Genetic Engeneering .... ST ... (All)

Wollo University
College of Natural Science

Department of Biotechnology
Module Name: Genomic Science and Bioinformatics

Module No.: 08
Module Code: Biot-M3081
Course Title: Genetic Engineering
Course Code: Biot-M3081 e r :
Year : 3rd a c h
T e .
Semester: 1 st
r se D
u i e
Chr/ECTS: 3/5 Co an
Chanie D. .... Dep't of Biotechnology ..........
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(Ass’t Prof.)
Chanie D. .... Dep't of Biotechnology .......... 2

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Chapter One
1. Introduction and History to Genetic
Engineering
1.1. Introduction:
 Biotechnology can be defined as “the use of living
organisms, cells or cellular components for the
production of compounds or precise genetic
improvement of living things for the benefit of man”.
 It is truly multidisciplinary in nature and it
encompasses several disciplines of basic sciences
and engineering.
o Microbiology, Biochemistry, Chemistry,
Genetics, Molecular biology, Immunology and
Physiology……… .
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 Biotechnology developmental stages grouped as;
I. Ancient Biotechnology: early history as related to
food and shelter; Includes domestication
II. Classical Biotechnology: built on ancient
biotechnology; Fermentation promoted food
production, and medicine
III. Modern biotechnology: manipulates genetic
information in organism, Genetic engineering
 Now a day, Biotechnology has five streams;

o Agricultural Biotechnology
o Industrial “
o Medical “
o Environmental “
o Genomics
Chanie D. ....and Bioinformatics
Dep't of Biotechnology .......... “ 5
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 The main distinction between a living and non-
living entity is the ability to replicate and
reproduce similar offsprings.
 Nucleic acid molecules (DNA and RNA) present
in a living organism acts as a genetic template to
pass the hereditary information from one
generation to the next.
 Nucleic acid molecules are organized as genes
which code for a particular phenotype via
 specific proteins and the expression of a gene is
regulated by both external and internal factors
which aid the developmental process of an
organism.

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Therefore,
 Gene is the region of DNA (or RNA in case of virus)
that controls a discrete hereditary characteristic,
usually corresponding to a single protein or RNA.
 Includes the entire functional unit, encompassing
coding (exons) and non-coding sequences (introns
and regulatory sequences).
Exons and introns which represent the
coding and non-coding regions are present
in a eukaryotic gene.
Introns are absent in prokaryotes.
The introns are removed by splicing and exons
are translated to tendems to yield functional
polypeptide. ………… post translation.
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 DNA ….. Genes …….. RNA …….. Proteins
 Within the cell the DNA performs two tasks:
Act as information repository
Pass on the information to the next generation
Mammalian red blood cells (RBCs)
discard nucleus during developmental
process and, thus lacks DNA in mature
state.
 Genes transcribed into RNA (mRNA, tRNA, rRNA),
then translated to protein.
 Proteins act as enzyme, hormone, receptors,
signaling molecules.

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 Genome is the complete set of genetic information of
a cell or an organism; in particular, the complete
sequence of DNA/RNA that carries this information.
 Depending on its localization, genome may be
nuclear or organellar /cytoplasm genome.
 Genome size of organisms differs significantly
between different species and governs the size and
complexity of organisms.
 Therefore, Genome contains genes

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 Cells are functional and structural unit of life.
 There are three main cell components;
o Cell membrane
o Cytoplasm
o Nucleus
 With in these cell components there are four major
Biomolecules and secondary metabolite products.
Chem’l ele’t …. Micro biomol. Macro biomol
 C, H, O, N, P …… Amino acids ….. Protein
 C, H, O ……… Fatty acids .……. Lipid
 C, H, O ……. Monosaccharide …. Carbohydrate
 C, H, O, N, P ….. Nucleotide ….. Nucleic acid
 There are linkage forces (covalent and non-covalent) b/n
those molecules Chanie D. .... Dep't of Biotechnology .......... 13
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1.2. History of Genetic Engineering
 There is a rich history of gene editing which shows
an industry determined to aid humanity through the
study of genetics.
 Understanding the genome editing history is
incredibly important to understanding the current
state of the field.
 Some important events include the discovery of the
o double helix,
o recombinant DNA (rDNA),
o human cancer therapies,
o the invention of CRISPR
(Clustered Regularly Interspaced Short Palindromic Repeats)
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Year Events
......... ……………………..
1953 Discovery of double helix
1958 DNA is made in a Test tube for the first time
1960 Discovering and Linking DNA
1962 Jellyfish Protein Turns Into a Tool to Observe Invisible
Cellular Processes
1967 DNA Ligation Links DNA Fragments Together
1968 Discovery of Restriction Enzymes
1970 Purification of Type II Restriction Enzymes
Gene Splicing Experiment Paves the Way for
1971 Recombinant DNA (rDNA)
Type II Restriction Enzymes Used for Mapping DNA
1972 Recombinant DNA (rDNA) is Created
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Year Events
1974 National Academy Moratorium on Genetic
Engineering Experiments
1975 Hybridoma Technology Revolutionizes Diagnostics
1981 The First Transgenic Animal
1982 First Genetically Engineered Human Drug
eg.Synthetic Insulin
1983 The Development of the Polymerase Chain Reaction
(PCR)
1985 Discovery of Zinc Finger Nuclease (ZFN)
1986 First Recombinant Vaccine for Humans is Approved
1988 The First Bt Corn Appears in Fields
1993 Discovery of the Principles of CRISPR
1994 A Tomato Engineered to Stay Ripe is Brought to
Market Chanie D. .... Dep't of Biotechnology .......... 21
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Year Events
1996 The Cloning of Dolly the Sheep
1999 History of Genetic Engineering in Humans is Made
when the First Human Chromosome is Sequenced
2001 The First Gene-Targeted Drug Therapy is Approved
2003 Sale of the Glo-Fish as a Pet for the Home
FDA Approval of the First Preventative Cancer Vaccine
2006
First Induced Pluripotent Stem Cells (iPSCs)
2010 The World's First Synthetic Life Form
2011 Discovery of TALENs (Transcription activator-like effector
nucleases)
2012 Discovery of CRISPR Genome Engineering Tool
2013 Showed CRISPR Utility in Eukaryotic Cells
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Year Events
2014 Identifying the Possibility of a Gene Drive
First GMO Salmon Sold in Canadian Markets
2015
A Human Embryo is Edited with CRISPR
2017 First CAR T (Chimeric antigen receptor) Therapy for Cancer is
Approved
2018 First Human Trials for CRISPR are Approved
2019 Prime Editing Makes Single Stranded Cuts a

Possibility
etc Etc.

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1.3. General Processes in Gene
Manipulation
 The basis of molecular genetics is the process of
genetic recombination, the breakage and re-union of
DNA molecules (RE & ligages)
 which is the fundamental importance to all living
organisms as a mechanism for adaptation and
variation.
 Therefore, Genetic Engineering is;
o defined as Isolation, introduction, expression of
foreign DNA in other organisms.
o used to refer the manipulation of existing genes
with the new or foreign genes isolated from other
organisms. Chanie D. .... Dep't of Biotechnology .......... 24
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 In other words it is the manipulation of Genetic
architecture of an organism using recombinant DNA
technology
 The general approach of genetic engineering
includes; (eg. plant)
 Select both the host organisms and the
gene of interest
 Isolation of gene of interest
 formation of recombinant gene using vector (if)
 Introduction of gene of interest into the
cells of concerned species /host/
 Integration of this gene into the nuclear or
organellar genomes
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of the
D. .... Dep't of Biotechnology .......... cells of the host
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 Expression of transferred gene in new genetic
background
 Regeneration of whole plant/ from GM cells/
 Finally transmission of transferred gene to sexual
progeny of these plants.
 The development of trasgenecies involves the following
important components……. Requirements…..
o Gene of interest of foreign gene
o Gene transfer system or vector
o Restriction enzyme
o Ligase enzyme
o Host
o Marker gene
o Efficient tissue culture technology /skill/
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1.4. Vectors in Recombinant DNA Technology
2.1. Introduction:
 Vector is a DNA molecule used for carrying an
exogenous DNA into a host organism and facilitates
stable integration and replication inside the host
system.
 Molecular cloning involves series of sequential steps
which includes;
o Choice of host organism and cloning vector
o Restriction digestion of DNA fragments both target
DNA and vector,
o Creation of recombinant DNA using ligase
o Introduction of rDNA into a cloning host organism for
multiplication.
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o Selection of organisms containing vector
sequences
o Screening for clones with desired DNA inserts
and biological properties
 In general, vectors should have following characteristics;
 Capable of replicating inside the host.
 Have compatible restriction site for insertion of
DNA molecule (insert).
 Capable of autonomous replication inside the
host (ori site).
 Smaller in size and able to incorporate larger
insert size.
 Have a selectable marker for selection
 Have screenable markers for
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of
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Plasmids
 Plasmids are naturally occurring extra chromosomal
double-stranded circular DNA molecules which can
autonomously replicate inside bacterial cells.
Plasmids range in size from about 1.0 kb to over
250 kb.
 Plasmids encode only few proteins required for their
own replication (replication proteins) and these proteins
encoding genes are located very close to the ori. and all
the other proteins required for replication,
e.g. DNA polymerases, DNA ligase, helicase, etc. are provided
by the host cell.
 Thus, only a small region surrounding the ori site is
required for replication.
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 Some of the phenotypes which the naturally
occurring plasmids confer on their host cells:
Antibiotic resistance
 Antibiotic production
 Degradation of aromatic compounds
 Haemolysin production
 Sugar fermentation
 Enterotoxin production
 Heavy metal resistance
 Bacteriocin production
 Induction of plant tumors
Hydrogen sulphide production
etc Chanie D. .... Dep't of Biotechnology .......... 31
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 Most plasmids exist as double-stranded circular
DNA molecules.
However, the inter-conversion of super coiled, relaxed
covalently closed circular DNA and open circular DNA
is possible.
 Not all plasmids exist as circular molecules, but
linear plasmids have been found in a variety of
bacteria.
 Therefore, Plasmids may be;
 Single stranded
 Double stranded
 Open circular
 Closed circular
 Linear
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Types of Plasmids
 Depending on the phenotype, plasmids are dividing
into the following six types;
1. Resistance (R) plasmid:
o carry genes which give resistance to the bacteria
from chemical agents (antibacterial agents).
o R plasmids are very important in clinical
microbiology in the treatment of bacterial
infections.
eg .RP4 plasmid, in Pseudomonas
2. Fertility (F) plasmid:
 are conjugative plasmid found in F+ bacterium with
higher frequency of conjugation.
 F plasmid carries transfer gene (tra) and has the
ability to form Conjugation Bridge (F pilus) with F−
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3. Col Plasmids:
have genes that code for colicins, proteins that
kill other bacteria.
eg. ColE1 of E.coli
4. Degradative Plasmids:
allow the host bacterium to metabolize unusual
molecules such as toluene and salicylic acid.
eg. TOL of Pseudomonas putida
5.Virulence Plasmids:
confer pathogenicity on the host bacterium.
eg: Ti plasmids of Agrobacterium
tumefaciens, which induce
crown gall disease on
dicotyledonous plants.
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6. Cryptic Plasmids:
do not have any apparent effect on the phenotype
of the cell harboring them.
They just code for enzymes required for their
replication and maintenance in the host cell.
 Based on the origin or source of plasmids, they have
been divided into two major classes: such as natural and
artificial.
i. Natural plasmids:
 They occur naturally in prokaryotes or
eukaryotes. eg: ColE1
ii. Artificial plasmids:
 They are constructed in-vitro by re-combining
selected segments of two or more other plasmids
(natural or artificial). eg: pBR322.
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Artificial Plasmids:
 Naturally occurring plasmids has several
limitations;
eg. some are stringent and not relaxed
(pSC101), some has poor marker genes
(ColE1), and some are too large (RSF2124).
To overcome the limitations of natural vectors,

artificial plasmid are designed by combining
different elements from diverse sources.
 Artificial plasmid vectors are classified into two
broad types based on their use:
 Cloning vector
Expression vector
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a. Cloning Vectors:
 A cloning vector is defined as a vector used for
replication of a cloned DNA fragment in a host cell.
 These vectors are frequently engineered to contain
“ori”- origin of replication sites particular to the host
organism.
 Examples of commonly used cloning vectors are:
pUC18, pUC19, pBluescript vectors etc
 Important features of a cloning vector used to carry DNA
molecules are as follows;
Stability in host cell:
o Vectors should be stabile in host cell after introduction
and should not get lost in subsequent generations.
o This permits replication of vectors producing large
copies of gene of....interest.
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 Ability to control their own replication:
o This property enables them to multiply and exist in
high copy number.
 Small size:
o Ideal vector should be less than or equal to 10kb.
o The small size is essential for easy introduction in cell
by transformation, transduction and electroporation.
 Multiple cloning sites:
o This property permits the insertion of gene of interest
and plasmid re-circularization.
 Should not be transferred by conjugation:
o This property of vector molecule prevents
recombinant DNA to escape to natural population of
bacteria.
 Selectable maker gene:
o Vector molecules should have some detectable traits.
o These traits
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Types of Cloning Vectors
 Cloning vectors extensively used in molecular cloning
experiments can be considered under following types:
o plasmid ,
o phage vector and
o cosmid.
 Different vectors have various
 insert size and
 in mode of replication inside the host.
 Eg. mammalian genes are usually too large
(~100 kb) and
 Thus, suffer from restrictions in complete inclusion
with the conventional cloning vectors having limited
insert size. Chanie D. .... Dep't of Biotechnology .......... 39
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 Vectors engineered more recently, known as artificial
chromosomes, have overcome this problem by
mimicking the properties of host cell chromosomes.
 They have much larger insert size than other
vectors.
eg;
o Bacterial Artificial chromosome (BAC)
75-300 kb
o Yeast Artificial chromosome (YAC)
100-1000 kb
o Mammalian Artificial Chromosome (MAC)
100 kb - 1Mb

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Examples of Cloning Vector
1. pBR322:

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2. pUC plasmids:

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Expression Vector
 A vector used for expression of a cloned DNA
fragment in a host cell is called as an expression
vector.
 These vectors are frequently engineered to contain
regulatory sequences that act as promoter and/or
enhancer regions and
 lead to efficient transcription of the insert
gene.
 Commonly used expression vector series are;
 pET vectors,
 pBAD vectors etc.
 For an expression vector following features are
essential; Chanie D. .... Dep't of Biotechnology ..........
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o Promoter:
 Promoter is a sequence which is recognized by
sigma subunit of RNA polymerase which is required
for initiation of transcription of gene of interest.
o Terminator:
 It is a DNA element present at the end of a gene
where transcription of gene ends.
 Terminator is short nucleotide sequences which
can base pair with itself to form hair pin loop.
o Ribosome binding site:
o It is a short nucleotide sequence recognized by the
ribosome as the point at which it should attach to
the messenger molecule.
o The initiation codon of the gene is always a few
nucleotides downstream of this.
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Specialized Expression Vectors
 In molecular biology, vectors are generally designed
for cloning a foreign gene into a host genome to
produce proteins which are not produced by host.
 Apart from these applications, different specialized
vectors have been constructed to achieve different
application in genetic and molecular biology studies
and are termed as specialized vectors.
 Some of the applications of specialized vectors are;
1. Promoter Probe Vectors:
 Specialized vectors used for identification of
efficient promoter region in a DNA segment are
termed promoter probe vectors.

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 Promoter-less reported genes (lacZ, GFP etc) are
used for construction of promoter probe vectors.
The expression of the reporter genes can be

monitored and quantified easily using various
biochemical or fluorescent techniques.
Some of the widely used promoter probe vectors
families are:
o pOT (eg. pRU1161, pRU1097 etc) and
o pJP2 (eg. pRU1156, pRU1157 etc).
 pOT vectors have higher copy number, but lower

stability as compared to pJP2 vectors.
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2. Gene Fusion Vectors:
 Fusion of one gene to another gene in order to
produce a fusion protein is widely used in molecular
biology studies.
 Fusion proteins are generated by cloning two or more
target genes with a reporter gene ( His-tag, gfp, rfp, lacZ
etc) by using gene fusion vectors.
 Fusion proteins may provide improved properties like;
o easy isolation and purification of target protein
(His-tag)
o easy monitoring of gene expression level (GFP,
RFP, lacZ),
o intracellular protein localization studies (GFP,
RFP, LUC)Chanie
etc.D. .... Dep't of Biotechnology .......... 47
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Group Discussion
What are plasmids?
 What are the important features of
 Cloning vectors
 Expression vectors
What was/were the need of
constructing;
o Artificial plasmid
o Artificial chromosome
o Specialized expression vectors
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Viral Vectors
o Virus particles has been modified to use as a
carrier of nucleic acid into a cell, termed as viral
vectors.
o Viral vectors are highly efficient in transferring
target DNA/RNA segment to the host cells with high
specificity.
o Viral vectors have wide application in gene therapy
and targeted drug delivery systems.
o Commonly used viral vectors are;
 Adenovirus, retrovirus, lentivirus, Adeno
associated virus (AAV), Herpes simplex
virus (HSV) etc.
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i. Simian Virus 40 (SV40)
ii. Baculoviral Vectors

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Yeast Vector System
 Cloning and expression of a gene using yeast
system has several advantages over E.coli system
due to presence of eukaryotic post-translational
modification machinery.
 Expression of complex proteins with proper
modification and folding can be achieved by yeast
eukaryotic system.
 Different types of yeast vector include;
o YIp (yeast integrative plasmid),
o YEp (yeast episomal plasmid),
o YRp (yeast replicating plasmid),
o YCp (yeast centromere plasmid) etc.
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Types, Biology and Salient Features of Vectors in
Recombinant DNA Technology
o In recombinant DNA technology, vectors are used as
carrier of foreign DNA into the host organism.
o Apart from bacterial plasmids, several other
modified vectors are constructed using molecular
tools.
o These “hybrid” vectors are designed by combining
different components from various origins.
 bacteriophage,
 F plasmid etc
o to create a capacity to load larger insert size and
higher transfection efficiency.
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Phage Vectors:
To insert DNA fragments of more than 10 kb,
normally plasmids are not the suitable vehicles,
As large inserts may trigger plasmid
rearrangement or affect plasmid replication.
This leads to development of a new class of
vectors based on bacteriophages.
Amongst various bacteriophages available such
as;
 λ,
T4,
 T5, and
T7 phages;
the λ phage gained favorable attention due to 53its
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A. λ phage:
 Bacteriophage λ contains ~49kb of DNA and has
a very efficient mechanism for delivering its
genome into a bacterium.
 Two key features contribute to its utility as a
vector to clone larger DNA fragments:
1. One-third λ genome is non-essential and could be
replaced with foreign DNA.
Approximately 24.6kb of λ genome can be deleted,
hence maximum insert size could be up to 26 kb.
2. Packing of DNA in phage could only take place if the
size is between 40 and 52 kb long, a constraint that
can be used to ensure packaging.
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Two types of vector have been developed using λ
genome;
1). Insertion vectors:
o Foreign DNA sequence is inserted into the λ genome
without any significant change of the wild type genome.
o Smaller insert size (up to ~10kb).
o They may contain a multiple cloning site inserted in lacZ
system for screening of recombinant bacterial colonies.
o Can be used to clone smaller DNA molecule.
Eg. λ ZAP, λ gt, etc.

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2). Replacement Vectors:
 Full length λ molecule having two identical restriction
sites flanked by “stuffer fragment”.
 Stuffer fragment is replaced by foreign DNA during
restriction cloning.
 The vector without the foreign insert cannot be
packaged due to the size limitation (smaller than the
required).
 Insert size ranges between 10-23 kb.
Eg. λ EMBL 3, λ EMBL 4, λ DASH etc.

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Features of λ Phage Vectors:
o The λ genome is linear, but in the ends has 12-
nucleotides overhangs, termed as cos (cohesive end)
sites, which are complementary to each other.
o A λ cloning vector can be circularized using cos site
which can be manipulated and replicated inside E. coli
via the process of transfection.
B. M13 Phage Vectors:
 M13 phage is filamentous phage that infects E. coli via
F-pilus.
 The genome is a single stranded circular DNA of size
~6.4kb surrounded by a proteinaceous coat.
 The DNA strand present in phage is called plus (+)
strand.
 After entering to E. coli host, it converts into double
stranded DNA molecule called replicative form (RF)57by
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 M13 phage as cloning vector can be obtained in both single
stranded as well as double stranded form.
 Replicative form double stranded vector are modified and

replicated inside E. coli host similar to a plasmid vector.
 Single stranded vectors can be isolated by collecting M13
phage.
 M13 vectors have useful application in following areas;
o DNA sequencing
o Mutagenesis study
o probe generation
o Phage display

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Cosmid:
 A cosmid, first described by Collins and Hohn in
1978, is a type of hybrid plasmid with a bacterial
“ori” sequence and a “cos” sequences derived
from the lamda phage. …. (plasmid + lemda phage)
 Cos site is the sequence required by a DNA molecule
in order to be recognized as a ‘λ genome' by the
proteins that package DNA into λ phage particles.
 Cosmid DNA containing particles are as transmittable
as real λ phages, but once inside the cell, the cosmid
cannot control synthesis of new phage particles and
instead replicates as a plasmid.
 They frequently also contain a gene for selection such as
antibiotic resistance.
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 Cosmids are widely exploited to build genomic libraries.
 The upper limit for the length of the cloned DNA is set by the
space available within the λ phage particle.
New DNA insert of size up to 44 kb can be inserted
before the packaging of the λ phage particle is
reached.
Limitation of Cosmid
 Slower replication
 Higher frequency of recombination inside bacterial host.
 Unstable inside E.coli host and thus easy to lose vector.
eg. pJB8:
 is 5.4 kb in size and carries the ampicillin-resistance gene
(ampR),
 a segment of λ DNA containing the cos site, and
 an Escherichia coli origin
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Biotechnology .......... (ori). 60
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Fosmid:
 Fosmids are similar to cosmids, however they
are primarily based on bacterial F-plasmid.
Simon and co-workers, in the year 1992, first
developed F-factor based vector named as pFOS
for stable propagation of cosmid sized human
genomic DNA inserts.
They carry the F plasmid origin of replication and
a λ cos site and Fosmids can carry up to 40 kb of
insert DNA.
The cloning vector is limited, as a host ( usually E.
coli ) can only contain one fosmid molecule.
Low copy number offers higher stability as
compared to high copy number cosmids.
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 Fosmids have high structural stability and have been
found to maintain human DNA effectively even after
100 generations of growth.
 It is ideal to use a fosmid vectors for constructing
genomic and meta-genomic libraries.
 Fosmids contain several functional elements;
o OriT (Origin of Transfer): The sequence which
marks the starting point of conjugative transfer.
o OriV (Origin of Replication): The sequence starting
with which the plasmid-DNA will be replicated in the
recipient cell.
o tra-region (transfer genes): Genes coding the F-
Pilus and DNA transfer process.
o IS (Insertion Elements): so-called "selfish genes"
(sequence fragments
Chanie D. .... Dep't ofwhich
Biotechnology can
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Phagemid:
M13 vectors is important vector, but it has
limitation with respect to size of DNA.
So to overcome this limitation, phagemid
vectors were developed by combining a part of
the M13 genome with plasmid DNA.
They contain an origin of replication (ori) for
double stranded replication inside E. coli host,
As well as an “f1 ori” to enable single stranded
replication and packaging into phage particles.
The components present in a phagemid vector
are:
o Origin of replication (ori) of a plasmid.
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o Intergenic region (IG region) which contains the
packaging signal for the phage particle and
also has replication origin inside phage.
o A gene encoding phage coat protein.
o A selection marker.
o Restriction enzyme recognition sites.

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Artificial Chromosomes
 Artificial chromosomes are DNA molecules
assembled in vitro from defined constituents that
can function like natural chromosomes.
 Types of artificial chromosomes:
o BACs: Bacterial artificial chromosomes
o YACs: Yeast artificial chromosomes
o MACs: Mammalian artificial chromosomes
o HACs: Human artificial chromosomes
o PACs: P1-derived artificial chromosomes

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o In general vectors may be;
 Prokaryotic cloning vector
• plasmid
 Bacteriophage vectors …………. Viral vectors
• Lamda phage
• M13 bacteriophage
Hybrid Vectors
• Cosmid
• Phagemid
• Fosmid
Eukaryotic cloning vectors
• Yeast derived plasmid vectors,
• Yeast episomal plasmids,
• YAC,
• Yeast integrative plasmids,
• Yeast replicative plasmids,
• Plant Chanie
viruses
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D. .... Dep't of Biotechnology ..........
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1.5. Cloning Host
 Molecular cloning is a set of experimental methods in
molecular biology that are used to assemble
recombinant DNA molecules and to direct their
replication within host organisms.
 It generally uses DNA sequences from two different
organisms:
 the species that is the source of the DNA to be
cloned, and …………… Donor host
 the species that will serve as the living host for
replication of the recombinant DNA. …Recipient host
 The bacterium Escherichia coli fulfils its used widely in
many cloning protocols.
 In addition to E. coli, other bacteria may be used as
hosts for gene cloning, including species of Bacillus,
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 One disadvantage of using a prokaryotic host is that
it lacks the membrane bound nucleus (and other
organelles), but found in eukaryotic cells.
This means that certain eukaryotic genes may not
function in a prokaryotic host cell as they would in
their normal environment,
 Which can hamper their isolation by selection
mechanisms that depend on gene expression.
If the production of a eukaryotic protein is the desired
outcome of a cloning experiment, it may not be easy to
ensure that a prokaryotic host produces a fully
functional protein.
 Eukaryotic cells range from microbes ( eg. yeast and
algae) to cells from complex multi-cellular organisms
(eg. plant and animal cells).
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 The method of introducing the recombinant DNA into
host cell depends on its type
 The process of transferring a DNA into a prokaryotic
host cell is called transformation and
 The process of transferring a DNA into a eukaryotic host
cell is called as transfection.

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Chapter Two
2. Enzymes in Genetic Engineering and Their
Mechanism of Action
 A recombinant DNA molecule is produced by joining
together two or more DNA segments usually originating
from different organisms.
 A recombinant DNA molecule is a vector into which the
desired DNA fragment has been inserted to enable its cloning in
an appropriate host.
 This is achieved by using specific enzymes for cutting the DNA
(restriction enzymes) into suitable fragments
 then for joining together the appropriate fragments by DNA
ligase.
 Different prokaryotic and Eukaryotic cells have been found to
contain different kinds
Chanie D. of
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 These includes;
Degrade /cutting/ DNA …… Nucleases
o Nuclease
 Endonuclease
 Exonuclease
o S1 nucleease
o DNA-ases
Join DNA fragments ………. Ligases
o DNA ligase
Modify end of DNA molecules …… Modifying E
o Alkaline phosphates
 Synthesis DNA ……………………….. Polymerase E
o DNA polymerase I
o Terminal transferase
o Reverse transcriptase
Topoisomerase Enzymes ………….... :
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DNA Degrade /Cutting/ Enzymes
a). Nucleases:
Nucleases are enzymes that degrade nucleic
acids
i.e. it hydrolysis /breaks down/ the polynucleotide
chain into its component nucleotides.
The nucleases are of two types.
o Endonuclease
o Exonuclease
 Endonucleases are enzymes that cleaves nucleic acids
at internal sites. eg. EcoR I, Hind III, BamH I etc
 When the endonucleases cut the DNA molecules only
on its specific site, which is a restriction sequences
(or), is called restriction endonucleases.
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Historically;
 In 1970 the first restriction endonuclease enzyme Hind
II was isolated.
 For the subsequent discovery and characterization of
numerous restriction endonucleases, in 1978 Daniel
Nathans, Werner Arber, and Hamilton O. Smith
awarded for Nobel Prize for Physiology or Medicine.
 Since then, restriction enzymes have been used as an
essential tool in recombinant DNA technology.
 These are popularly called
o Molecular knives (or)
o Molecular scissors (or)
o Molecular scalpels (or)
o Biological scissors.WU 76
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Nomenclature of Restriction Enzymes
o Restriction endonucleases are named according to the
organism in which they were discovered, using a
system of letters and numbers.
o The Roman numerals are used to identify specific
enzymes from bacteria that contain multiple restriction
enzymes indicating the order in which restriction
enzymes were discovered in a particular strain.
o A system based on the proposals of Smith and Nathans
has been followed, which includes.
 The first letter of this code is derived from first letter of
genus name.
 The second and third letters are from the species
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If a particular strain has more than one restriction
enzyme, these will be identified by sequential
roman numbers I, II, III, etc.
 Eg.
 EcoRI for the first enzyme of Escherichia coli and
serotype R;
 H. influenzae strain ‘d’ are named Hind II, Hind III

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Classification of Restriction Endonucleases
 There are three major classes of restriction
endonucleases based on;
o their composition
o types of sequences recognized /restriction site/,
o nature of the cut made in the DNA, and
o enzyme structure:
o Co-factor requirements
 These are;
o Type I restriction enzymes
o Type II ; ;
o Type III ; ;
Type I Restriction Enzymes:
 These enzymes have both restriction and modification
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 Restriction depends upon the methylation status of the
target DNA.
 Cleavage occurs approximately 1000 bp away from the
recognition site.
 The recognition site is asymmetrical and is composed of two
specific portions in which one portion contain 3-4 nucleotides
while another portion contain 4-5 nucleotides and
 Both the parts are separated by a non-specific spacer of
about 6-8 nucleotides.
 They require S-adenosylmethionine (SAM), ATP, and
magnesium ions (Mg2+) for activity.
 These enzymes are composed of mainly three subunits,
• specificity subunit that determines the DNA recognition
site,
• restriction subunit, and
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Type II Restriction Enzymes:
 Restriction and modification are mediated by separate
enzymes so it is possible to cleave DNA in the absence
of modification.
 Although the two enzymes recognize the same target
sequence, they can be purified separately from each other.
 Cleavage of nucleotide sequence occurs at the restriction
site.
 These enzymes are used to recognize rotationally
symmetrical sequence which is often referred as
palindromic sequence.
In a palindrome the nucleotide base sequences in the
second half of a DNA strand is the mirror image of the
sequence in its first half.
Eg: i). 5’ GAA ? AAG 3’ Single strand
ii). 5’ Chanie
GAA
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AAG 3’ Double strand DNA 82
 They require only Mg2+ as a cofactor and ATP is not
needed for their activity.
 Type II endonucleases are widely used for mapping
and reconstructing DNA in vitro.
 because they recognize specific sites and cleave just at
these sites.
Type III Restriction enzymes:
 These enzymes recognize and methylate the same
DNA sequence, but cleave 24–26 bp away.
 They have two different subunits, in which one subunit
(M) is responsible for recognition and modification of
DNA sequence and other subunit (R) has nuclease
action.
 Mg+2 ions, ATP Chanieare needed
D. .... Dep't of Biotechnology for
.......... DNA cleavage and
83
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Restriction Patterns:
 The restriction enzymes cut DNA molecule by cleavage
which occur in two types;
Blunt end:
 Certain restriction enzymes Alu 1 (Arthrobacter luteus)
make cuts across both strands of DNA at the same
position so that the
 resulting termini or ends have blunt end in which the
two strands end at the same point.
Sticky end:
 In this style the restriction endonuclease Eco R I, Bam,
Hind I, Hind III make single strand cuts that produce
ends sticky ends in which
 two strands of DNA are cleaved at different locations
generating fragments with
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protruding
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Application of Restriction Enzymes:
 In various applications related to genetic
engineering, DNA is cleaved by using these
restriction enzymes.
 They used;
 in the process of insertion of genes into plasmid
vectors during gene cloning and protein
expression experiments.
 to distinguish gene alleles by specifically
recognizing single base changes in DNA known
as single nucleotide polymorphisms (SNPs).
 for Restriction Fragment Length Polymorphism
(RFLP) analysis for identifying individuals or
strains of Chanie
a particular species
D. .... Dep't of Biotechnology .......... 87
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Exonucleases:
o Degrade nucleic acids starting at one or both the
ends of polynucleotide chain.
o They hydrolyse the phosphodiester bonds of the
terminal nucleotide.
o It will cut either the 3’-OH end of the
phosphodiester back bone of polynucleotide
chains or the free 5’-P end and digest the
polynucleotide in 5’-3’ end direction.
o In both the cases, the enzymes travel along the
chain is stepwise manner liberating.
o Single nucleotide monophosphate molecules
degrade the entire polymer.
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For example:
 Phage exonuclease:
o used to 5’ end modification
 Exonuclease III:
o used for 3’ end modification
 S1 nuclease:
o It converts cohesive ends of duplex DNA
to blunt or flush ends or trimming away
single standard ends.
o It is used when annealing of two
incompatible ends requires over lapping
ends to be removed.
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Applications:
The enzyme is used
 To remove single strand tails from DNA
fragment to produced blunt ends
To digest hair pinloop formed in the synthesis
of double stranded cDNA
 For DNA mapping.
To analyze RNA-DNA hybrid structure.
Deoxy Ribose nuclease I (DNA ase I)
It is an endonuclease enzyme which digest either
Ss/Ds DNA and produces mixture of mono and
oligo nucleotides.
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Enzymes that join DNA fragments
 The enzymes used to join DNA fragments are called
DNA ligases.
1) DNA ligase:
This enzyme is used to join the recombinant DNA
fragment.
 Ligase requires 3’-OH and 5’-phosphate group
for ligation.
It joins the DNA fragment or seals the nicks
between adjacent nucleotides in double stranded
DNA reduces
There are 2 types of ligases.
o E. coli DNA ligase
o Chanie
T4 D.bactreiophage
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DNA ligase.
.... Dep't of Biotechnology .......... 92
Applications
 The DNA ligases are used for
o Joining DNA fragments to produce the rDNA
molecule
o Ligation of vector and inserting the rDNA
o Ligation of linkers /adoptor/ molecule at the
blunt ends of fragments
o Sealing nicks in Ds DNA

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Enzymes that modify ends of DNA Molecule
a. Alkaline Phosphatase:
 It catalyzes the removal of 5' phosphate
groups from the DNA and thus modify the
termini of DNA.
By treatment with Alkaline phosphatase, both
re-circularization and plasmid dimmer
formation are prevented because ligase cannot
join the ends.
b. Kinase:
Bacteriophase T4 polynucleotide kinase
catalyses the transfer of Y32p (gamma-
phosphate) of ATP to a 5' terminus of DNA or
RNA. Chanie D. .... Dep't of Biotechnology .......... 94
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Enzymes that Synthesis DNA
a. DNA Polymerase I:
 DNA polymerase I of E.coli, also known as DNA dependent
DNA polymerase, directs the synthesis of complementary
nucleic acids using single stranded DNA as a template.
 DNA synthesis requires a pre-existing DNA or RNA
primer with a 3' hydroxy to initiate de novo synthesis.
b. Terminal Transferases:
 An enzymes that adds nucleotides to the 3’ terminal of
DNA molecule.
 It has been purified from calf thymus.
 If the restriction enzyme produces blunt ends component,
single standard ends must be added to DNA fragments in
vitro.
 This is accomplished by
Chanie D. .... Dep't
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using..........the enzyme terminal
of Biotechnology 95
c. Reverse Transcriptase :
It is used to synthesise cDNA by using mRNA as
template.
 DNA copy of an RNA molecule is produced by the
enzyme reverse transcriptase generally obtained from
Avian mycle blastoris virus (AMV)
 This enzyme performs similar reaction as DNA
polymerase and has an absolute required for a primer
with a free 3’-OH.
[RNA … Reverse Transferases … SsDNA … DNA
polymerase ….. Ds DNA]
Application:
 Synthesis of cDNA from mRNA in vitro.
 Helps in the formation of second strand in cDNA
synthesis
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Enzymes that Degrade RNA
Ribonuclease (RNA ases):
 It degrades RNA portion of RNA-DNA ligase application
 Key enzymes in cDNA cloning as it is used to remove the
mRNA from RNA-DNA hybrid.
 Used to detect the presence of RNA-DNA hybrids and used
to remove poly A tails of mRNA.
Linkers:
 These are short, chemically synthesized, self complementary,
double stranded oligo nucleotides, which contain one or more
restriction endonuclease sites.
 Linkers are joined with blunt ended DNA fragments, cleavage
of the linker with the appropriate restriction enzyme creates
suitable cohesive Chanie
protruding ends. ..........
D. .... Dep't of Biotechnology 97
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Adaptors:
 Adoptors are short, chemically synthesized DNA double
strands which
 can be used to link the ends of two DNA molecules that
have different sequences at their ends.

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Chapter Three
3. Nucleic acid Hybridization
and Amplification
3.1. Introduction:
 Nucleic acid hybridization is the formation of a stable
duplex between two complementary strands of nucleic
acid by means of hydrogen bonding between base pairs.
 Nucleic acid hybridization is a process used to identify
specific DNA sequences.
 It is also permits detection of mutations such as deletion,
insertion and copy number variation for disease diagnosis.
 Amplification is a process by which a nucleic acid molecule
is enzymatically copied to generate a progeny population with
the same sequence Chanieas
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theof Biotechnology
D. .... Dep't parental .......... one . 102
3.2. Polymerase Chain Reaction (PCR) and
its Applications
 Polymerase chain reaction (PCR) is a widely
employed technique in molecular biology
 to amplify single or a few copies of DNA,
 generating millions of copies of a particular
DNA sequence.
 The PCR results in the selective amplification of a
target region of a DNA or RNA molecule.
 PCR has been extensively exploited in;
Amplification,
target gene detection,
sequencing etc.
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 The method consists of thermal cycles of repeated
heating followed by cooling of the reaction mixture
to achieve melting and primer hybridization to enable
enzymatic replication of the DNA.
 The DNA polymerases initially employed for in vitro
experiments were unable to withstand these high
temperatures.
 In 1976, Chien et al discovered a novel DNA polymerase
from the extreme thermophile Thermus aquaticus which
naturally dwell in hot water spring (122 to 176°F).
 The enzyme was named as Taq DNA polymerase
which is stable up to 95°C.
 In 1985, Kary Mullis invented a process PCR using
the thermo-stableChanie
Taq
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Basic Protocol for PCR
Components and Reagents:
 A basic PCR set up requires the following essential
components and reagents;
1. Template DNA containing the DNA region (target) to be
amplified.
2. Primers that are complementary to the 5' ends of each
of the sense (Forward primer) and anti-sense strand of
the DNA target (Reverse primer).
3. Taq polymerase or other thermostable, high fidelity DNA
polymerase (Pfu polymerase isolated from Pyrococcus
furiosus).
4. Deoxyribonucleotide triphosphates (dNTPs), which are
the building-blocks forofaBiotechnology
Chanie D. .... Dep't newly..........
synthesized DNA strand.
105
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5. Buffer solutions to provide a suitable chemical
condition for optimum activity and stability of
the DNA polymerases
eg. magnesium and potassium
6. Divalent cations (eg. magnesium or manganese
ions).
They act as a co-factor for Taq
polymerase which increases its
polymerase activity.
Generally Mg2+ is used, but Mn2+ can be
applied to achieve PCR-mediated DNA
mutagenesis.
This is because higher Mn2+
concentration leads to higher error rate
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Procedure:
 Typically, PCR is designed of 20-40 repeated thermal cycles,
with each cycle consisting of 3 discrete temperature steps:
o Denaturation,
o Annealing and
o Extension
 The thermal cycles are often proceeded by a
temperature at a high range (>90°C), and
 followed by final product extension or brief storage at 4
degree Celsius.
 In PCR cycles, the temperatures and the duration of
each cycle is determined based on various parameters
like;
 the type of DNA polymerase used,
 the melting temperature (Tm) of the primers,
 concentration of divalent ions and dNTPs in the
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 The various steps in PCR involved are;
a). Initial Denaturation:
It involves heating of the reaction to a
temperature of 94-96°C for 7-10 minutes (or
98°C if extremely thermostable polymerases are
used).
For specifically engineered DNA polymerases
(Hot start Taq polymerases) activity requires higher
range of temperature.
The initial heating for such a long duration also
helps in gradual and proper unfolding of the
genomic DNA and subsequent denaturation, and
thus exposing target DNA sequence to the
correspondingChanie
primers.
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b). Denaturation:
 Denaturation requires heating the reaction
mixture to 94-98°C for 20–30 seconds.
 It results in melting of the DNA template by
disrupting the hydrogen bonds between
complementary bases, yielding single-stranded
DNA molecules.

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c). Annealing:
Following the separation of the two strands of
DNA during denaturation, the temperature of the
reaction mix is lowered to 50-65°C for 20-50
seconds to allow annealing of the primers to the
single-stranded DNA templates.
Typically the annealing temperature should be
about 3-5°C below the Tm of the primers.
Stable complimentary binding are only formed
between the primer sequence and the template
when there is a high sequence complimentarily
between them.
The polymerase enzymes initiate the replication
from 3' end of Chanie
theD. primer towards the 5'end of it.111
.... Dep't of Biotechnology ..........
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d). Extension /Elongation:
o Extension/elongation step includes addition of
dNTPs to the 3' end of primer with the help of
DNA polymerase enzyme.
o The type of DNA polymerase applied in the
reaction determines the optimum extension
temperature at this step.
o DNA polymerase synthesizes a new DNA strand
complementary to its template strand by addition of
dNTPs, condensing the 5'-phosphate group of the dNTPs
with the 3'-hydroxyl group at the end of the nascent
(extending) DNA strand.
o Conventionally, at its optimum temperature, DNA
polymerase can add up to a thousand bases per minute.
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e). Final Elongation & Hold:
 Final elongation step is occasionally performed for
5-15 minutes at a temperature of 70-74°C after the
last PCR cycle to ensure amplification of any
remaining single-stranded DNA.
 Final hold step at 4°C may be done for short-term
storage of the reaction mixture.
 To check the desired PCR amplification of the target
DNA fragment (also sometimes referred to as the amplicon
or amplimer), agarose gel electrophoresis is employed
for separation of the PCR products based on size.
 The determination of size (s) of PCR products is
performed by comparing with a DNA ladder, which
contains DNA fragments of known size, run on the
gel along side theChanie
PCR products.
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Principles of PCR
 The PCR involves the primer mediated enzymatic
amplification of DNA.
 It is based on using the ability of DNA polymerase to
synthesize new strand of DNA complementary to the
offered template strand.
 Primer is needed because DNA polymerase can add
a nucleotide only onto a preexisting 3′-OH group to
add the first nucleotide.
 DNA polymerase then elongate its 3’ end by adding
more nucleotides to generate an extended region of
double stranded DNA.

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Variation in PCR
 In addition to the amplification of a target DNA sequence by
the typical PCR procedures, several specialized types of
PCR have been developed for specific applications.
1. Real-time PCR
2. Quantitative real time PCR (Q-RT PCR)
3. Reverse Transcriptase PCR (RT-PCR)
4. Multiplex PCR
5. Nested PCR
6. Long-range PCR
7. Single-cell PCR
8. Fast-cycling PCR
9. Methylation-specific PCR (MSP) ……..etc
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Application of PCR
 Infectious disease diagnosis, progression and
response to therapy;
 facilitates the detection of DNA or RNA of pathogenic
organisms
 determine the body's immune response to a pathogen
 to enumerate the amount of virus in a person's blood
(‘viral load’).
eg. for HIV,HPV, etc
 Diagnosis of genetic diseases
o diagnosing genetic diseases, whether due to innate
genetic changes or as a result of a natural genetic
mutations.
o for instance Single-strand conformation polymorphism
(SSCP) is important to detect the disease.
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 Genetic Counseling:
to check the account of genetic disease beforehand to
make a decision on having children.
 Forensic sciences:
Genetic fingerprint is one of the most exploited
application of PCR (also known as DNA profiling).
 plays a role in analysis of genomic or mitochondrial
DNA, in which investigators used samples from hair
shafts and bones when other samples are not
accessible.
 Research in Molecular Biology:
 PCR is an essential technique in cloning procedure
which allows generation of large amounts of pure DNA
from tiny amount of template strand and further study
of a particular gene.
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 Some alterations to the PCR protocol can
generate mutations
 also investigate “ON or OFF” of particular
genes at different stages in tissues (or even in
individual cells).
 Others:
 The genes associated with a variety of
diseases have been identified using PCR
 PCR has been used to identify and to explore
relationships among species in the field of
evolutionary biology.
 It commonly used by Paleontologists to
amplify DNA from extinct species or
cryopreserved fossils of millions years.
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 What is PCR?
 Requirements for PCR
 Phases of PCR
 How do you know
whether a given gene
amplify or not and also its
size?
 What is/ are application of
PCR
 Compare and contrast
PCR & Molecular cloning.
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3.3. Methods of Nucleic Acid Hybridization
and Detection
 Nucleic acid hybridization is a process used to
identify specific DNA sequences.
 Specific DNA probes are denatured and annealed to
sample DNA that has also been denatured.
Probes used in hybridization reactions are usually
chemically synthesized DNA or RNA that has been
labeled with a fluorescent dye or radioactive isotope
such as 32P.
 There are two different types of nucleic acid
hybridization and detection techniques generally
used, which are called
o NorthernChanie
blotting
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 Southern Blotting
o is used to identify specific sequences on
target DNA using labeled probes
complementary to a DNA sequence in the
sample.
 Procedure:
 DNA digestion
 Restriction digest to make different sized fragments
 Agarose gel electrophoresis to separate by size
 Since only single strands bind to the filter, the DNA
must be denatured.
 Denaturation to permit binding to the filter (NaOH)
 Transfer to filter paper (capillary flow)
 Hybridization to probe
 Washing Chanie D. .... Dep't of Biotechnology ..........
124
 Visualization of probe WU
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Blotting

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 Northern Blotting
 is used to identify a specific RNA molecule in a mixture
of different RNA by using labeled DNA probes.

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Another type of blotting technique is called Western
Blotting, is used for identifying specific protein molecules
using protein probes labeled with a fluorescent dye or with
a radioisotope such as 35S.

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4.5. Probe and Target Sequences
 A probe is a single-stranded sequence of DNA or
RNA used to search for its complementary sequence
in a sample genome.
 The probe is placed into contact with the sample
under conditions that allow the probe sequence to
hybridize with its complementary sequence.
 Probe is a nucleic acid that can be labeled with a
marker which allows identification and quantitation.
 Types of labels
o Radioactive (32P, 35S, 14C, 3H)
o Fluorescent
» FISH: fluorescent in situ hybridization
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Probe Target Sequences
 A probe is a single-stranded sequence of DNA or
RNA used to search for its complementary sequence
in a sample genome.
 Gene sequencing can be accomplished using
several different DNA sequencing methods,
depending on the scale.
 Highly targeted single-gene sequencing is accurate,

fast, and affordable with Sanger sequencing.
 Fast and affordable sequencing of multiple genes
and panels can be accomplished with the Ion PGM™
System combined with Ion AmpliSeq™ technology.
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Chapter Four
4. Construction of DNA Libraries
4.1. Introduction:
 Every gene manipulation procedure requires genetic
materials 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
Chanie D. .... Dep'tconcentrations
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 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
Chromosome
3
Cell 1
Nucleic
Cell 2 Acid 4
DNA
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DNA Isolation and Extraction (eg. plant)
Plant material is the most difficult for high quality
DNA extraction.
To prepare the tissue for extraction involves the
use of
o liquid nitrogen flash freezing
o followed by grinding the frozen tissue
with mortar and pestle. …… dried tissue
Liquid nitrogen is difficult and dangerous in open
to handle in laboratory class room.
Therefore, simple and modified plant DNA
extraction from fresh tissue.
The protocols and results are present below;
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Extraction Reagents and Buffers
EBA EBB TE Others
Extraction Buffer A (EBA) Per 100 mL:

o 2% (w/v) hexadecyltrimethylammonium bromide (CTAB)
…………………………………………2.0 g
o 100 mM Tris (pH 8.0) (Use 1 M stock) ……………. 10 mL
o 20 mM EDTA (Use 0.5 M stock) …………………….. 1 mL
o 1.4 M NaCl……………………………………………... 8.2 g
o 4% (w/v) polyvinylpyrrolidone (PVP) ……………….. 4.0 g
o 0.1% (w/v) ascorbic acid ...……………………………0.1 g
o 10 mM β-mercaptoethanol (BME) (use 14.3M stock)……70 µL
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Extraction Buffer B (EBB) Per 100 mL
o 100 mM Tris-HCl (pH 8.0) (Use 1 M stock) ….... 10 mL
o 50 mM EDTA (Use 0.5 M stock) …………………2.5 mL
o 100 mM NaCl ………………………………………. 0.6 g
o 10 mM β-mercaptoethanol (BME) (Use 14.3 M stock) …. 70 µL
TE Buffer Per 100 mL

10 mM Tris (pH 8.0) (Use 1 M stock) ………….. 1.0 mL
1 mM EDTA (Use 0.5 M stock) ………… ………. 50 µL
Other Required Reagents
 20% (w/v) sodium dodecyl sulphate (SDS)
 5 M potassium acetate (Stored at –20 oC)
 3 M sodium acetate (pH 5.2)
 70% ethanol (stored at -20 oC)
 Absolute isopropanol (stored at -20 oC)
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Protocol:
1. Prepare the plant specimen and sample
2. Weigh out 0.3 g of plant tissue
3. Place it on clean glass slide and chop into
smaller pieces
4. Transfer the tissue for further grinding
5. Add 300 µL EBA , 900 µL EEB and 100 µL SDS.
6. Vertex and incubate at 65 ºC for 10 mins
7. Place tube on ice and add 410 µL cold potassium
acetate. Mix by inversion and turn back to ice
8. Centrifuge at 13,200 rpm for 15 mins.
9. Transfer 1 mL of the supernatant to a new 1.5 mL micro-
centrifuge tube.
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10. Add 540 µL of ice cold absolute isopropanol and
incubate in ice for 20 mins.
11. Centrifuge at 10,200 rpm for 10 mins.
12. Discard the supernatant and wash the pellet
once in 500 µL 70 % ethanol and let dry.
13.Re-suspend the dry pellet in 600 µL of TE.
14.Add 60 µL 3M sodium acetate and 360 µL
absolute ice cold isopropanol and incubate on
ice for 20 mins.
15. Repeat step 8-14 twice
16. Re-suspend the pellet in 50 µL TE and carry out
agarose gel.
17.Analyze the result
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Isolation and Purification of Genomic DNA
From Bacteria
 Genomic DNA is found in the nucleus of all living cells
with the structure of double-stranded DNA remaining
unchanged, except virus.
 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 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
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from the cell extract
i. Growth and Harvest of Bacterial Culture:
o Bacterial cell culture is more convenient than any
other microbe;
 as it requires only liquid medium
e ta containing essential nutrients at
M s
en e
g optimal concentrations, for the growth
Pure Culture and division of bacterial cells.
growth the bacterial cells are usually grown
on a complex medium like Luria-
Bertani (LB), in which the medium
composition is difficult to decipher.
the cells are separated by
centrifugation and re-suspended in 1%
or less of the initial culture volume. 143
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ii. 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 using the following ways;
o Physical method by mechanical forces.
o Chemical method by metal chelating
agents i.e. EDTA and surfactant i.e. SDS or enzyme (eg.
lysozyme).
o Lysozyme:
• Present in egg-white, salivary secretion and
tears.
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• Catalyzes the breakdown of cell wall i.e. the
peptidoglycan layer.
o EDTA (Ethylene diamine Tetra-acetic Acid)
 A chelating agent necessary for destabilizing
the integrity of cell wall.
 Inhibits the cellular enzymes that degrade
DNA.
o SDS (Sodium Dodecyl Sulphate)
Helps in removal of lipid molecules and
denaturation of membrane proteins.
Generally, a mixture of EDTA and
lysozyme is used.
Cell lysis is followed by centrifugation to
pellet down the cell wall
fractions leaving
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iii. Purification of DNA:
o In addition to DNA, a cell extract contains
significant quantities of protein and RNA which
can be further purified by following methods;
a. 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.
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The RNA can be effectively removed by using
Ribonuclease, an enzyme which rapidly degrades
RNA into its ribonucleotide subunits.
o Repeated phenol extraction is not
desirable, as it damages the DNA.
b. 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. WU
iV. 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.
o Advantages:
 It leaves short-chain and monomeric nucleic
acid components in solution.
Ribonucleotides produced by the ribonuclease
treatment are separated from DNA.
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Summarize the following for
Bacterial DNA Extraction
Reagents
 Equipments
 Procedures
 Timing and
 Concentration of DNA
o Measurement of quantity & Purity

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 To extract the DNA from bacteria includes the ff;
Reagents:
1. EDTA-Saline (0.01 M and NaCl 0.15 M; pH = 8.0 x 800 µl
2. Lysozyme (300 mg/ml x 10 µl).
3. Mutanolysin (1,000 U/ml x 10 µl).
4. RNase A, DNase-free (100 mg/ml x 7.0 µl).
5. SDS 25% (w/v) x 80 µl).
6. Sodium chloride (5 M x 250 µl).
7. Chloroform: isoamyl alcohol (24:1) x 2.0 ml].
8. Sodium acetate (3 M x 90 µl).
9. Absolute isopropanol (600 µl).
10. Low-TE 1 mMChanie
and EDTA
D. .... Dep't 0.1 ..........
of Biotechnology mM; pH = 7.0 – 8.0
151 x
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Equipment:
Sterile 1.5 ml micro-centrifuge tubes.
Sterile 2.0 ml micro-centrifuge tubes.
Vortex.
Heating block or water bath.
Micro-centrifuge
Orbital shaker.
Glass rods

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Procedures:
1. Prepare specimen and sample (… ??????)
2. Suspend a fully-loaded inoculating loop of bacterial biomass
in a 2.0 ml tube with 800 µl of EDTA-Saline;
3. Vortex at maximum speed to mix thoroughly.
4. Add 10 µl of lysozyme.
5. If Gram-positive bacteria, add 10 µl of mutanolysin.
6. Mix by vortexing for a few seconds to suspend the biomass.
7. Add 7 µl of RNase A.
8. Incubate at 37°C for at least 15 – 45 min and vortex every
15 min.
9. Add 80 µl of SDS.
10. Vortex at maximum speed for a few seconds; the viscosity is
increased.
11. Incubate at 65°C for 10 min and vortex once during this
time. Chanie D. .... Dep't of Biotechnology ..........
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153
12. Spin down briefly and add 250 µl of sodium chloride 5 M.
13. Vortex at maximum speed for a few seconds and spin down
briefly.
14. Add 400 µl of chloroform: isoamyl alcohol.
15. Vortex at maximum speed for a few seconds; shake for 15 min
at 1,400 rpm on an orbital shaker.
16. Centrifuge at > 13,000 x g for 15 min.
17. Transfer the top-layer to a new tube (2 ml) and avoid the
protein layer.
18. Add 400 µl of chloroform:isoamyl alcohol again; shake
vigorously by hand and centrifuge again.
19. Repeat step 18 until there is no protein layer.
20. To 1 ml of solution, add 90 µl of sodium acetate 3 M.
21. To 1 ml of solution, Chanie
addD. 600
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.... Dep't µl of cold..........
of Biotechnology isopropanol (4°C). 154
22. Precipitate the DNA by inverting the tube several times by
hand, when threads of DNA are seen, shake the tube harder
to clump the DNA threads.
23. Spool the DNA using a glass rod and leave it to dry
completely, for at least 5 min at room temperature.
24. Alternatively, for steps 22-23, if there is no formation of
threads, spin down at > 13,000 x g for 10 min,
25. Carefully discard the supernatant and let the DNA pellet dry
completely at room temperature.
25. Suspend the spooled or pelleted DNA in 100 µl of ‘low’-TE.
26. Incubate the re-suspended DNA over-night at 4ºC for
complete re-suspension.

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Isolation and Purification of Plasmid DNA
A. Growth of the Bacterial cell
 It involves growth of the bacterial cells in a media
containing essential nutrients.
B. Harvest and Lysis of Bacteria
 Lysis of bacteria results in the precipitation of
DNA and cellular proteins.
 Addition of acetate-containing neutralization
buffer results in the precipitation of large and less
super coiled chromosomal DNA and proteins
leaving the small bacterial DNA plasmids in
solution.
C. Purification of Plasmid DNA
 This step is same for both plasmid and genomic, but
former involves an additional step
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i.e. the separation of plasmid DNA from the
large bacterial chromosomal DNA.
Methods for Separation of Plasmid DNA:
o Separation of plasmid DNA is based on the
several features like size and conformation of
plasmid DNA and bacterial DNA.
o Plasmids are much smaller than the bacterial
main chromosomes,
o The separation of small molecules from larger
ones is based on the fact that plasmids and the
bacterial chromosomes are circular,
o but bacterial chromosomes break into linear
fragments during the preparation of the cell
extract resulting in separation of pure plasmids.
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a. Separation Based on Size Difference
It involves lysis of cells with lysozyme and in the
presence of sucrose (prevents the immediate bursting of
cell).
 Cells with partially degraded cell walls are formed
that retain an intact cytoplasmic membrane called
as spheroplasts.
 Cell lysis is then induced by the addition of a non-
ionic detergent (eg. Triton X-100) or ionic detergents
(eg.SDS) causing chromosomal breakage.
 Bacterial chromosome attached to cell membrane,
upon lysis gets removed with the cell debris.
 A cleared lysate consisting almost entirely of
plasmid DNA is formed with very little breakage of
the bacterial DNA.
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• ..

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b. Separation Based on Conformation
o Plasmids are super-coiled molecules formed by partial
unwinding of double helix of the plasmid DNA during the
plasmid replication process by enzymes called
topoisomerases.
o The super-coiled conformation can be maintained when
both polynucleotide strands are intact, hence, called
covalently closed-circular (ccc) DNA.
o If one of the polynucleotide strands is broken, the double
helix reverts to its normal relaxed state taking an
alternative conformation, called open-circular (oc).
o Super coiling is important in plasmid preparation due to

the easy separation of super-coiled molecules from non-
super-coiled ones.
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160
o The commonly used methods of separation
based on conformation are;
Alkaline Denaturation Method:
 This method is based on maintaining a very narrow pH
range for the denaturation of non-super-coiled DNA but
not the super-coiled plasmid.
 Addition of sodium hydroxide to cell extract or cleared
lysate (pH 12.0-12.5) results in disruption of the hydrogen
bonds of non-super-coiled DNA molecules.
 As a result, the double helix unwinds and two
polynucleotide chains separate.
Further addition of acid causes the aggregation
of these denatured bacterial DNA strands into a
tangled mass which can be pelleted by
centrifugation, leaving plasmid DNA in the
supernatant. WU
…

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Ethidium Bromide-cesium chloride density gradient
centrifugation
 Density gradient centrifugation can separate DNA,
RNA and protein. It is a very efficient method for obtaining
pure plasmid DNA.
 A density gradient is produced by centrifuging a
solution of cesium chloride at a very high speed which
pulls the CsCl ions towards the bottom. This process is
referred as isopycnic centrifugation.
 The DNA migrates to the point at which it has density
similar to that of CsCI i.e.1.7 g/cm3 in the gradient.
 In contrast, protein molecules having lower buoyant
densities float at the top of the tube whereas RNA gets
pelleted at the bottom.
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 Density gradient centrifugation in the presence of
ethidium bromide (EtBr) can be used to separate
super-coiled DNA from non-super coiled molecules.
 Ethidium bromide is an intercalating dye that binds to
DNA molecules causing partial unwinding of the
double helix.
 Super-coiled DNA have very little freedom to unwind
due to absence of free ends and bind to a limited
amount of EtBr resulting in very less decrease in
buoyant density (0.085 g/cm3 ) than that of linear DNA
(0.125 g/cm3 ).
 As a result, they form a distinct and separated from
the linear bacterial DNA.
 The EtBr boundChanie
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Isolation and Purification of RNA
 RNA (Ribonucleic acid) is a polymeric substance
consisting of a long single-stranded chain of
phosphate and ribose units with the nitrogen bases
adenine, guanine, cytosine and Uracil bonded to the
ribose sugar present in living cells and many
viruses.
 The steps for preparation of RNA involve
homogenization,
 phase separation,
 RNA precipitation,
 washing and
re-dissolving RNA.
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o The method for isolation and purification of RNA are;
1. Organic extraction method
2. Filter-based, spin basket formats
3. Magnetic particle methods
4. Direct lysis method.
Organic Extraction Method:
This method involves phase separation by
addition and centrifugation of a mixture of a
solution containing phenol, chloroform and a
chaotropic agent (guanidinium thiocyanate) and
aqueous sample.
Guanidium thiocyanate results in the denaturation
of proteins and RNases, separating rRNA from
ribosomes. Chanie D. .... Dep't of Biotechnology .......... 167
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 Addition of chloroform forms a colorless
 upper aqueous phase containing RNA,
 an inter-phase containing DNA and
 lower phenol-chloroform phase
containing protein.
 RNA is collected from the upper aqueous phase by
alcohol (2-propanol or ethanol) precipitation followed by
rehydration.
One of the advantages of this method is the
stabilization of RNA and rapid denaturation of
nucleases.
 Also, it has several drawbacks such as;
 it is difficult to automate,
 needs labor and manual intensive processing,
 use ofChanie D. .... Dep't of Biotechnology ..........
chlorinated organic reagents.
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168
Direct Lysis Methods:
 This method involves use of lysis buffer under
specified conditions for the disruption of sample
and stabilization of nucleic acids.
 If desired, samples can also be purified from
stabilized lysates.
This method eliminates the need of binding and
elution from solid surfaces and thus avoids bias
and recovery efficiency effects.
 This method is important as
 Extremely fast and easy.
 Highest ability for precise RNA
representation.
 Easy to work on very small samples.
 Amenable
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to simple automation.
4.2. Quantification and Storage of Nucleic
Acids
4.2.1. Preparation:
 Nucleic acid molecules like DNA, RNA are basic,
essential and primary molecules for all molecular biology
related research.
 Before detect the nucleic acids the isolation of pure form
of nucleic acid is needed.….. Preparation step
 Thus, establishment of pure DNA or RNA is the first step
before detection.
 Contaminants such as other species, cellular proteins,
lipids, etc should be avoid.
 The optimal Nucleic acid protocol should provide
 Reproducible result
 No degradation
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4.2.2. Quantification of Nucleic Acids:
 Quantification of nucleic acids is done to determine
the average concentrations of DNA or RNA present
in a mixture, as well as their purity.
 The accurate measurement is based on sensitivity,
specificity and interference by contaminants.
 Various methods that can be employed to quantify
the nucleic acid concentration are;
 Spectrophotometric analysis
 Nanodrop
 Fluorescence based method
 Fluorescence in situ hybridization (FISH)
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1. Spectrophotometric Analysis:
o It is a simple and accurate method to assess the
concentration and purity of nucleic acids based on
their absorption at different wavelengths.
o According to Beer Lambert's Law, when light is
passed through a substance of concentration c at
a path length L (typically 1 cm), the absorbance is
directly proportional to the concentration of the
substance and the path length
i.e. A= ∈cl
o Where;
• A= absorbance of nucleic acids at a particular wavelength
• ∈=Molar extinction coefficient (M-1 cm-1) or specific absorption coefficient
(μg/ml)-1 cm-1
• l = Path length of the spectrophotometer cuvette
• c= concentration of a substance
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The value of for the DNA bases is
Bases ∈(at 260 nm) ( M-1 cm-1)
Adenine (A) 15,200

Cytosine (C) 7,050
Guanine (G) 12,010
Thymine (T) 8,400

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Quantitation with a Spectrophotometer
 Use H2O or 1X TE as a solvent to suspend the
nucleic acids, and place each sample in a quartz
cuvette.
 Zero the spectrophotometer with a sample of
solvent.
 For more accurate readings of the nucleic acid
sample of interest, dilute the sample to give
readings between 0.1 and 1.0.
 For a 1-cm path length, the optical density at 260
nm (OD260) equals 1.0 for the following solutions:
 a 50 μg/mL solution of dsDNA
 a 33 μg/mL solution of ssDNA
 a 20-30 μg/mL
Chanie D. ....solution
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of oligonucleotide
Dep't of Biotechnology .......... 174
Example:
A sample of dsDNA was diluted 50X. The diluted
sample gave a reading of 0.65 on a
spectrophotometer at OD260.
To determine the concentration of DNA in the
original sample, perform the following calculation:
dsDNA concentration = 50 μg/mL × OD260 × dilution factor
o dsDNA concentration = 50 μg/mL × 0.65 × 50
o dsDNA concentration = 1.63 mg/mL
 Total yield is obtained by multiplying the DNA
concentration by the final total purified sample
volume.
DNA yield (µg) = DNA concentration × total sample
volume
Chanie D. .... (ml)..........
Dep't of Biotechnology
175
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 To evaluate DNA purity, measure absorbance from
230nm to 320nm to detect other possible
contaminants.
 The most common purity calculation is the ratio of
the absorbance at 260nm divided by the reading at
280nm
 The purity of the nucleic acids can be determined by;
A260/A280 = A260 : A280
DNA purity (A260/A280) = (A260 reading – A320 reading) ÷
(A280 reading – A320 reading)
 Value of this ratio is 2.0, 1.8 and 0.6 for pure RNA,
DNA and protein respectively.
 A ratio of less than 1.7 or1.8 signifies that the DNA
sample is contaminated with protein or phenol and
the preparation is not proper.
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2. Nanodrop Method:
o The NanoDrop Lite is designed to measure the
absorbance and calculate the concentration of
nucleic acids (260 nm) and
purified proteins (280 nm).
o This would include
 dsDNA,
 ssDNA,
 RNA and
 purified proteins.
o The Nanodrop does an excellent job at measuring
across a wide spectrum that spans UV and visible
light.
o To get the result, you have to tell the software before
beginning measurements so it ..........
Chanie D. .... Dep't of Biotechnology
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can report an accurate
177
o Unlike spectrophotometric method requiring 1-2
ml of the sample, this technique involves micro
volume (1-2μl) quantification of nucleic acid
sample.
o The nucleic acids having the concentration
range from 2-15,000 ng/μl can be assessed
by this method.
o For this, 1-2μl of sample is loaded between
the two optical surfaces and
o the software automatically calculates the
concentration and purity of the nucleic acid
o and displays sample quality (purity) as a spectral
output.
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3. Quantification Using Fluorescent Dyes:
 This method is simple and more sensitive than
spectrophotometric method which measures the
fluorescence intensity of the dyes that fluoresce
upon interaction with the nucleic acids.
Various fluorescent dyes such as EtBr, Hoechst
33258, picogreen, DAPI can be used for
quantification of nucleic acids.
 Quantification of nucleic acids separated by gel
electrophoresis can be done by comparing the
stained nucleic acids with stained standards of
known concentration separated on the same gel.
The dye: DNA complex shows greater fluorescence
than the unbound
Chanie D. .... Dep'tdye by intercalating
of Biotechnology .......... between 180
the
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 The fluorescence intensity of the band estimates
the concentration/amount of DNA.
The intensity of the stain is dependent at least in
part on the base pair composition of the nucleic
acid.

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4. Fluorescence in situ hybridization (FISH):
 Fluorescent in situ hybridization (FISH) can be used to test
for the presence or absence of specific chromosome regions
 This involves using a specific DNA probe which recognizes
the region to be tested.
 In situ hybridization (ISH) is a type of hybridization that uses
a labeled complementary DNA, RNA or modified nucleic
acids strand to localize a specific DNA or RNA sequence in a
portion or section of tissue (in situ).
 In a cytometric system, quantification of FISH signals and
accurate estimation of fluorescence intensity of specific DNA
sequences can be performed using an epi-fluorescence
microscope with a multi-wavelength illuminator, fitted with a
cooled charge couple
Chaniedevice
D. .... Dep't of(CCD)
Biotechnology camera.
.......... 182
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Quantitation by FISH was first applied as a basis for
rudimentary cytogenetic assays.
 Detection using CCD camera enables the quantitative
analysis of mRNA as well.
 The two factors affecting the fluorescence assay
are reproducibility and
 signal irregularity as well as background noise
which may vary from sample to sample and cell to
cell.
 Various approaches have been developed to
reduce background noise such as use of reducing
agents
eg. sodium borohydride and pre-treatment by light
irradiation, and image analysis methods.
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 The output thus consist of a true signal and various
noise components, the profile for each of them can
be estimated and deleted by digital methods like
independent component analysis.
 Further, FISH can be employed for interpretation
using various approaches such as multi-color
cytometry algotithms, dot-counting approaches, use
of diagnostic probe sets etc.

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4.2.3. Storage of Nucleic Acids
 Maintaining the long-term integrity of nucleic acids in the
laboratory has traditionally required the use of freezers.
 However, novel nucleic acid stabilization technologies may
allow for the storage of DNA and RNA at room temperature
in a cost-effective, environmentally friendly manner.
 The purified DNA can be stored at -20°C or -70°C under
slightly basic conditions
e.g. (Tris- Cl, pH 8.0)
as acidic conditions result in hydrolysis of DNA
 RNA preservation under frozen conditions is helpful.
 Purified RNA can be stored at -20°C or -80°C in RNase-
free solution such as;
 0.1 mM EDTA
 TE Buffer
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 The RNA Storage Solution (1mM sodium citrate,
pH 6.4 ± 0.2):
o It is a buffer that delivers greater RNA
stability than 0.1 mM EDTA or TE.
o The presence of sodium citrate and low pH
minimizes base hydrolysis of RNA.
o Sodium citrate acts both as a chelating and
buffering agent.
 This RNase-free solution is compatible with all RNA
applications including;
in vitro translation,
reverse transcription,
nuclease protection assays and
 northernChanie
analysis.
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4.3. Construction of cDNA Library
 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.
 Using 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
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187
 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.

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o The construction of cDNA library involves following
steps;
1. 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;
o Chromatographic purification of mRNA using oligo-dT column,
which retains mRNA molecules, resulting in their enrichment.
o Spinning down mRNA by density gradient centrifugation.
o 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
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 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. Chanie
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2. 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
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 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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3. Incorporation of cDNA into a Vector:
The blunt-ended cDNA termini are modified in
order to ligate into a vector to prepare ds-cDNA
for cloning.
 Since blunt-end ligation is inefficient, short restriction-site
linkers are first ligated to both ends.
Linker:
It is a double-stranded DNA segment with a
recognition site for a particular restriction enzyme.
 It is 10-12 base pairs long prepared by hybridizing
chemically synthesized complementary
oligonucleotides.
The blunt ended ds-DNAs are ligated with the linkers
by the DNA ligase from T4 Bacteriophage.
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 The resulting double-stranded cDNAs with linkers at
both ends are treated with a restriction enzyme
specific for the linker generating cDNA molecules
with sticky ends.
o Problems arise, when cDNA itself has a site for the
restriction enzyme cleaving the linkers.
 This can be overcome using an appropriate
modification enzyme (methylase) to protect any
internal recognition site from digestion which
methylates specific bases within the restriction-site
sequence, thereby, preventing the restriction
enzyme binding.
 Ligation of the digested ds-cDNA into a vector is the
final step in the construction of a cDNA library.
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4. Cloning of cDNAs:
 cDNAs are usually cloned in phage insertion
vectors.
 Bacteriophage vectors offer the following
advantageous over plasmid vectors,
 are more suitable when a large number of
recombinants are required for cloning low
abundant mRNAs as recombinant phages are
produced by in vitro packaging.
 can easily store and handle large numbers of
phage clones
 Plasmid vectors are used extensively for
cDNA cloning, particularly in the isolation of
the desired cDNA sequence involving the
screening of a relatively small number of
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Problems in cDNA Preparation
 Large mRNA sequence results in inefficient
synthesis of full-length cDNA.
o This cause problems during expression as it
may not contain the entire coding sequence of
the gene.
 This arises because of the poor process
of RTase purified from avian
myeloblastosis virus (AVM) or
 produced in E.coli from the gene of
Moloney murine leukemia virus (MMLV).
 Use of S1 nuclease, the enzyme used to trim the
ds-cDNA, may remove some important 5´
sequences. Chanie D. .... Dep't of Biotechnology .......... 205
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Strategies to overcome the limitations in
cDNA preparation
 Strategies that can be employed to overcome the
above limitations are listed as follows;
 A specially designed E. coli vector can be used to
avoid incomplete copying of the RNA.
 The use of single strand specific nuclease can be
avoided by adding a poly-C tail to the 3´-end of the
single stranded cDNA produced by copying of the
mRNA by the enzyme terminal deoxynucleotidyl
transferase.
Complementary oligonucleotide (Poly-G) is now
used as a primer for the synthesis of
complementary strand to yield ds-cDNA without a
hairpin loopChanie
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enhancing
D. .... Dep't of Biotechnologythe
.......... full-length cDNA
206
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
Chanieprocess.
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Disadvantages of cDNA libraries:
o cDNA libraries contain only the parts of genes found
in mature mRNA.
• However, the sequences before and after the
gene, for example, those involved in the
regulation of gene expression, will not occur in
a cDNA library.
o Construction of a cDNA library cannot be used for
isolating the genes expressed at low levels as there
will be very little mRNA for it in any cell type and
may completely Chaniebe
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out
D. .... Dep't maneuvered
of Biotechnology .......... by the more
208
4.4. Construction of Genomic Library
 A genomic library is an organism specific collection of
DNA covering the entire genome of an organism.
 It contains all DNA sequences such as;
o expressed genes,
o non-expressed genes,
o exons and introns,
o promoter and terminator regions and
o intervening DNA sequences.
 Construction of a genomic DNA library involves
 isolation,
 purification and
 fragmentation of genomic DNA
 cloning ofChanie
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the D. ....fragmented DNA
Dep't of Biotechnology .......... 209
 The eukaryotic cell nuclei are purified by digestion
with
o protease (inorganic)
o organic (phenol-chloroform) extraction
 The derived genomic DNA is too large to incorporate
into a vector and needs to be broken up into
desirable fragment sizes.
 Fragmentation of DNA can be achieved by
o physical method and
o enzymatic method.
 The library created contains representative copies of
all DNA fragments present within the genome.

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Mechanisms for Cleaving DNA
(a). Physical Method:
 It involves mechanical shearing of genomic DNA using
a narrow-gauge syringe needle or
 Sonication to break up the DNA into suitable size
fragments that can be cloned.
 Typically, an average DNA fragment size of about
20 kb is desirable for cloning into λ based vectors.
 This method requires large quantities of DNA.
(b). Enzymatic Method:
 It involves use of restriction enzyme for the
fragmentation of purified DNA.
 This method is limited by distribution probability of site
prone to the action of restriction enzymes which will
generate shorterChanie
DNA fragments than the desired size.
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The two factors which govern the selection of the
restriction enzymes are;
o type of ends (blunt or sticky) generated by the
enzyme action and
o susceptibility of the enzyme to chemical
modification of bases like methylation which can
inhibit the enzyme activity.
The fragments of desired size can be recovered
by either;
o agarose gel electrophoresis or
o sucrose gradient technique and ligated to
suitable vectors.

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Partial Restriction digestion
 is achieved using restriction enzymes that
produce blunt or sticky ends as follows;
i. Restriction Enzymes that Generating blunt ends
o The genomic DNA can be digested using
restriction enzymes that generate blunt ends
eg. HaeIII and AluI
o Blunt ends are converted into sticky ends prior to
cloning.
o These blunt ended DNA fragments can be ligated
to oligonucleotides that contain the recognition
sequence for a restriction enzyme called linkers or
o possess an overhanging sticky end for cloning
into particular Chanie
restriction sites called adaptors. 215
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ii. Restriction enzymes that generate sticky ends
Genomic DNA can be digested with commonly
available restriction enzymes that generate sticky
ends.
o eg. digestion of genomic DNA with the
restriction enzyme Sau3AI (recognition
sequence 5'-GATC-3') generates DNA
fragments that are compatible with the
sticky end produced by BamHI (recognition
sequence 5'-GGATCC-3') cleavage of a
vector.
Once the DNA fragments are produced, they are
cloned into a suitable vector.

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Cloning of Genomic DNA:
 Various vectors are available for cloning large
DNA fragments.
o λ phage, yeast artificial chromosome,
bacterial artificial chromosome etc. are
considered as suitable vectors for larger
DNA and
o λ replacement vectors like
 λ DASH and EMBL3 are preferred
for construction of genomic DNA
library.
o T4 DNA ligase is used to ligate the selected
DNA sequence into the vector.
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Number of Clones Required for a Library:
 The number of clones to be pooled depends
upon the size of the genome (f) and average size
of the cloned DNA.
 Let (f) be the fraction of the genome size
compared to the average individual cloned
fragment size, would represent the lowest possible
number of clones that the library must contain.
 The minimum number of clones required can be
calculated as;
f = genome size / fragment size
 For the E. coli genome (4.6 Mb) with an average
cloned fragment size of 5 kb, f will be 920.
 Bigger the library better will be the chance220 of
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Amplified Library:
 The primary library created is usually of a low titer
and unstable.
 The stability and titer can be increased by
amplification.
o For this, the phages or bacterial colonies are
plated out several times and the resulting
progenies are collected to form an amplified library.
 The amplified library can then be stored almost
indefinitely due to long shelf-life of phages.
 It usually has a much larger volume than the primary
library, and consequently may be screened several times.
 It is possible that the amplification process will result
in the composition of the amplified library not truly
reflecting the primary one.
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Subgenomic Library:
Subgenomic library is a library which represents
only a fraction of the genome.
Enhancing the fold of purification of target DNA is
crucial for subgenomic DNA libraries which can be
achieved by multiple, sequential digestion when
information of the restriction map of the sequences of
interest is known.
After initial purification of a given fragment, the

purification can further be increased by re-
digestion with another enzyme generating a
smaller (clonable) fragment relative to original
DNA.
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Advantages of Genomic Libraries:
 Identification of a clone encoding a particular gene of
interest.
 It is useful for prokaryotic organisms having relatively
small genomes.
 Genomic libraries from eukaryotic organisms are
very important to study the genome sequence of a
particular gene, including its regulatory sequences
and its pattern of introns and exons.
Disadvantages of Genomic Library:
o Genome libraries from eukaryotes having very large
genomes contain a lot of DNA which does not code
for proteins and also contain non-coding DNA such
as repetitive DNA and regulatory regions
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Applications of Genomic Library:
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.
Used for;
o genome mapping,
o sequencing

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Gr
ou
pD
isc
us
sio
n
Mention the difference
b/n cDNA and
Genomic DNA

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4.5. Screening and Preservation of DNA
Libraries
 Library screening is the process of identification of
the clones carrying the gene of interest.
 Screening relies on a unique property of a clone in a
library.
 The DNA libraries consist of a collection of probably
many thousand clones in the form of either plaques
or colonies on a plate.
 Screening of libraries can be done by following
approaches based on;
o Detecting a particular DNA sequence and
o Gene expression.
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Chapter Five
5. Gene Transfer Techniques
5.1. Introduction
 The process of transfer, integration and expression of
transgene in the host cells is known as genetic
transformation.
 Various genetic transfer techniques are grouped into two
main categories.
1. Vector mediated (Indirect gene transfer,
Biological method).
2. Vector less (Direct gene transfer,
physical & chemical method)
 There are different Biotechnology Techniques (PBT) to
transfer gene from Chanie
oneD. ....toDep'tthe other...........
of Biotechnology 228
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A. Selective Breeding: a c h
i se
B. Cloning (via Tc) H ow
 e? V s e
o n ica l e n
C. Nature's way (using microbes) d ert
t alg
V izon
D. Cellular target practice: r
Ho sfer
r a n
1. Microinjection of single cells t
2. Biolistic gene transfer
3. Electroporation of cells grown without a
cell wall (protoplast)
4. Agrobacterium-mediated transfer
5. Protoplast fusion (PTC)
6. ETC.
 In general, those gene transfer techniques are
Natural and Artificial transformation.
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5.2. Indirect Transformation
Agrobacterium-Mediated Transformation
 The Agrobacterium system was historically the first
successful plant transformation system, marking the
break through in plant Genetic engineering in 1983.
 The Agrobacterium is naturally occurring gram negative
soil bacterium (known as natural gene engineers) with two
common species
o A. tumifacience and
o A. rhizogenes
 A.tumifacience induces tubers called crown galls,
where as A. rhizogenes causes hairy root diseases.
 Large plasmids in these bacteria are called tumer
inducing (Ti plasmid) and root inducing (Ri plasmid)
respectively. Chanie D. .... Dep't of Biotechnology ..........
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232
A. tumifacience
A. rhizogenes

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 The Ti plasmid has two major segments of interest in
transformation that is T DNA and Virs region.
 The T DNA region of the Ti plasmid is the part which is
transferred to plant cell and incorporated into nuclear
genome of cells.
 The transfer of T DNA is mediated by genes in the
another region of Ti plasmid called Virs genes (virulence
genes).
Ti plasmids are used as gene vectors for delivering
useful foreign genes into target plant cells and tissues.
The foreign gene is cloned in the T-DNA region of Ti-
plasmid in place of unwanted sequences.
 To transform plants, leaf discs (in case of dicots) or
embryogenic callus (in case of monocots) are collected
and infected with Agrobacterium carrying recombinant
disarmed Ti-plasmid
Chanievector.
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Agrobacterium Culture/Co-cultivation:
 The infected tissue is then cultured (co-cultivation)
on shoot regeneration medium for 2-3 days during
which time the transfer of T-DNA along with foreign
genes takes place.
 After this, the transformed tissues (leaf discs/calli)
are transferred onto plant regeneration medium
supplemented with usually lethal concentration of an
antibiotic to selectively eliminate non-transformed
tissues.
 After 3-5 weeks, the regenerated shoots are
transferred to root-inducing medium
 3-4 weeks after, complete plants are transferred to
soil following the hardening (acclimatization) of
regenerated plants.
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Expression of the T- DNA Genes:
The molecular techniques like PCR and southern
hybridization /blotting/ are used to detect the
presence of foreign genes in the transgenic
plants.

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Group Discussion
Steps for Transgenic plant
formation
 Components of regeneration
& Rooting media.
Why to use antibiotics in
such culture medium?

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5.3. Direct Transformation
 Introduction of DNA into plant cells without the
involvement of biological agents such as
Agrobacterium and leading to stable transformation is
called direct gene transfer.
 The following are direct gene transformation methods;
Chemical methods
Electroporation
Particle bombardment
 Lypofection
 Micro injection
Macro injection
Pollen transformation
 Delivery via growing pollen tubes
Laser induced transformation
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1). Chemical Methods:
o It is based on ability of protoplast to uptake the foreign
DNA from surrounding solution.
o An isolated plasmid DNA is mixed with protoplast in the
presence of the;
o Poly ethylene glycol (PEG),
o Polyvinyl alcohol (PVA) and
o Ca(PO4) which enhance the uptake of DNA
by protoplast.
o After 15-20 min of incubation, the protoplasts are cultured.
o On the presence of appropriate selective agents, the
protoplast are regenerated and
o the transgenic plants are further characterized for
conformation. Chanie D. .... Dep't of Biotechnology ..........
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242
2). Electroporaton:
 Induction of DNA into cell by exposing them for a very brief period
to high voltage electrical pulses to induce transiant pores in the
plasma lemma is called Electroporation.
 A suspension of protoplast with a desired DNA is
prepared. Then, a high voltage current is applied through
the protoplast DNA suspension.
 The electric current leads to the formation of small
temporary holes in the membrane of the protoplasts
through which the DNA can pass.
 After entry into the cell, the Foreign DNA gets incorporated
with the host genome, resulting the genetic transformation.
 The protoplasts are then cultured to regenerate in to whole
plants. Chanie D. .... Dep't of Biotechnology ..........
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243
3) Lypofection:
 Introduction of DNA into cells via lyposomes
 is known as lipofection, lioposomes are small lipid
artificial vesicles.
 The DNA enclosed in the lipid vesicles when mixed
with protoplast under appropriate condition penetrates
into the protoplast where lipase activity of the
protoplast dissolves the lipid vesicles and DNA gets
released for integration into the host genome.
 This method has not been commonly used as it is
difficult to construct the lipid vesicles.
 The success depends upon the protoplast
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4) Particle bambordment / microprojectile / biolistic /
gene gun / particle acceleration
 The process of particle acceleration or biolistics acceleration of
DNA into cells with sufficient force such that a part of it gets
integrated into DNA of target cells.
 The process of transformation employes foreign DNA coated
with minute 0.2-0.7 μm gold or are tungstun particles to
deliver into target plant cells using pressurized helium gas or
electro static energy.
 Because of the physical nature of process there is no
biological limitation to the active DNA delivery that makes
it, genotype independent.
 This method allows the transport of genes into many cells of
nearly any desired position in an experimental system without
too much manual labour.
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5) Microinjection:
The DNA solution is injected directly inside the
cell using capillary glass micropipetts with the
help of micromanipulators of a microinjection
assembly.
 It is easier to use protoplast than cells since cell
wall interferes with the process of microinjection.
The protoplast are usually immobilized in
agarose or on a glass slides coated with
polylysine or by holding them under suction by a
micropipette.
6. Pollen transformation:
o Involves the gene transfer by soaking the pollen
grains in DNA solution prior to their use 247for
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7. DNA Delivery via growing
pollen tubes:
 The stigmas were cut after
pollination exposing the
pollen tubes, the DNA was
introduced onto the cut
surface that presumably
diffused through the
germinating pollen tube into
the ovule.
 This method is simple easy and
very promising provided
consistent result and stable
transformations are achieved
 The mechanism of DNA transfer
into zygote through this method is
not yet established.
8. ETC WU
Chapter Six
6. Transgenic Science and Genetic
Improvement
6.1. Introduction:
 The Plants, Animals and Microorganisms obtained
through genetic engineering contain a gene (or)
genes usually from an unrelated organisms.
 Such genes are called transgenes and organisms
containing transgenes are known as transgenic or
GMO.
 There is differences b/n GMOs and Transgenic
organisms.
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6.2. Transgenic Science in Plant Improvement
and Bio-pharming
 In case of plant, transgenic crop varieties resistant to
herbicides, insects or viruses, delay ripening, and slow fruit
softening were developed.
 Genetic engineering has several potential applications in crop
improvements as follows;
1. Distant Hybridization:
 With the advancement of genetic engineering, it is now
possible to transfer genes between distantly related
species.
 The barriers of gene transfer between species or even genera
have been overcome.
 The desirable genes can be transferred even from lower
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2. Development of Transgenic Plants:
 Genetically transformed plants which contain foreign
genes are called transgenic plants.
 Resistance to diseases, insects and pests, herbicides,
drought; metal toxicity tolerance; induction of male sterility
for plant breeding purpose; and improvement of quality
can be achieved through this recombinant DNA
technology.
eg. BT-cotton, golden rice, plant edible vaccine

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3. Development of Root Nodules in Cereal Crops:
 Leguminous plants have root-nodules which contain
nitrogen fixing bacteria Rhizobium. This bacteria
converts the free atmospheric nitrogen into nitrates in
the root nodules.
 This bacteria in nodules converts the free
atmospheric nitrogen into nitrates in the root nodules.
 The bacterial genes responsible for this nitrogen
fixation can be transferred now to cereal crops like
wheat, rice, maize, barley etc.
 Through the techniques of genetic engineering thus
making these crops too capable of fixing atmospheric
nitrogen. Chanie D. .... Dep't of Biotechnology ..........
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252
4. Development of C4 Plants:
 Improvement in yield can be achieved by improving the
photosynthetic efficiency of crop plants.
 The photosynthetic rate can be increased by
conversion of C3 plants into C4 plants, which can be
achieved either through protoplasm fusion or
recombinant DNA technology.
 C4 plants have higher potential rate of biomass
production than C3 plants.
 Most C4 plants (sorghum, sugarcane, maize, some
grasses) are grown in tropical and subtropical zones.
 Not only in plants, but also it used to for the
improvement of animal
Chanie
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D. .... Dep't oftreats;
Biotechnology .......... 253
Importance of Transgenic Plants
1. Resistance to Viral Diseases
 Plant viruses often cause considerable crop damage and
significantly reduce yields.
 Researchers have used the techniques of genetic engineering
to develop non-conventional types of virus-resistant transgenic
plants.
eg. Transgenes derived from the rice tungro spherical-virus genome
allow the plant to develop defense system
 These methods used “immunization” with viral coat protein
genes, other viral genes, or viral gene antisense sequences to
confer resistance.
i). Coat Protein Mediated Resistance (CP-MR):
 Introduction of viral coat
Chanie D.
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.... Dep't protein
of Biotechnologygene
.......... into plants makes
254 it
 If susceptible strain of a crop is inoculated with a
mild strain of a virus, the susceptible strain
develops resistance against more virulent strain.
eg. tobacco expressing TMV
ii). Satellite RNA Mediated Resistance:
 Satellite RNAs are RNA molecules that are
dependent up on helper virus for its replication
and transmission, even though they are
unrelated to viral genome.
 The presence of SAT RNA leads to reduction in
severity of disease symptoms and thus, have
been used to develop resistance against specific
viruses.
Eg: Engineering cucumber using cucumber
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2. Resistance to Fungal Diseases
i). Antifungal Protein Mediated Resistance:
Introduction of two genes coding for chitinase
and glucanase makes the plant resistant to
fungal infection by degrading the major
constituents of fungal cell wall.
eg. Co expression of chintinase and glucanase
genes in tobacco and tomato plants confers
higher level of resistance than either gene alone.
ii). Antifungal Compound Mediated Resistance:
The low molecule weight compounds such as
Phytoalexins possess antimicrobial properties and
play an important role in plant resistance to fungal
and bacterial pathogens.
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3. Resistance to Bacterial Diseases
 The expression of a bacteriophage T4 lysozyme in
transgenic potato tubers led to increased resistance
in Erwinia carotovora.
 The expression of Barley thionin gene significantly
enhanced the resistance of transgenic tobacco to
bacteria pscudomonas syringae.

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4. Resistance to Insect Pests
 Several insect resistant transgenic varieties have been
tested and released in crops like tomato, potato,
cotton, and maize.
 The genetic transfer of proteins with insecticidal
properties would mean environmentally friendly insect
control ……. Eco-friend.
 If crop plants could be genetically engineered to
produce functional insecticides,
 Then, it might be possible to develop crops that would
be intrinsically tolerant of insect predators and
 Would not need to be sprayed (often six to eight times
during a growing season ) with costly and hazardous
chemical pesticides..… No pollution and economic loss258
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5. Quality Improvement
 Using the rDNA technology, we can develop the
plants with ff characters;
Development of stress-and senescence-tolerant
plants
For fruit ripening condition
For the purpose of flower pigmentation
Modification of plant nutritional content
Modification of food plant taste and appearance
o Preventing discoloration
o Sweetness
Oil bodies and Pharmaceuticals
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Examples of
Transgenic Plants
A. Transgenic Corn
(Bt Corn):
 This is developed by
inserting the gene of Bt
into Corn and it can
produce the toxic
substance, natural
pesticide production.
 Now, they don’t have to
be sprayed with cancer
causing pesticides.
 No environmental
pollution and economic
cost. Chanie D. ..... Biotechnology ..... WU 260
B. Venomous Cabbage:
 Gene from a scorpion tails
inserted into cabbage.
 Cabbage now produces that
chemical and limit pesticide
use while still preventing
insects from damaging crops.
 the toxin is modified, so it isn’t
harmful to humans.
C. Banana Vaccines:
o virus is injected into a banana
and it produces the virus
proteins, not disease.
o When people eat, their
immune systems creates
antibodies to fight Chanie
theD.disease
..... Biotechnology ..... WU 261
D. Golden Rice:
 Golden rice created by
transforming two β-carotene
genes;
 a plant phytoene synthase
(psy)
 a bacterial phytoene
desaturase (crt I)
E. Flavr-SAVR:
 This is produced antisense
technology.
 The polygalactouronase gene,
responsible for fruit decay is
silenced.
F. ETC
Chanie D. ..... Biotechnology ..... WU 262
A n t is
ense
R NA te nt
with
gene chno
ticall logy
P ro b m e
y mo
dified
plant
lems
i g n
ss
g A
di n
ea
e R
om
H Chanie D. .... Dep't of Biotechnology ..........
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263
6.3. Transgenic Science for Animal
Improvement and Bio-pharming
Transgenic Animals:
 are produce for high milk yield, better
quality wool, egg laying frequency, etc.
 Transgenic mice are used as animal
models for understanding many
human diseases and finding
therapeutic drugs.
 Transgenic fish are used as model for
studying the effect of growth hormone
transgene on growth rate, for
understanding the effect of pollutants
or mutagens. Chanie D. .... Dep't of Biotechnology .......... 264
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 Genes inserted into animals, so they produce what
humans need.
 Steps:
1. Construction of transgenes ………… rDNA
Promoter
Gene to be expressed …. GOI
Termination sequence
2. Introduction of foreign genes into the animal
o Microinjection
o Embryonic stem cell mediated
o Retrovirus mediated
3. Screening of transgenic positives
Eg. PCR
4. Further Animal breeding is done to obtain
maximum.
 Three basic methods of producing transgenic animals are
1. DNA Microinjection (pronuclear microinjection)

2. Embryonic stem cell mediated gene transfer

3. Retrovirus mediated gene transfer

Examples of Transgenic Animals
A. Transgenic Cows:
 Gene inserted to increase milk production.

B. Spider Goat:
o gene from spider inserted into goat.
o Goats makes silk of the spider web in their milk.
o Flexible, stronger than steel. Used in bullet proof
jackets.

C. Glow-in-the-Dark Cats
 Scientist used a virus to insert
DNA from jellyfish
 The gene made the cat
produce a fluorescent protein in
its fur.
D. Super Mouse
o Inserting a human
growth hormone gene
into mouse genome
E. Super fish
o growth hormone gene
inserted into fertilized
egg cell
F. Glo fish
o Introducing a fluorescent
protein gene from
jelyfish to embryoChanieofD. ..... Biotechnology ..... WU 271
6.4. Bacterial Gene
Transformation
 Bacteria, such as
E.coli, can take up
and express foreign
DNA, usually in the
form of a plasmid.
 After Transformation,
gene cloning is done
to make lots of
copies of a desired
gene.

Steps of Bacterial Gene
Transformation
1. Choose a bacterial host
eg. E. coli is a model organism
2. Choose a plasmid to transform
 Single restriction site
 Ori rep
 Gene marker
3. Prepare bacterial cells for transformation
a. Treat with calcium chloride – softens
the phospholipid bilayer of the cell
membrane, which allows the
plasmid to pass through
b. Electroporation – brief electric pulse
c. Directly inject plasmid into bacterial
host
4. Plate transformation on
appropriate media
a. Contains nutrients for
bacteria and
b. antibiotic to
distinguish transformed
bacteria from non
transformed bacteria
5. Incubate plates overnight
eg. E.coli grows at body temp.
(37 °C)
6. Analyze plates

6.5. Gene Mapping in Plants and Animals
 A genome map is the graphical description of the location
of genes and DNA markers on the genome of an organism.
……. Landmark of genomes
 Gene mapping describes the methods used to identify
the locus of a gene and the distances between genes.
 The essence of all genome mapping is to place a collection of
molecular markers onto their respective positions on the
genome.
 Molecular come in all forms and genes can be viewed as one
special type of genetic markers in the construction of genome
maps, and mapped the same way as any other markers.
 There are two distinctive types of "Maps" used in the field of
genome mapping: Chanie D. .... Dep't of Biotechnology .......... 275
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 Genetic mapping is based on
 Using genes
 Molecular markers
 Physical mapping is also based on
o Restrction mapping
o Cytogenetic mapping
o STS content mapping
o Radiation hybrid mapping
 A genetic map is constructed using recombination
frequency calculated from the progenies and it is an
indirect method of locating the positions of genes or
DNA markers. The unit of measurement is cM, whereas
 Physical mapping pertains to locating the position of
DNA sequences directly on the chromosome of a large
DNA fragment. The unit of measurement is the base
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276
 Genetic and physical maps illustrate the
arrangement of genes and DNA markers on a
chromosome.
 Genes that are sufficiently close together on a
chromosome will tend to "stick together," and
 the versions (alleles) of those genes that are
together on a chromosome will tend to be inherited
as a pair more often than not. This phenomenon is
called genetic linkage.
 When genes are on separate chromosomes, or

very far apart on the same chromosomes,
they assort independently.

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 Homologous chromosomes are paired chromosomes that
carry the same genes, but may have different alleles of those
genes.

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 Genes are on the same chromosome, but very far
apart, they assort independently due to crossing
over (homologous recombination).
 At this time, new gametes are formed. Among those;

o Parental gametes
o Recombinant gametes (Cross over type)
280
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 Now, we can calculate the recombination frequency
between two gene crosses. …………. RF
 Let’s use the ff (two genes in the fruit fly ( Drosophila),

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 To ensure that the alleles provided by the non-tester
parent and fully determine the phenotype, or
appearance, of the offspring, double heterozygous cross
with a tester.

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 Now, RF = Recombinant / Total offspring x 100
= (151 + 154 / 151 + 154 + 1195 + 1339) x 100
= (305/ 2839) x 100
= 10.7 %
 Using RF, we can figure out the order of genes on a
chromosome.
 Eg.

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 In linkage map, distances can expressed using,
 Centimorgan ….. cM
 Map unit ……. Mu
 RF
 Sometimes, the directly measured recombination
frequency between two genes is not the most
accurate measure of their map distance.
 That's because, in addition to the single crossovers,
double crossovers can also occur.

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I. Determine the parental genotype
 The genotype found most frequently is the parental
genotypes. Therefore, “ABD” and “abd” are the
parental type on the above table.
II. Determine the gene order
 After determined the parental type, we should
determine the double crossover types are always in
the lowest frequency.
 The double crossover event moves the middle allele
from one sister chromatid to the other
 On the double cross over gametes, those which is
d/t from the other with relative to the parental type
is the middle gene.
 Therefore, ABD” and “abd” are the parental and AbD
and aBd are double cross over types. So that gene
“B” is the middle gene. Thus, the order of the gene
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III. Determine the linkage distance
 To find the linkage distance b/n genes, we count
both cross b/n genes (single and double crosses).
 Based on the table above, the distance b/n
A_B = 45 + 50 + 2 + 3 x 100% = 10% = 10 mu.
1000
B_D = 75 + 70 + 2 + 3 x 100% = 15% = 15 mu
1000
A_D = 45 + 50 + 75 + 70 + 2 + 3 x 100% =24.55%
1000 = 24.5 mu
 Hence, the distance b/n “A” and “D” is also “A_B” +
“B_D” this is the real distance.
 But the double crossover detected the linkage
distance 24.5 mu. Therefore, 25 mu – 24.5 mu = 0.5
which is called Chanie
variation.
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Interference and Concidence
 The formation of one chiasmata actualy reduces the
probabilities of another chiasmata forming in an
immediately adjacent regions of the chromosome.
 The strength of interference varies in d/t segments of a
chromosome and usually expressed in terms of concidence
is the ratio of observed to the expected double cross over.
Cc = % observed DCo
% expected DCo
Concidence + interference = 1.0
 Therefore, the degree of interference is measured by the
coefficient of concidence.
 If interference is Chanie
complete (1.0) no crossovers will292be
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Example one:
Given the map distance “A_B” = 10 and “B_C”
= 20, then 0.1x0.2 = 0.02 0r 2% double cross
over are expected if there is no interference.
Suppose we observe 1.6% double crossover in
a test cross experiment.
Solution;
Concidence = 1.6%/2% = 0.8
This means that we observed only 80% of the
expected double crossovers, and
Interference = 1- Cc
= 1- 0.8 = 0.2
Thus, 20% of the expected double
crossoversChanie
didn’t form due to interference. 293
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Eg. Illustration showing a genetic map of the chromosomes from the fruit fly
(Drosophila melanogaster). The names of the genes are shown to the right
of each chromosome. The numbers to the left of each
chromosome represent the distance between these genes.

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Physical Mapping:
 Physical mapping gives an estimation of the
(physical) distance between specific known DNA
sequences on a chromosome.
 The distance between these known DNA
sequences on a chromosome is expressed as the
number of base pairs between them.
 There are several different techniques used for
physical mapping.
 These include:
o Restriction mapping (fingerprint and optical
mapping)
o Fluorescent in situ hybridization (FISH)
mapping
o Sequence tagged site (STS) mapping.
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1. Restriction Mapping:
 This uses specific restriction enzymes to cut an unknown
segment of DNA at short, known base sequences called
restriction sites.
 Restriction enzymes always cut DNA at a specific
sequence of DNA (restriction site).
eg. the restriction enzyme EcoRI always cuts
at the sequence GAATTC/CTTAAG.
Therefore, if we use EcoRI to cut the DNA
we know that the DNA sequence either side
of the cut will be AATT.
 A restriction map shows all the locations of that
particular restriction site (GAATTC) throughout the
genome. Chanie D. .... Dep't of Biotechnology .......... 297
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 A physical map is generated by aligning the
different restriction maps along the chromosomes.
 There are two specific types of Restriction
mapping; optical and fingerprint.
Illustration showing the restriction site for the restriction enzyme

EcoRI. Restriction enzymes
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a. Fingerprint mapping:
o In fingerprint mapping
the genome is broken
into fragments.
o The DNA copies
(clones) are then cut by
restriction enzymes and
the lengths of the
resulting fragments are
estimated using a lab
method
called electrophoresis.
o Electrophoresis separates
the fragments of DNA
according to size resulting
Illustration showing how a DNA fingerprint
in a distinct banding
isDep't
Chanie D. .... created by electrophoresis.
pattern. WU
o The fingerprint map
is constructed by
comparing the
patterns from all
the fragments of
DNA to find areas
of similarity.
o Those with similar

patterns are then
grouped together to
form a map.

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b. Optical Mapping
 Optical mapping uses single
molecules of DNA that are
stretched and held in place
on a slide.
 Restriction enzymes are added to
cut the DNA at specific points
leaving gaps behind.
 The fragments are then stained
with dye and the gaps are
visualized under a fluorescence
microscope.
 The intensity of the fluorescence is
used to construct an optical map
of single molecules.
 These can then be combined and
overlapped to give a global
overview of the genome and aid Illustration showing the process of
with assembling a Chanie sequenced optical ..........
D. .... Dep't of Biotechnology mapping. 301
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genome.
2. Fluorescent in situ Hybridization (FISH) Mapping
 This uses fluorescent probes to detect the location of
DNA sequences on chromosomes.
 First, the probes are prepared.
 The probes are then labelled with fluorescent dye
before being mixed with the chromosome DNA so that it
can bind to a complementary strand of DNA on the
chromosome.
 The fluorescent tag allows the scientist to see the
location of the DNA sequence on the chromosome.
Eg. The ff lustration showing how FISH can be used to produce a
genetic map. The photograph on the left shows Chromosome 17 from
four British peppered moths with fluorescent probes indicating the
physical positions of specific genes. The illustration on the right
shows the relative positions of the genes on the chromosome.
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3. Sequence-Tagged Site (STS) Mapping
 This technique maps the positions of short DNA
sequences that are easily recognizable and only occur
once in the genome.
 These short DNA sequences are called sequence-tagged
sites (STSs).
 To map a set of STSs a collection of overlapping DNA
fragments from a single chromosome or the entire
genome is required.
 To do this, the genome is first broken up into fragments.
 The fragments are then replicated up to 10 times in
bacterial cells to create a library of DNA clones.
 The PCR is then used to determine which fragments
contain STSs. Chanie
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 Special primers are
designed to bind either
side of the STS to
ensure that only that
part of the DNA is
copied.
 If two DNA fragments
are found to contain the
same STS then they
must represent
overlapping parts of the
genome.
 If one DNA fragment
contains two different
STSs then those two
STSs must be near to
each other in the
genome.
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Chapter Seven
7. Other Applications of GE
5.1. Introduction:
 Biotechnology are an industrial process that uses the
scientific research on DNA for practical benefits.
 Biotechnology is synonymous with genetic engineering
because the genes of an organism are changed during
the process and the DNA of the organism is
recombined.
 Recombinant DNA technology is used to make
microbes, plants, and animals that carry genes from
other species.
 It can be used in Chanie the
D. .... Dep'tprenatal diagnosis of human
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 In general, genetic engineering (rDNA technology) is
applicable in the area of;
o Agriculture
o Medicine
o Industry
o Environment

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7.2. Application of Genetic Engineering in
Medicine
o In the field of medicine, biotechnology, especially genetic
engineering plays an important role in the production of
 antibiotics,
 hormones,
 vaccines and
 interferon

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1. Production of Antibiotics:
 Penicillium and Streptomyces fungi are used
for mass production of famous antibiotics
penicillin and streptomycin.
Genetically, efficient strains of these fungi have
been developed to greatly increase the yield of
these antibiotics.
How to produce?
2. Solution of Disputed Parentage:
o Disputed cases of parentage can now be solved
most accurately by recombinant technology than
by blood tests.
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How ?
310
3. Production of Insulin:
o Insulin, a hormone, used by diabetics, is usually
extracted from pancreas of cows and pigs.
o This insulin is slightly different in structure
from human insulin.
o As a result, it leads to allergic reactions in about
5% patients.
o Human gene for insulin production has been
incorporated into bacterial DNA and
o Such genetically engineered bacteria are used for
large scale production of insulin.
o This insulin does not cause allergy.
How to Develop it?
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4. Production of Vaccines:
 Vaccines are now produced by transfer of antigen
coding genes to disease causing bacteria.
 Such antibodies provide protection against the infection by
the same bacteria or virus.
 How?
 How to
develop
Edible
vaccine?

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5. Production of Interferon:
 Interferon’s are virus-induced proteins produced
by virus-infected cells.
Interferon are anti-viral in action and act as first
line of defense against viruses causing serious
infections, including breast cancer and lymph
nodes malignancy.
Natural interferon is produced in very small quality
from human blood cells and very costly also.
It is now possible to produce interferon by
recombinant DNA technology at much cheaper
rate.

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6. Production of Enzymes:
Some useful enzymes can also be produced by
recombinant DNA technique.
 For instance, enzyme urikinase, which is
used to dissolve blood clots, has been
produced by genetically engineered
microorganisms.
7. Gene Therapy:
 Genetic engineering may one day enable the
medical scientists to replace the defective genes
responsible for hereditary diseases.
eg.(haemophilia, phenylketonuria,
alkaptonuria) with normal genes.
 This new system of therapy is called gene
therapy. WU
8. Diagnosis of Disease:
 Recombinant DNA technology has provided a broad
range of tools to help physicians in the diagnosis of
diseases.
 Most of these involve the construction of probes:
short Segments of single stranded DNA attached to
a radioactive or fluorescent marker.
 Such probes are now used for identification of
infectious agents, for instance,
food poisoning Salmonella, Pus forming
Staphylococcus, hepatitis virus, HIV, etc.
 By testing the DNA of prospective genetic disorder
carrier parents, their genotype can be determined
and their chances of producing an afflicted child can
be predicted. Chanie D. .... Dep't of Biotechnology .......... 316
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Some medically useful recombinant products and their
applications:

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7.3. Industrial Application of
Genetic Engineering
 Genetic engineering has been
especially valuable for producing
recombinant microorganisms that
have a wide variety of industrial uses.
Eg. production of modified bacteria
that devour hydrocarbons.
 These microbes are used to destroy
oil slicks and to clean up sites
contaminated with toxic wastes.
 Genetically engineered microbes are
used to produce enzymes used in
laundry detergents and contact lens
solutions.
 Recombinant microbes also are used
to make substances that can be
converted to polymers Chanie D. ..... Biotechnology ..... WU 318
7.4. Application of Genetic Engineering in
Environment
 Genetic engineering is exploiting the huge potential
of microorganisms, plants, animals for the restoration
of the environment.
 It is actively involved in the development of
microorganisms and biocatalysts for remediation of
contaminated environments, and in development of
eco-friendly processes
eg. developing recombinant strain for bio-fuel
production etc.
 A number of genetically engineered microorganisms are
developed which are involved in the bio degradation of
waste materials.
eg. Camphor and Octane degrading genes
 The presence of heavy metals and other toxic organic
materials present in the effluent is a major cause of
concern for the aquatic life.
eg. Eutrophication
 New recombinant strains of Pseudomonas have been
developed which transforms a number of toxic
chemicals.
Eg. hydrocarbons, chlorinated, solvents,
polychlorobiphenyls and metals in a less toxic form.
 Increased level of carbon dioxide is directly linked to
global warming and greenhouse effect. So efforts are
being made to reduce the atmospheric CO2
concentration.
eg. Microalgae like mutants of Anacystis nidulans and
Oocystis sp. are designed for ribulose biphosphate
carboxylase (RUBP-case).
 Genetically modified organisms
are used in clearing up of oil spills
which is a major environmental
hazard.
Eg. Pseudomonas have been
developed to break down a variety
of hydrocarbons present at the oil
spill site.
 The problem of soil pollution
caused due to increased use of
herbicides, pesticides and
insecticides can also be solved by
using recombinant
microorganisms.
eg. toxic genes from Bacillus thuringiensis is
cloned areused as biological
pesticides. Chanie D. ..... Biotechnology ..... WU 322
Chapter Eight
8. Gene Therapy, Introduction and Methods

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Wollo University

College of Natural Science

Module Name: Genomic Science and Bioinformatics

Chanie D. .... Dep't of Biotechnology .......... 2

 Now a day, Biotechnology has five streams;

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2019 Prime Editing Makes Single Stranded Cuts a

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To overcome the limitations of natural vectors,

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The expression of the reporter genes can be

 pOT vectors have higher copy number, but lower

ii. Baculoviral Vectors

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 Replicative form double stranded vector are modified and

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 Highly targeted single-gene sequencing is accurate,

EBA EBB TE Others

Extraction Buffer A (EBA) Per 100 mL:

TE Buffer Per 100 mL

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o Super coiling is important in plasmid preparation due to

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Adenine (A) 15,200

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After initial purification of a given fragment, the

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