs. It is the combination of pharmaceuticals
and genomics.
 The aim of this field is to develop such drugs which are inserted in the individual
according to the genetic information present in the individual.
Genetic Testing
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 Genetic testing is a technique of genetics which is used to determine the genetic diseases
in parents, sex and carrier screening.
 The method of genetic testing is to use DNA probes which have the sequences similar to
the mutated sequences.
 This technique is also used to identify the criminals and to test the paternity of the child.
Importance of Genetically Engineered Crops
 Genetically engineered crops have desirable genes (as of insect/pest resistance, giving
better yield) incorporated in them.
 Genetically modified crops have
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 More tolerance to abiotic stresses such as cold, drought, salinity, heat, etc.
 Insect/pest resistance
 Reduced post-harvest losses
 Efficient mineral usage by plants
 enhanced nutritional value (e.g., Vitamin A rich rice)
State ideal features of microorganisms used in Biotechnology

Answer
The ideal features of microorganisms used in Biotechnology
a. The nutritional requirement of the organism must be from a very cheap source.
b. The strain should be able to protect itself against contamination.
c. It must be stable and amenable for genetic manipulation.
d. It must have a high productivity i.e the rate at which substrates are converted to products
must be high. Moreover, it must give a yield of product per unit time.
e. It must easy to extract the desired product after the bioconversion process.
f. It must be suitable for the type of process to be employed.
g. It must not react with the equipment
Itemize major points to describe the term “Strain improvement and its purposes
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Answer
Improvement of strains can be put down in simple term as follows:
(i) Regulating the activity of the enzymes secreted by the organisms;
(ii) In the case of metabolites secreted extracellularly, increasing the permeability of the
organism so that the microbial products can find these ways more easily outside the cell;
(iii) Selecting suitable producing strains from a natural population;
(iv) Manipulation of the existing genetic apparatus in a producing organism;
(v) Introducing new genetic properties into the organism by recombinant DNA technology or
genetic engineering.
Purposes of strain improvement
i. Increase productivities
ii. To change unused co-metabolites
iii. To improve the use of carbon and Nitrogen sources
iv. To improve morphology of cells to be a better cells in order to separate the cells and its
products.
Describe parasexuality, protoplast fussion and metabolic engineering as methods of strain

improvement.
Answer: Parasexuality & Heterokaryosis is a rare form of sexual reproduction which occurs in
some fungi.
 In parasexual recombination of nuclei in hyphae from different strains fuse, resulting in
the formation of new genes.
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 Heterokaryosis - co-existence of genetically- different nuclei in cytoplasm continuity
with one another.
 Plays major role - variability and sexuality in fungi.
 Due to Anastomosis (fusion of hyphae)- Fusion is mostly intra-specific.
 Nuclear migration from the point of fusion to the remainder of the mycelium takes place -
heterokaryotic mycelium. eg- development of heterokaryon in basidiomycota.
 Parasexuality is important in those fungi such as Penicillium chrysogenum and
Aspergiluss niger in which no sexual cycles have been observed.
 It has been used to select organisms with higher yields of various industrial products such
as phenoxy methyl penicillin, citric acid, and gluconic acid.
 Parasexuality has not become widely successful in industry because the diploid strains
are unstable and tend to revert to their lower-yielding wild-type parents.
 More importantly is that the diploids are not always as high yielding as the parents.
Protoplast fusion
 Protoplasts are cells devoid of their cell walls and may be prepared by subjecting cells to
the action of wall degrading enzymes in isotonic solutions.
 It is a versatile tool for genetic manipulation and breeding in industrial microorganisms.
Cell fusion followed by nuclear fusion occurs between protoplast and hence
recombination occurs.
 Protoplasts are the cells of which cell walls are removed and cytoplasmic membrane is
the outer most layer in such cells. Protoplast can be obtained by specific lytic enzymes to
remove cell wall. Protoplast fusion is a physical phenomenon.
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 During fusion two or more protoplasts come in contact and adhere with one another
either spontaneously or in presence of fusion inducing agents.
 By protoplast fusion it is possible to transfer some useful genes from one species to
another. Protoplast fusion is an important tool in strain improvement. These are the
powerful techniques for engineering of microbial strains for desirable industrial
properties.
 By protoplast fusion it is possible to transfer some useful genes such as disease
resistance, nitrogen fixation, rapid growth rate, more product formation rate,
protein quality, frost hardiness, drought resistance, herbicide resistance, heat and
cold resistance from one species to another.
 Protoplast fusion an important tools in strain improvement for bringing genetic
recombinations and developing hybrid strains in filamentous fungi.
 Protoplast fusion has been used to combine genes from different organisms to create
strains with desired properties.
 Fusion of freely isolated protoplasts from different sources with the help of fusion
inducing chemical agents is known as induced fusion.
 Techniques for induced fusion are: PEG treatment. NaNO3 treatment; Calcium ion
treatment; and electrical impulse—are needed to achieve the phenomenon of induced
fusion.
 The protoplast mixture is treated with 28-50 % PEG for 15-30 min, followed by gradual
washing of the protoplasts to remove PEG; protoplast fusion occurs during the washing.
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 The washing medium may be alkaline (pH 9-10) and contain a high Ca2+ ion
concentration; this approach is a combination of PEG and high pH-high Ca2+ treatments,
and is usually more effective than either treatment alone.
 PEG is negatively charged and may bind to cations like Ca2+, which, in turn, may bind to
the negatively charged molecules present in (plasma membrane). vi. During the washing
process, PEG molecules may pull out the plasmamembrane components bound to them.
This would disturb plasma lemma organisation and may lead to the fusion of protoplasts
located close to each other. vii. Protoplasts are formed from bacteria, fungi, yeasts and
Actinomycetes when dividing cells are caused to lose their cell walls.
 Metabolic engineering is the science that combines systematic analysis of metabolic and
other pathways with molecular biological techniques to improve cellular properties by
designing and implementing rational genetic modifications.
 Altering metabolic pathways to improve cell properties and produce or overproduction of
certain target metabolites.
Specifically:
 Improving the production of chemicals already produced by the host organism,
 Extending the range of substrate for growth and product formation
 Adding new catabolic activities for degradation of toxic chemicals
 Producing chemicals new to the host organism were produced
 Creating situations that contributed to a drastic modification of the overall cellular
properties
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 Site-directed mutation: Mutations are caused by radiation, viruses, transposes and
mutagenic chemicals, as well as errors that occur during meiosis or DNA replication.
 Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide
directed mutagenesis is a molecular biology technique often used in bio molecular
engineering in which a mutation is created at a defined site in a DNA molecule.
 Site-directed mutagenesis is the technique for generating amino acid coding changes in
the DNA (gene). Site-directed mutagenesis is incorporation of a desired amino acid (of
one's choice) in place of a specific amino acid in a protein or a polypeptide.
Discuss Microbial Growth Curve
 When microorganisms are introduced into fresh culture medium, usually no immediate
increase in cell number occurs, so this period is called the lag phase
 there is no net increase in mass, the cell is synthesizing new components.
 A lag phase prior to the start of cell division can be necessary for a variety of reasons.
The cells may be old and depleted of ATP, essential cofactors, and ribosomes; these must
be synthesized before growth can begin.
 The medium may be different from the one the microorganism was growing in
previously. Here new enzymes would be needed to use different nutrients.
 Possibly the microorganisms have been injured and require time to recover.
 This phase may be quite long if the inoculum is from an old culture or one that has been
refrigerated. Inoculation of a culture into a chemically different medium also results in a
longer lag phase.
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 On the other hand, when a young, vigorously growing exponential phase culture is
transferred to fresh medium of the same composition, the lag phase will be short or
absent.
Exponential growth
 During the exponential or log phase, microorganisms are growing and dividing at the
maximal rate possible given their genetic potential, the nature of the medium, and the
conditions under which they are growing.
 The microorganisms are dividing and doubling in number at regular intervals.
 The population is most uniform in terms of chemical and physiological properties during
this phase; therefore exponential phase cultures are usually used in biochemical and
physiological studies.
Stationary phase
 Because this is a closed system, eventually population growth ceases and the growth
curve becomes horizontal.
 This stationary phase usually is attained by bacteria at a population level of around 109
cells per ml.
 Microbial populations enter the stationary phase due to nutrient limitation;
 If an essential nutrient is severely depleted, population growth will slow.
 Aerobic organisms often are limited by O2 availability.
 May also cease due to the accumulation of toxic waste products.
 Finally, there is some evidence that growth may cease when a critical population level is
reached.
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 Procaryotes have evolved a number of strategies to survive starvation,which are
morphological changes gene expression and physiology.
 Starving bacteria frequently produce a variety of starvation proteins, which make the
cell much more resistant to damage in a variety of ways.
Death phase
 For many years, the decline in viable cells following stationary cells was described
simply as the ―death phase.‖
 It was assumed that detrimental environmental changes like nutrient deprivation and the
buildup of toxic wastes caused irreparable harm resulting in loss of viability
MATHEMATICS OF MICROBIAL GROWTH
Let N0 the initial population number
Nt the population at time t
n the number of generations in time t
Nt N0 2n.
Solving for n, the number of generations, where all logarithms
are to the base 10,
log Nt log N0 + n.log 2, and
 The rate of growth during the exponential phase in a batch culture can be expressed in
terms of the mean growth rate constant (k). (K) is the number of generations per unit
time, often expressed as the generations per hour.
K= =
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 The time it takes a population to double in size—that is, the mean generation time or
mean doubling time (g) can now be calculated. If the population doubles (t = g), then, Nt
= 2N0
Substitute 2N0 into the mean growth rate equation and solve for k
K= =
K=
KINETICS OF MICROBIAL GROWTH
Consider that a medium is inoculated with 100 cells that divide after every 30 minutes. The cell
population will be 200 after 30 minutes, 400 after 60 minutes and so on. Since the population
n
doubles after every generation, the final population is always 2 where n is the number of
generations. Thus, the resulting population increase is logarithmic.
 The rate of growth during log phase is proportional to the cell concentration and can be
described by the following equation:
αх or,
= μx (1)
 where μ is the proportionality constant, known as specific growth rate. Eq. (1) can be
written as:
xdx = μ dt
where x can represent either the cell number (N) or the cell mass (x).
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Assuming x = x at t = 0 and x = x at time t, the integration of Eq. (2) gives:
0 t
ln = μt (3)
μt
or, x = x e (4)
t 0
where e is the base of natural logarithm.
Eq. (4) shows that the cell population increases exponentially during the log phase. Eq. (3) can
be written as:
log x – log x = μt
t 0
or, log x = log x + μt (5)

t 0
After taking natural logarithms, eq. (5) can be written as
2.303 log x = 2.303 log x + μt

t 0
or, log x = log x + 303.2μ t (6)

t 0
 Thus if log x is plotted against t, then a straight line is obtained whose slope is equal to μ
t
/ 2.303, from which μ can be easily calculated.
 In a batch culture where the nutrients are in excess, the cells grow at their maximum
specific growth rate (μ ) during the log phase. The values of μ for a few
max max
microorganisms.
 It is to be emphasized that the value of specific growth rate is not constant but depends on
the composition of the medium and the environmental conditions under which the
microorganism is growing.
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 The generation time or the doubling time of a growing culture can be calculated easily as
follows:
 If t is time taken for a cell population to double in numbers, then the initial cell
d
population N after time t will be 2N . Substituting the value of N and t in equation (3),
o d o
we get the following relation: µ
 ln =μt
d
or, μ t = ln 2
d
or, t = = (7)
d
Thus, if the value of μ is calculated by the procedure mentioned above, it is very easy to find the
doubling or the generation time of the microbial culture.
The generation times vary markedly with the species of the microorganisms and can vary from
about 10 minutes for a few bacteria to a few days for large bacteria and many eukaryotic
organisms. It may be remembered that the generation times are in general much longer in nature
than in a cultured environment.
 A continuous culture is that culture where a steady exponential phase from growth of
culture retards due to the depletion of nutrients, rather than by accumulation of toxic
products.
 It is prevented by the addition of fresh medium to the fermentor and removal of spent
medium and microbial biomass.

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 Microbial culture grows continuously in log phase. One of the nutrients of the medium is
kept limited.
 The growth rate is controlled by manipulating the inflow of the fresh medium while
population is regulated through changing the concentration of the limiting nutrients.
 Limiting nutrient is a nutrient essential for growth present in the medium at a limiting
concentration e.g C-source, N-source, P-source may serve as limiting nutrient.
 Final cell population depends on the concentration of the limiting nutrients, therefore the
growth rate of the microorganism depend on the rate at which new medium is fed into the
culture vessel.
 The growth of the new biomass is equivalent to the rate at which culture is being diluted.
 µ=D Steady state
 µ˃D Starvation
 µ<D Washout
 The relationship between the specific growth rate and the concentration of the growth
limiting substrates can be described by monod equation.
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Fig. Continuous culture
The system continuously monitors the culture density in the growth vessel and controls
the dilution rate to maintain the culture density at a constant rate. If the culture density
becomes too high the dilution rate is increased, and if it becomes too low the dilution rate
is decreased.
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What are the factors to be considered, in designing and constructing a fermentor
 The vessel must be capable of being operated aseptically for a number of days and should
be reliable in long term operations.
 Adequate aeration and agitation should be provided to meet the metabolic requirements
of microorganisms.
 Power consumption should be as low as possible.
 It must have a system of temperature control.
 It must have a system of pH control.
 Sampling facilities should be provided.
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 Evaporation losses from the fermenter should not be excessive.
 The vessel should be designed to require the minimal use of labour in operation cleaning,
harvesting and maintenance.
 The vessel should be suitable for a range of processes.
 It should have smooth internal surface.
 The cheapest material which enables satisfactory result to be achieved should be used.
 The vessel should be of similar geometry to both smaller and larger vessels in the pilot plant to
facilitate scale-up.
 There should be adequate service provision for industrial parts.
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Upstream process
• Three main areas:
• A) Producer microorganism
• This include processes for
• obtaining a suitable microorganism
• strain improvement to increase the productivity and yield
• maintenance of strain purity
• preparation of suitable inocullum
• B ) Fermentation media
• C) Fermentation Process
DOWN PROCESSING
• The processes that follows fermentation:
• A) Cell harvesting
• B) Cell disruption
• C) Product purification from cell extracts or the growth medium
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Write short notes chemical mutagens as methods for the improvement of microbial strains
for over production of desired protein products.
Answer
 Some chemical mutagens, such as nitrous acid and nitrosoguanidine work by causing
chemical modifications of purine and pyrimidine bases that alter their hydrogen bonding
properties.
 For example, nitrous acid converts cytosine to uracil which then forms hydrogen bonds
with adenine rather than guanine. These chemicals act on the non-dividing cell and
include nitrous acid, alkylating agents and nitrosoguanidine (NTG) (also known as
MNNG).
 Nitrous acid: This acid is rather harmless and the mutation can be easily performed by
adding 0.1 to 0.2 M of sodium nitrate to a suspension of the cells in an acid medium for
various times. The acid is neutralized after suitable intervals by the addition of
appropriate amounts of sodium hydroxide. The cells are plated out subsequently.
 Alkylating agents: These are compounds with one or more alkyl groups which can be
transferred to DNA or other molecules. Many of them are known but the following have
been routinely used as mutagens: EMS (ethyl methane sulphonate), EES (ethyl ethane
sulphonate) and DES (Diethyl sulphonate).
 They are liquids and easy to handle. Cells are treated in solutions of about 1%
concentration and allowed to react from ¼ hour to ½ hour and thereafter are plated out.
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Experimentation has to be done to decide the amount of kill that will provide a suitable
amount of mutation.
 While some are carcinostatic (i.e., stop cancers), some are carcinogenic and must be
handled carefully.
 NTG – nitrosoguanidine: also known as M-methyl-N-nitro-M-guanidine - MNNG: It is
one of the most potent mutagens known and must therefore should be handled with care.
Amounts ranging from 0.1 to 3.0 mg/ml have been used but for most mutations the lower
quantity is used.
 It is reported to induce mutation in closely linked genes. It is widely used in industrial
microbiology.
 Nitrogen mustards: The most commonly used of this group of compounds is methyl-bis
(Beta-chlorethyl) amine also referred to as ‗HN2‗. Nitrogen mustards were used for
chemical warfare in World War I. Other members of the group are ‗HN,‗ ‗HN1‗, or
‗HN3‗ from the wartime code name for mustard gas, H.
 The number after the H denotes the number of 2-chloroethyl groups which have replaced
the methyl groups in trimethylamine. A spore or cell suspension is made in HN2 (methyl-
bis [Beta-chloroethyl amine]) and after exposure to various concentrations for about 30
minutes each, the reaction is ended by a decontaminating solution containing 0.7 %
NaHCO3 and 0.6 % glycine.
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 The solution is then plated out for survivors. Between 0.05 and 0.1% HN2 solutions in
2% sodium bicarbonate solutions have been found satisfactory for Streptomyces.
 Sometimes the exposure time may be extended.
Base Analogues
 These are compounds which because they are similar to base nucleotides in composition
may be incorporated into a dividing DNA in place of the natural base. However, this
incorporation takes place only in special conditions.
 The best examples include 2-amino purine, a compound that resembles adenine, and 5-
bromouracil (5BU), a compound that resembles thymine. The base analogs, however, do
not have the hydrogen-bonding properties of the natural base.
 Base analogues are not useful as routine mutagens because suitable conditions for their
use may be difficult to achieve. For example, with BU, incorporation occurs only when
the organisms is starved of thymine.
Frame-Shift Mutations.
 Frameshift mutagens (also known as intercalating agents) Frameshift or intercalating
agents are planar three-ringed molecules that are about the same size as a nucleotide base
pair.
 During DNA replication, these compounds can insert or intercalate between adjacent
base pairs thus pushing the nucleotides far enough apart that an extra nucleotide is often
added to the growing chain during DNA replication.
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 A mutation of this sort changes all the amino acids downstream and is very likely to
create a nonfunctional product since it may differ greatly from the normal protein.
Furthermore, reading frames (i.e., the DNA base sequences) other than the correct one
often contain stop codons which will truncate the mutant protein prematurely.
 Dye acridine (proflavine) inserted between base pair, will cause a frame-shift and will
misread its genetic information.
 Acridines are among the best known of these mutagens, which cause a displacement or
shift in the sequence of the bases.
 Although strongly mutagenic for some bacteriophages, acridines have not been found
useful for bacteria.
The physical mutagens for improving microorganisms used in Molecular
 The properties of any microorganism depend on the sequence of the four nucleic acid
bases on its genome: adenine (A), thymine (T), cytosine (C), and guanine (G).
 The arrangement of these DNA bases dictates the distribution of genes and hence the
nature of proteins synthesized. A mutation can therefore be described as a change in the
sequence of the bases in DNA (or RNA, in RNA viruses).
 Ionizing radiations: X-rays, gamma rays, alpha-particles and fast neutrons are ionizing
radiations and have all been successfully used to induce mutation.
 Ionizing radiations are so called because they knock off the outer electrons in the atoms
of biological materials (including DNA) thereby causing ionization in the molecules of
DNA.
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 As a result, highly reactive radicals are produced and these cause changes in the DNA.
Some authors do not advise the use of ionizing radiations unless all other methods fail.
 Ultraviolet light: The mutagenic range of ultraviolet light lies between wave length 200
and 300 nm. ‗Low pressure‘ UV lamps used for mutagenesis emit most of their rays in
the 254 nm region.
 The suspension of cells or spores to be mutagenized is placed in a Petri dish 2-3 cm
below a 15 watt lamp and stirred either by a rocking mechanism or by a magnetic stirrer.
 The organisms are exposed for varying periods lasting from about 300 seconds to about
20 minutes depending on the sensitivity of the organisms.
 Since UV damage can be repaired by exposure to light in a process known as photo-
reactivation all manipulations should be conducted under a special light source such as 25
watt yellow or red bulbs.
 The main effect of ultraviolet light on DNA is the formation of covalent bonds between
adjacent pyrimidine (thymine and cytosine) bases.
 Thymine is mainly affected, and hence the major effect of UV light is thymine
dimerization, although it can also cause thymine-cytosin and cytosin-cytosin dimers.
 Dimerization causes a distortion of the DNA double strand and the ultimate effect is to
inhibit transcription and finally the organism dies.
 Some chemical mutagens, such as nitrous acid and nitrosoguanidine work by causing
chemical modifications of purine and pyrimidine bases that alter their hydrogen bonding
properties.
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 For example, nitrous acid converts cytosine to uracil which then forms hydrogen bonds
with adenine rather than guanine. These chemicals act on the non-dividing cell and
include nitrous acid, alkylating agents and nitrosoguanidine (NTG) (also known as
MNNG).
ISOLATION AND PURIFICATION OF NUCLEIC ACIDS
Introduction
Every gene manipulation procedure requires genetic material like DNA and RNA. Nucleic acids
occur naturally in association with proteins and lipoprotein organelles. The dissociation of a
nucleoprotein into nucleic acid and protein moieties and their subsequent separation, are the
essential steps in the isolation of all species of nucleic acids. Isolation of nucleic acids is
followed by quantitation of nucleic acids generally done by either spectrophotometric or by
using fluorescent dyes to determine the average concentrations and purity of DNA or RNA
present in a mixture. Isolating the genetic material (DNA) from cells (bacterial, viral, plant or
animal) involves three basic steps-
 Rupturing of cell membrane to release the cellular components and DNA
 Separation of the nucleic acids from other cellular components
 Purification of nucleic acids
Isolation and Purification of Genomic DNA
Genomic DNA is found in the nucleus of all living cells with the structure of double-stranded
DNA remaining unchanged (helical ribbon). The isolation of genomic DNA differs in animals
and plant cells. DNA isolation from plant cells is difficult due to the presence of cell wall, as
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compared to animal cells. The amount and purity of extracted DNA depends on the nature of the
cell.
The method of isolation of genomic DNA from a bacterium comprises following steps:
1. Bacterial culture growth and harvest.
2. Cell wall rupture and cell extract preparation.
3. DNA Purification from the cell extract.
4. Concentration of DNA solution.
Growth and harvest of bacterial culture
Bacterial cell culture is more convenient than any other microbe, as it requires only liquid
medium (broth) containing essential nutrients at optimal concentrations, for the growth and
division of bacterial cells. The bacterial cells are usually grown on a complex medium like Luria-
Bertani (LB), in which the medium composition is difficult to decipher. Later, the cells are
separated by centrifugation and resuspended in 1% or less of the initial culture volume.
Preparation of cell extract
Bacterial cell is surrounded by an additional layer called cell wall, apart from plasma membrane
with some species of E. coli comprising multilayered cell wall. The lysis of cell wall to release
the genetic material i.e. DNA can be achieved by following ways-
• Physical method by mechanical forces.
• Chemical method by metal chelating agents i.e. EDTA and surfactant i.e. SDS or enzyme (e.g.
lysozyme).
Lysozyme
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 Present in egg-white, salivary secretion and tears.
 catalyzes the breakdown of cell wall i.e. the peptidoglycan layer.
EDTA (Ethylene diamine tetra-acetic acid)
 A chelating agent necessary for destabilizing the integrity of cell wall.
 inhibits the cellular enzymes that degrade DNA.
SDS (Sodium dodecyl sulphate)
 It helps in removal of lipid molecules and denaturation of membrane proteins.
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Generally, a mixture of EDTA and lysozyme is used. Cell lysis is followed by centrifugation to
pellet down the cell wall fractions leaving a clear supernatant containing cell extract.
Purification of DNA
In addition to DNA, a cell extract contains significant quantities of protein and RNA which can
be further purified by following methods-: Organic extraction and enzymatic digestion for the
removal of contaminants. It involves the addition of a mixture of phenol and chloroform (1:1) to
the cell lysate for protein separation. The proteins aggregate as a white mass in between the
aqueous phase containing DNA and RNA, and the organic layer. Treatment of lysate with
pronase or protease, in addition to phenol/chloroform, ensures complete removal of proteins
from the extract. The RNA can be effectively removed by using Ribonuclease, an enzyme which
rapidly degrades RNA into its ribonucleotide subunits. Repeated phenol extraction is not
desirable, as it damages the DNA.
Using ion-exchange chromatography
This involves the separation of ions and polar molecules (proteins, small nucleotides and amino
acids) based on their charge. DNA carrying negative charge binds to the cationic resin or matrix
which can be eluted from the column by salt gradient. Gradual increase in salt concentration
detaches molecules from the resin one after another.
Concentration of DNA samples
Concentration of DNA can be done using ethanol along with salts such as sodium acetate,
potassium acetate etc. These salts provide metal ions like sodium ions (Na+), potassium ions
(K+) which help in aggregation and hence precipitation of DNA molecules.
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STEPS INVOLVED IN RECOMBINANT DNA TECHNOLOGY
1. Isolation of DNA - Genomic DNA, cDNA, Chemically Synthesis
2. Fragmentation of the DNA using the enzyme Restriction endonucleases
3. Isolation of the desired DNA fragment
4. Isolation of Vector - EColi Bactria (Plasmid PBR, PUC etc, Cosmid, Phage, YAC,
BAC etc)
5. Amplification of the gene of interest
6. Ligation of the DNA fragment into a suitable vector by the enzyme DNA ligases
7. Transfer of DNA into the host cell – transformation, transduction, electroporation,
liposome fusion, etc
8. Culturing the host cells on a suitable medium on a large scale
9. Extraction of the desired product
10. Downstream processing of the products
The basic steps of a gene cloning experiment are diagrammatically. At each of the above steps a
suitable strategy is employed depending upon the objectives of the cloning experiment, types of
vectors, host organism and available information about the gene (s) to be cloned. For example, a
gene may be isolated either from a cDNA (complementary DNA) library/ genomic library or it
may be chemically synthesized if the gene product is known.
a. Preparation of vector and insert DNA
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This is usually done by cleaving both vector and foreign DNA with a suitable RE to generate
complementary/compatible ends.
Figure: Recombinant DNA Technology
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b. Construction of recombinant DNA (rDNA)
The joining of foreign DNA to vector is done with the help of enzyme DNA ligase. This step is
also known as ligation. T4 phage encoded DNA ligase is most commonly used because of its
high efficiency of ligation as well as ability to join even the blunt ends. This DNA ligase is
obtained from E.coli that has been infected with T4 phage. The enzyme joins 5‘ P and 3‘ OH
ends of DNA molecules and requires ATP for this reaction.
STRATEGY FOR EFFICIENT LIGATION OF VECTOR AND INSERT DNA
To achieve good efficiency of ligation of foreign DNA into a vector and avoid high background
of non-recombinants, following strategies are used:
1. Remove 5' phosphate groups from the cut vector by alkaline phosphatase to prevent self-
ligation.
2. Sometimes the foreign DNA may get inserted in inverse direction when both the vector and
foreign DNA are cut with one RE. To avoid this problem, ends of the vector are generated
with two different REs. The ends of the insert are also obtained in the same way so that the
foreign DNA is ligated in only one direction.
3. If vector and insert DNA are cut with an enzyme which leaves blunt DNA ends, the
background of non-recombinant plasmids can be high. The problem can be solved by using
high concentrations of both DNAs (vector and insert) and of the DNA ligase enzyme.
Another way is to convert blunt ended molecules into those with cohesive ends. This can be
achieved by adding homopolymer tails or addition of linkers/adapters.
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Homopolymer Tailing
 A homopolymer is a polymer in which all the subunits are same e.g. a DNA strand made
up of many deoxy- guanosine residues i.e. poly (dG).
 Homopolymer tailing is done with the help of enzyme terminal deoxynucleotidyl
transferase which adds nucleotides to 3‘ OH termini of a double stranded DNA molecule.
 The enzyme has the property to add same type of nucleotides when the reaction is
carried out in the presence of only one type of deoxynucleoside triphosphate resulting in
formation of homopolymer tails.
 Complementary homopolymer tails are added to the vector and the DNA insert e.g. if
poly (G) tail is added to vector then poly C is added to the insert.
Linkers
 Linkers are short pieces of double stranded DNA, of known nucleotide sequence that are
blunt ended but have R.E. site in them. They are chemically synthesized.
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 Linkers are attached to the blunt ended DNA inserts with the help of enzyme DNA
ligase.
 These are then cut with a suitable R.E. to produce cohesive ends.
Adaptors
 Adaptors, like linkers are short synthetic oligonucleotides that have one blunt end and
one sticky end.
 The blunt end of the adaptor is joined to the DNA insert to produce DNA molecules with
sticky ends.
 These sticky ends have a known sequence which is complementary to the ends generated
by cutting the vector with a R.E. so that these can be joined to the vector.
 Also two different adaptors may be joined to the two ends of DNA insert for its ligation
in correct orientation.
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Transformation
It is the process by which plasmids (or other DNA) are introduced into a host cell. The
transformation of different host cells is described below:
i) Transformation of E.coli
The bacterial cells are made competent by incubation in the presence of divalent cations (usually
2+
Ca ) and a brief heat shock (42°C) is given which induces the E. coli cells to take up the foreign
DNA. The efficiency of transformation is calculated as the number of transformants/μg of
inputDNA. Alternatively transformation can also be done by Electroporation. Here the foreign
DNA and bacterial cells are mixed together and a brief pulse of high voltage is applied. This
increases permeability of the bacterial cells and allows uptake of foreign DNA.
A recombinant DNA constructed with phage vector can be introduced in E.coli either by
transfection or in vitro packaging.
Transfection
This process is same as transformation except that purified phage DNA i.e. recombinant phage
vector is used in place of plasmid for introduction into the host.
In vitro packaging
Recombinant DNA molecules constructed with lambda phage vectors are mixed in a test tube
with in vitro packaging extracts that contain phage head and tail structures. Assembly of the
phage particles occurs automatically in the test tube and mature assembled phages are allowed to
infect E.coli cells. This method is highly efficient for introduction of foreign DNA.
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TOOLS OF RECOMBINANT DNA TECHNOLOGY
Following basic tools are required for RDT:
1. Restriction endonucleases and other enzymes
2. Vector
3. Host Cell
Restriction endonucleases and other enzymes
The in vivo function of restriction enzymes is to protect bacteria from invasion by viruses. There
are three types of restriction endonucleases namely, type I, type II and type III. Out of these only
types II or class II enzymes are important for gene cloning. These are the enzymes that recognize
specific sequences in double stranded DNA and cleave within these sequences i.e. they are very
specific in their action.
E Escherichia (Genus)
Co coli (species)
R RY13 (strain)
1 First identified order in bacterium
First order in
identified bacterium
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USES OF RESTRICTION ENZYMES
RFLP analysis (Restriction Fragment Length Polymorphism)
DNA sequencing
DNA storage – libraries
Transformation
Large scale analysis – gene chips
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Other Enzymes used in RDT:
i) DNA ligase is used for joining DNA molecules.
ii) Alkaline phosphatase is used for dephosphorylation of the vector i.e. removal of 5‘
phosphate to avoid recircularization of the cut vector.
iii) S1 nuclease is used for cutting single stranded nucleic acids.
iv) Terminal transferase is used for adding homopolymer tails.
v) Reverse transcriptase is used for cDNA synthesis.
vi) DNA polymerases are used for DNA replication.
vii) RNase H is used for removal of RNA from RNA/DNA hybrid.
GENERAL FEATURES OF CLONING VECTORS AND HOST BACTERIA
Features of cloning vector
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1. Origin sequence (ori) required for replication.
2. Selectable trait that enables E. coli that carry the plasmid to be separated from E.
coli that do not (e.g., antibiotic resistance, grow cells on antibiotic; only those
cells with the antibiotic resistance grow in colony).
3. Unique restriction site such that an enzyme cuts the plasmid DNA in only one
place. A fragment of DNA cut with the same enzyme can then be inserted into
the plasmid restriction site.
4. Simple marker that allows you to distinguish plasmids that contain inserts from
those that do not (e.g., lacZ+ gene)
Host Cell (Competent Cells)
A good host should have the following properties:
1. It should be easy to transform.
2. It should not hinder replication of recombinant vector.
3. It should not have restriction and methylase activities.
4. It should be deficient in recombination function so that the introduced recombinant vector
is not altered.
5. It should be easy to grow.
6. The recombinant vector should be easily retrieved from the transformed host.
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Application of Recombinant DNA Technology
i. Gene Mapping
ii. Genetic disorder
iii. Monoclonal antibodies Productions
iv. Gene therapy
v. Gene fingerprinting
vi. Vaccine Production
vii. Pharmaceutical Products
POLYMERASE CHAIN REACTION
Reaction components
1. Nucleic acid template 2. Oligonucleotide Primers 3. Deoxynuc1eotide
Triphosphates
4. Taq Polymerase 5. Magnesium Chloride 6. Buffer
 There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done
on an automated cycler (Thermocycler), which can heat and cool the tubes with the
reaction mixture in a very short time.
 Denaturation at 94°C .The unknown DNA is heated to about 94°C, which causes the
DNA to denature and the paired strands to separate.
 Annealing at 54°C. A large excess of primers relative to the amount of DNA being
amplified is added and the reaction mixture cooled to allow double-strands to anneal;
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because of the large excess of primers, the DNA single strands will bind more to the
primers, instead of with each other.
 Extension at 72°C. This is the ideal working temperature for the polymerase. Primers that
are on positions with no exact match, get loose again (because of the higher temperature)
and do not give an extension of the fragment.
 The bases (complementary to the template) are coupled to the primer on the 3‘ side (the
polymerase adds dNTP‘s from 5‘ to 3‘, reading the template from 3‘ to 5‘side, bases are
added complementary to the template). The process of the amplification in the PCR
process is shown
Application of PCR
i. Diagnosis of pathogens
ii. Diagnosis of specific mutation
iii. In prenatal diagnosis
iv. DNA fingerprinting
v. In Research
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vi. IN molecular archaeology
vii Diagnosis of plant pathogens
GEL ELETROPHORESIS
 Gel electrophoresis acts as a molecular sieve.
 The gel is an aqueous matrix of agarose or polyacrylamide. DNA molecules are loaded
into a slot or well at one end of the gel.
 When an electric field is applied, the negatively charged DNA molecules migrate toward
the positive electrode.
 Shorter DNA molecules are less hindered by the agarose or polyacrylamide matrix and
migrate faster than longer DNA molecules, which must wind their way around obstacles
and through the pores in the gel matrix.
 Agarose gel electrophoresis is a method of gel electrophoresis used
in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed
population of macromolecules such as DNA , RNA or proteins in a matrix of agarose.
 Agarose is a natural linear polymer extracted from seaweed that forms a gel matrix by
hydrogen-bonding when heated in a buffer and allowed to cool.
 They are the most popular medium for the separation of moderate and large-sized nucleic
acids and have a wide range of separation.
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Principle of Agarose Gel Electrophoresis
 Gel electrophoresis separates DNA fragments by size in a solid support medium such as
an agarose gel. Sample (DNA) are pipetted into the sample wells, followed by the
application of an electric current at the anodal, negative end which causes the negatively-
charged DNA to migrate (electrophorese) towards the bottom (cathodal, positive) end.
 The rate of migration is proportional to size: smaller fragments move more quickly, and
wind up at the bottom of the gel.
 DNA is visualized by including in the gel an intercalating dye, ethidium bromide.
 DNA fragments take up the dye as they migrate through the gel.
 Illumination with ultraviolet light causes the intercalated dye to fluoresce.
Requirements/ Instrumentation of Agarose Gel Electrophoresis
The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively
simple and include:
1. An electrophoresis chamber and power supply
2. Gel casting trays, which are available in a variety of sizes and composed of
UVtransparent plastic. The open ends of the trays are closed with tape while the gel is
being cast, then removed prior to electrophoresis.
3. Sample combs, around which molten medium is poured to form sample wells in the gel.
4. Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).
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5. Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to
―fall‖ into the sample wells, and one or two tracking dyes, which migrate in the gel and
allow visual monitoring or how far the electrophoresis has proceeded.
6. Staining: DNA molecules are easily visualized under an ultraviolet lamp when
electrphoresed in the presence of the extrinsic fluor ethidium bromide. Alternatively,
nucleic acids can be stained after electrophoretic separation by soaking the gel in a
solution of ethidium bromide. When intercalated into doublestranded DNA, fluorescence
of this molecule increases greatly. It is also possible to detect DNA with the extrinsic
fluor 1-anilino 8-naphthalene sulphonate.
7. Transilluminator (an ultraviolet light box), which is used to visualize stained DNA in
gels.
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1. To prepare gel, agarose powder is mixed with electrophoresis buffer to the desired
concentration, and heated in a microwave oven to melt it.
The concentration of Agarose Gel
 The percentage of agarose used depends on the size of fragments to be resolved.
 The concentration of agarose is referred to as a percentage of agarose to volume of buffer
(w/v), and agarose gels are normally in the range of 0.2% to 3%.
 The lower the concentration of agarose, the faster the DNA fragments migrate.
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 In general, if the aim is to separate large DNA fragments, a low concentration of agarose
should be used, and if the aim is to separate small DNA fragments, a high concentration
of agarose is recommended.
2. Ethidium bromide is added to the gel (final concentration 0.5 ug/ml) to facilitate
visualization of DNA after electrophoresis.
3. After cooling the solution to about 60oC, it is poured into a casting tray containing a
sample comb and allowed to solidify at room temperature.
4. After the gel has solidified, the comb is removed, taking care not to rip the bottom of the
wells.
5. The gel, still in plastic tray, is inserted horizontally into the electrophoresis chamber and
is covered with buffer.
6. Samples containing DNA mixed with loading buffer are then pipetted into the sample
wells, the lid and power leads are placed on the apparatus, and a current is applied.
7. The current flow can be confirmed by observing bubbles coming off the electrodes.
8. DNA will migrate towards the positive electrode, which is usually colored red, in view of
its negative charge.
9. The distance DNA has migrated in the gel can be judged by visually monitoring
migration of the tracking dyes like bromophenol blue and xylene cyanol dyes.
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Applications of Agarose Gel Electrophoresis
Agarose gel electrophoresis is a routinely used method for separating proteins, DNA or RNA.
 Estimation of the size of DNA molecules
 Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
 Separation of restricted genomic DNA prior to Southern analysis, or of RNA prior to
Northern analysis.
 The agarose gel electrophoresis is widely employed to estimate the size of DNA
fragments after digesting with restriction enzymes, e.g. in restriction mapping of cloned
DNA.
 Agarose gel electrophoresis is commonly used to resolve circular DNA with different
supercoiling topology, and to resolve fragments that differ due to DNA synthesis.
 In addition to providing an excellent medium for fragment size analyses, agarose gels
allow purification of DNA fragments.
 Since purification of DNA fragments size separated in an agarose gel is necessary for a
number molecular techniques such as cloning, it is vital to be able to purify fragments of
interest from the gel.
Advantages of Agarose Gel Electrophoresis
 For most applications, only a single-component agarose is needed and no polymerization
catalysts are required. Therefore, agarose gels are simple and rapid to prepare.
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 The gel is easily poured, does not denature the samples.
 The samples can also be recovered.
Disadvantages of Agarose Gel Electrophoresis
 Gels can melt during electrophoresis.
 The buffer can become exhausted.
 Different forms of genetic material may run in unpredictable forms.
Give a detailed account on the Sanger and Coulson’s and Sanger method of gene
sequencing
Answer
 This procedure is performed by four reaction mixtures at the same time
 In all of these mixtures, DNA polymerase is used to make the complement of a particular
sequence
 It is primed by a fragment that contains the complementary sequence to a known
sequence
 What makes this method special is its use of specific ―terminators‖ – each reaction
contains a small amount of a 2‘-3‘ –dideoxy analog of one of the nucleotides (a different
nucleotide for each reaction mixture – remember, there are 4 reaction mixtures, so 1
mixture will have the adenine form of the dideoxy analog, another will have the guanine
form, and the other two will have thymine and cytosine)
 Normally, the polymerase adds a complementary base, then moves to the next nucleotide
and does the same

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 However, in addition to the regular nucleotides that the DNA polymerase usually add,
they can also add these 2‘-3‘ –dideoxy analogs, as long as the base is the same. For
example, if it needs to add an adenine to complement a thymine, it can also add a 2‘-3‘ –
dideoxy analog of adenine (but it can not add a dideoxy version of guanine to
complement thymine)
 However, if the dideoxy analog version of the nucleotide base is added instead of the
pure nucleotide base, the polymerase is no longer able to add a nucleotide after it (this
phenomenon is known as chain termination). The reason is that this dideoxy analog
version of the nucleotide base does not have the OH group at the 3‘ end, and thus,
polymerase can no longer add bases
 The concentration of this dideoxy analog is low enough though that this ―chain
termination‖ will only take place occasionally (ie most of the times polymerase will add
the regular nucleotide, but sometimes it will add this dideoxy version)
 Thus, different length fragments will be produced, but all of them will be terminated by a
dideoxy analog base, and as such, these fragments of different length will correspond to
the positions of its complement base on the regular
 Four sets (one for each base) of these chain terminated fragments then undergo
electrophoresis, and the base sequence of the new DNA is read from the autoradiogram
of the four lanes (remember, this is the new DNA – the sequence of the old DNA would
be the complement to this new DNA)
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 Fluorescent tags can also be used – it can either be attached to the priming fragment (a
different color for each of the four reaction mixtures), or to the dideoxy analog itself (a
different color for each of the four analogs)
 If added to the priming fragment, the four reactions are combined, then subjected to
electrophoresis, and the separated bands of the DNA are detected by their fluorescence as
they emerge from the gel
 If added to the dideoxy analog, only 1 reaction mixture is required
 Fluorescent labels are considered good because it eliminates the use of radioactive
reagents and can be readily automated.
Figure Sanger and Coulson Method of Sequencing
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CONSTRUCTION OF cDNA LIBRARY
Introduction
In higher eukaryotes, gene expression is tissue-specific. Only certain cell types show moderate to
high expression of a single gene or a group of genes. For example, the genes encoding globin
proteins are expressed only in erythrocyte precursor cells, called reticulocytes. Using this
information a target gene can be cloned by isolating the mRNA from a specific tissue. The
specific DNA sequences are synthesized as copies from mRNAs of a particular cell type, and
cloned into bacteriophage vectors. cDNA (complementary DNA) is produced from a fully
transcribed mRNA which contains only the expressed genes of an organism. Clones of such
DNA copies of mRNAs are called cDNA clones.
A cDNA library is a combination of cloned cDNA fragments constituting some portion of the
transcriptome of an organism which are inserted into a number of host cells. In eukaryotic cells,
the mRNA is spliced before translation into protein. The DNA synthesized from the spliced
mRNA doesn‘t have introns or non-coding regions of the gene. As a result, the protein under
expression can be sequenced from the DNA which is the main advantage of cDNA cloning over
genomic DNA cloning.
Construction of a cDNA Library
The construction of cDNA library involves following steps-
1. Isolation of mRNA
2. Synthesis of first and second strand of cDNA
3. Incorporation of cDNA into a vector
4. Cloning of cDNAs
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Isolation of mRNA
It involves the isolation of total mRNA from a cell type or tissue of interest. The amount of
desired mRNA can be increased by following ways-
• Chromatographic purification of mRNA using oligo-dT column, which retains mRNA
molecules, resulting in their enrichment.
• Spinning down mRNA by density gradient centrifugation.
• mRNA preparation from specialized cell types, e.g. developing seeds, chicken oviduct,
erythrocytes, β cells of pancreas etc.
The 3′ ends of eukaryotic mRNA consist of a string of 50 – 250 adenylate residues (poly A Tail)
which makes the separation easy from the much more prevalent rRNAs and tRNAs in a cell
extract using a column containing oligo-dTs tagged onto its matrix.
When a cell extract is passed through an oligo-dT column, the mRNAs bind to the column due to
the complementary base-pairing between poly (A) tail and oligo-dT. Other RNAs (ribosomal
RNAs and transfer RNAs) flow through as unbound fraction. The bound mRNAs can then be
eluted using a low-salt buffer.
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Fig. Isolation of mRNA using oligo-dT column chromatography
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Synthesis of first and second strand of cDNA
 mRNA being single-stranded cannot be cloned as such and is not a substrate for DNA
ligase. It is first converted into DNA before insertion into a suitable vector which can be
achieved using reverse transcriptase (RNA-dependent DNA polymerase or RTase)
obtained from avian myeloblastosis virus (AMV).
 A short oligo (dT) primer is annealed to the Poly (A) tail on the mRNA.
 Reverse transcriptase extends the 3´-end of the primer using mRNA molecule as a
template producing a cDNA: mRNA hybrid.
 The mRNA from the cDNA: mRNA hybrid can be removed by RNase H or Alkaline
hydrolysis to give a ss-cDNA molecule.
 No primer is required as the 3´end of this ss-cDNA serves as its own primer generating a
short hairpin loop at this end. This free 3´-OH is required for the synthesis of its
complementary strand.
 The single stranded (ss) cDNA is then converted into double stranded (ds) cDNA by
either RTase or E. coli DNA polymerase.
 The ds-cDNA can be trimmed with S1 nuclease to obtain blunt–ended ds-cDNA
molecule followed by addition of terminal transferase to tail the cDNA with C‘s and
ligation into a vector.
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Applications of cDNA libraries/cloning
 Discovery of novel genes.
 in vitro study of gene function by cloning full-length cDNA.
 Determination of alternative splicing in various cell types/tissues.
 They are commonly used for the removal of various non-coding regions from the library.
 Expression of eukaryotic genes in prokaryotes as they lack introns in their DNA and
therefore do not have any enzymes to cut it out in transcription process. Gene expression
required either for the detection of the clone or the polypeptide product may be the
primary objective of cloning.
 To study the expression of mRNA
Steps involved in the construction and Selection of Genomic Library.
Answer
 For construction of a genomic library, total DNA of the cell is isolated. Since DNA in all
cells of an organism is same, genomic libraries can be prepared from any cell of an
individual.
 Genes isolated from genomic libraries contain both coding (exons) as well as non-coding
(introns) sequences.
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 The DNA is then cut into small pieces with a restriction enzyme. Usually partial
digestion with a tetra cutter such as Sau3A or Mbo I is done to obtain overlapping
fragments of about 20 kb size DNA.
 Each piece of DNA is then joined to a vector or cloning vehicle.
 Vectors derived from λ phage are commonly used for preparation of genomic libraries.
 The recombinant DNA is then introduced into bacterial cells such as E. coli.
 The bacteria are plated on agar plates containing suitable medium on which they grow
and form colonies or plaques (when vector is a phage).
 Each colony/plaque represents a recombinant clone carrying a different piece of genomic
DNA.
 A collection of all these clones is called a genomic library. To isolate genes, genomic
library is screened with the help of probes.
 Genomic libraries are useful to study detailed structures of genes, to identify regulatory
regions, i.e. DNA sequences needed for correct expression of the gene.
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Figure. Construction of Genomic Library
Application of GENOMIC LIBRARIES
 To determine the complete genome sequence of a given organism.
 To study and generate transgenic animals through genetic engineering, serving as a
source of genomic sequence.
 To study the function of regulatory sequences in vitro.
 To study the genetic mutations.
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• Used for genome mapping, sequencing and the assembly of clone contigs
SCREENING OF DNA LIBRARIES

i. Inactivational selection using tetR and ampR
ii. Blue and White selection
iii. Direct selection
iv. Plaque and colony hybridization
v. Plaque lifting Method
Direct selection for the desired gene
 To avoid the growth of the untransformed bacterial cells, plasmid vectors are engineered
with selectable marker gene for resistance to the antibiotics.
 The media in which the transformed bacterial cells are grown is supplied with that
antibiotic whose resistance gene is present in the plasmid.
 Due to this only transformed cells show antibiotic resistance will grow in the media
supplied with antibiotic and untransformed cells cannot grow as they do not carry
antibiotic resistance gene.
 Transformed bacterial cells may contain either recombinant plasmid DNA (vector
containing foreign DNA insert) or non-recombinant plasmid DNA (self ligated vector
only).
 Both type of transformed bacterial cells will show antibiotic resistance and grow on the
agar media plate.
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Colony hybridization
 The recombinant bacterial colonies or phage plaques to be screened are transferred from the
culture plate on to a nitrocellulose filter paper by replica plating.
 The filter with colony replicas is treated with NaOH to lyse the cells/ phages and to denature
DNA.
0
 The filter is then baked at 80 C for two hours in vacuum oven to fix the DNA.
 The filter is allowed to hybridize with a labeled probe.
 The filter is washed to remove the unbound excess probe, dried and then subjected to
autoradiography if the probe is radioactively labeled.
 The washing conditions are kept stringent (high temperature and low salt concentration)
when the probe is homologous and non-stringent (lower temperature and high salt
concentration) in case of heterologous probe.
 Colonies which develop positive X-ray image are compared with master plate and picked up
and multiplied on the nutrient medium
Inactivational selection
 In this approach one of the genetic traits is disrupted by inserting foreign DNA Antibiotic
resistant genes act as good insertion in activation system.
 Plasmid Pbr322 contains two antibiotic resistant genes one for ampicllin (AmpR gene and
the other for tetracycline (tetR gene)
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 If the target DNA is inserted into tetR gene using BamH1 the poperty of resistance to
tetracycline will be lost such recombinant will test-sensitive when such recombinant
(containing target DNA tetR gene) are grown onto medium containing tetracycline
 They will not grow because their tetR gene have been inactivated but they are resistant to
ampicilling because ampR is functional on the other hand
 The self ligated recombinant will show resistance to tetracycline and ampicilline
therefore they would grow on the medium containing both antibiotics.
“Blue-White Screening,‖
Blue-white screening or ―lac selection‖ (also called α-complementation) can be used to
distinguish between recombinant transformants and non- recombinant transformants.
Bacterial colonies are allowed to grow on selective media containing antibiotic and X-gal (5-
bromo-4-chloro-indolyl-β-D-galactopyranoside), a colorless chromogenic compound.
 The plasmid vector contains a gene that codes for ampicillin resistance.
 The host bacterium cannot survive on a medium that contains ampicillin unless it has
taken up the plasmid.
 The plasmid vector has a second gene that codes for the enzyme Beta-galactosidase the
enzyme cleaves lactose into glucose and galactose).
 The Beta-galactosidase gene has several sites that can be cut by restriction enzymes.
 The plasmid vector and foreign DNA containing the gene of interest are digested with
the same restriction enzyme.
 . Fragments of the foreign DNA may insert into the Beta-galactosidase gene, rendering it
non-functional.
Ajao Page 66
 The recombinant plasmid is inserted into ampicillin-sensitive bacteria by transformation.
 The recombinant bacteria are grown on a special medium called X-gal, which (in addition
to normal growth nutrients) contains ampicillin, and a substrate for Beta-galactosidase.
 Only cells that contain the plasmid vector will grow on this medium, because they now
have the gene for ampicillin resistance.
 If foreign DNA was not successfully inserted into the Beta-galactosidase gene, the gene
will code for functional enzyme that will hydrolyze a component of the X-gal medium to
produce a blue colored compound;
 This will manifest in the form of blue colonies. (Non-recombinant plasmid).
 If foreign DNA was successfully inserted into the Beta-galactosidase gene, it will be
non-functional and colonies of these cells will appear white on the X-gal medium.
(Recombinant plasmid).
 Even with the successful production of recombinant cells, it is still not known if the
desired gene of interest was inserted into the Beta-galactosidase gene, or whether some
other DNA fragment was inserted. Further testing is therefore required.
Ajao Page 67
**** If the gene of interest codes for the production of an identifiable product, the bacterial
isolate only needs to be grown in culture and tested. Otherwise, the gene itself must be identified
in the bacterium via a procedure known as colony hybridization.
Ajao Page 68
Fig. Schematic process for screening libraries by Plaque hybridization
Ajao Page 69

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2019

Department of Biosciences and

Biotechnology, Kwara State

DR. A.T. AJAO

LECTURE NOTES ON MICROBIAL GENETICS AND

MOLECULAR BIOLOGY & PRINCIPLES OF BIOTECHNOLOGY

(MCB 311 & MCB 309)

(beetles) lepidopterans (tobaccobudworm, armyworm) & dipterans (flies, mosquitoes).

alkaline pH of the gut which solubilise the crystals.

swelling and lysis and death of the insect.

of cryIAb controls corn borer.

Nematode resistance in tobacco plants:

 RNA interference (RNAi) strategy is used to prevent this infestation.

of a specific mRNA (silencing) due to a complementary dsRNA molecule.

genetic elements (transposons) that replicate via an RNA intermediate.

thus, silenced the specific mRNA of nematode.

The major different types of applications of Environmental Biotechnology are:

toxic or the pollution effect caused.

2. Bioenergy like Biogas, biomass, fuels, and hydrogen

3. Bioremediation for cleaning up the hazardous substances into non-toxic compounds…

4. Biotransformation to change of the complex/toxic to simple non-toxic compounds.

functioning in the favor of the body.

 Pharmacogenomics is another genetically modified technique which is used to study the

genetic information of an individual.

 It analyzes the body's response to certain drugs. It is the combination of pharmaceuticals

according to the genetic information present in the individual.

in parents, sex and carrier screening.

the mutated sequences.

Importance of Genetically Engineered Crops

better yield) incorporated in them.

 Genetically modified crops have

 Reduced post-harvest losses

 Efficient mineral usage by plants

 enhanced nutritional value (e.g., Vitamin A rich rice)

State ideal features of microorganisms used in Biotechnology

The ideal features of microorganisms used in Biotechnology

b. The strain should be able to protect itself against contamination.

c. It must be stable and amenable for genetic manipulation.

f. It must be suitable for the type of process to be employed.

g. It must not react with the equipment

(i) Regulating the activity of the enzymes secreted by the organisms;

(iii) Selecting suitable producing strains from a natural population;

(iv) Manipulation of the existing genetic apparatus in a producing organism;

Purposes of strain improvement

ii. To change unused co-metabolites

iii. To improve the use of carbon and Nitrogen sources

Describe parasexuality, protoplast fussion and metabolic engineering as methods of strain

 In parasexual recombination of nuclei in hyphae from different strains fuse, resulting in

the formation of new genes.

with one another.

 Plays major role - variability and sexuality in fungi.

 Due to Anastomosis (fusion of hyphae)- Fusion is mostly intra-specific.

heterokaryotic mycelium. eg- development of heterokaryon in basidiomycota.

 Parasexuality is important in those fungi such as Penicillium chrysogenum and

Aspergiluss niger in which no sexual cycles have been observed.

as phenoxy methyl penicillin, citric acid, and gluconic acid.

are unstable and tend to revert to their lower-yielding wild-type parents.

the action of wall degrading enzymes in isotonic solutions.

 It is a versatile tool for genetic manipulation and breeding in industrial microorganisms.

remove cell wall. Protoplast fusion is a physical phenomenon.