Gene Transfer in Plants

Plant Biochemistry and Plant Biotechnology
3rd Year Courses Course Code: Bot. 301

Course Title: Plant Biochemistry and Biotechnology
Lecture: 150 Full Marks: 100
Year: III Pass Marks: 35
Unit B: Plant Biotechnology

4. Gene transfer in plants: (10+1+3 hrs) = 14 hrs
A) Concept of gene cloning:
Basic requirements for gene cloning in plants; gene isolation and cloning; Concept
of vectors; marker and reporter genes and their roles in plant transformation;
identification and analysis of cloned genes (colony hybridization, immunological
detection, PCR, blotting).
A) Concept of gene cloning
Gene cloning (DNA cloning)
The production of exact copies (clones) of a particular gene or DNA sequence
using genetic engineering techniques is called gene cloning.
The term gene cloningDNA cloningmolecular cloningrecombinant

DNA technology‖ all refer to same technique.
When DNA is extracted from an organism, all its genes are obtained. In gene
(DNA) cloning a particular gene is copied forming ―clones.
Cloning is one method used for isolation and amplification of gene of interest.
DNA cloning can be achieved by two different methods:
1. Cell based DNA cloning
2. Cell-free DNA cloning (PCR)

The traditional technique for gene cloning involves the transfer of a DNA
fragment of interest from one organism to a self-replicating genetic element,
such as a bacterial plasmid.
This technique is commonly used today for isolating long or unstudied genes
and protein expression.
A more recent technique is the use of polymerase chain reaction (PCR) for
amplifying a gene of interest.
The advantage of using PCR over traditional gene cloning, is the decreased
time needed for generating a pure sample of the gene of interest.
However, gene isolation by PCR can only amplify genes with predetermined
sequences. For this reason, many unstudied genes require initial gene cloning
and sequencing before PCR can be performed for further analysis.

Requirements for Gene Cloning (Cell-based)

DNA fragment containing the desired genes to be cloned.
Restriction enzymes and ligase enzymes.
Vectors – to carry, maintain and replicate cloned gene in host cell.
Host cell– in which recombinant DNA can replicate
Principle of Gene Cloning
A fragment of DNA, containing the gene to be cloned, is inserted into a suitable
vector, to produce a recombinant DNA molecule.
The vector acts as a vehicle that transports the gene into a host cell usually a
bacterium, although other types of living cell can be used.

Within the host cell the vector multiplies, producing numerous identical copies not
only of itself but also of the gene that it carries.
When the host cell divides, copies of the recombinant DNA molecule are passed
to the progeny and further vector replication takes place.
After a large number of cell divisions, a colony, or clone, of identical host cells is
produced.
Each cell in the clone contains one or more copies of the recombinant DNA
molecule; the gene carried by the recombinant molecule is now said to be
cloned.
Plasmids (Vector)
The term plasmid was originally introduced by Lederberg in 1952 to describe
any extra chromosomal hereditary determinant.
Plasmids are those accessory DNA circles which are found in bacteria in
addition to the main chromosome.
They are genetic elements which are made up of DNA.
They are smaller than nuclear DNA and separate from the chromosome.
They are capable of replication and contain only few hundreds of nucleotide
sequence.
Restriction Enzymes
In nature, bacteria use restriction enzymes to cut foreign DNA, such as from
phages or other bacteria.
Most restrictions enzymes are very specific, recognizing short DNA nucleotide
Sequences and cutting at specific point in these sequences.
Steps in Gene Cloning

The basic 7 steps involved in gene cloning are:
Isolation of DNA [gene of interest] fragments to be cloned.
Insertion of isolated DNA into a suitable vector to form recombinant DNA.
Introduction of recombinant DNA into a suitable organism known as host.
Selection of transformed host cells and identification of the clone containing the
gene of interest.
Multiplication/Expression of the introduced Gene in the host.
Isolation of multiple gene copies/Protein expressed by the gene.
Purification of the isolated gene copy/protein.

A. Isolation of the DNA fragment or gene
 The target DNA or gene to be cloned must be first isolated.
 A gene of interest is a fragment of gene whose product (a protein, enzyme or a
hormone) interests us. For example, gene encoding for the hormone insulin.
 The desired gene may be isolated by using restriction endonuclease (RE)
enzyme, which cut DNA at specific recognition nucleotide sequences known as
restriction sites towards the inner region (hence endonuclease) producing blunt
or sticky ends.
 Sometimes, reverse transcriptase enzyme may also be used which
synthesizes complementary DNA strand of the desired gene using its mRNA.
B. Selection of suitable cloning vector
 The vector is a carrier molecule which can carry the gene of interest (GI) into a
host, replicate there along with the GI making its multiple copies.
 The cloning vectors are limited to the size of insert that they can carry.
Depending on the size and the application of the insert the suitable vector is
selected.
 The different types of vectors available for cloning are plasmids,
bacteriophages, bacterial artificial chromosomes (BACs), yeast artificial
chromosomes (YACs) and mammalian artificial chromosomes (MACs).
 However, the most commonly used cloning vectors include plasmids and
bacteriophages (phage λ) beside all the other available vectors.

C. Essential Characteristics of Cloning Vectors
All cloning vectors are carrier DNA molecules. These carrier molecules should
have few common features in general such as:
 It must be self-replicating inside host cell.
 It must possess a unique restriction site for RE enzymes.
 Introduction of donor DNA fragment must not interfere with replication property
of the vector.
 It must possess some marker gene such that it can be used for later
identification of recombinant cell (usually an antibiotic resistance gene that is
absent in the host cell).
 They should be easily isolated from host cell.

D. Formation of Recombinant DNA

The plasmid vector is cut open by the same RE enzyme used for isolation of
donor DNA fragment.
The mixture of donor DNA fragment and plasmid vector are mixed together.
In the presence of DNA ligase, base pairing of donor DNA fragment and plasmid
vector occurs.
The resulting DNA molecule is a hybrid of two DNA molecules – the GI and the
vector. In the terminology of genetics this intermixing of different DNA strands is
called recombination.
Hence, this new hybrid DNA molecule is also called a recombinant DNA
molecule and the technology is referred to as the recombinant DNA
technology.
E. Transformation of recombinant vector into suitable host

 The recombinant vector is transformed into suitable host cell mostly, a bacterial
cell.
 This is done either for one or both of the following reasons:
To replicate the recombinant DNA molecule in order to get the multiple
copies of the GI.
To allow the expression of the GI such that it produces its needed protein
product.
 Some bacteria are naturally transformable; they take up the recombinant
vector automatically.
 For example: Bacillus, Haemophillus, Helicobacter pylori, which are naturally
competent.
Some other bacteria, on the other hand require the incorporation by artificial
methods such as Ca ++ ion treatment, electroporation, etc.

F. Isolation of Recombinant Cells
The transformation process generates a mixed population of transformed and
non-transformed host cells.
The selection process involves filtering the transformed host cells only.
For isolation of recombinant cell from non-recombinant cell, marker gene of
plasmid vector is employed.
For examples, PBR322 plasmid vector contains different marker gene (Ampicillin
resistant gene and Tetracycline resistant gene). When pst1 RE is used it knock
out Ampicillin resistant gene from the plasmid, so that the recombinant cell
become sensitive to Ampicillin.

G. Multiplication of Selected Host Cells
Once transformed host cells are separated by the screening process; becomes
necessary to provide them optimum parameters to grow and multiply.
In this step the transformed host cells are introduced into fresh culture media .
At this stage the host cells divide and re-divide along with the replication of the
recombinant DNA carried by them.
If the aim is obtaining numerous copies of GI, then simply replication of the host
cell is allowed. But for obtaining the product of interest, favourable conditions
must be provided such that the GI in the vector expresses the product of
interest.
H. Isolation and Purification of the Product
The next step involves isolation of the multiplied GI attached with the vector or of
the protein encoded by it.
This is followed by purification of the isolated gene copy/protein.
Applications of Gene Cloning

A particular gene can be isolated and its nucleotide sequence determined
Control sequences of DNA can be identified & analyzed
Protein/enzyme/RNA function can be investigated
Mutations can be identified, e.g. gene defects related to specific diseases
organisms can be ‗engineered‘ for specific purposes, e.g. insulin production,
insect resistance, etc.
Concept of vectors
Cloning vector
Cloning vector is a small DNA molecule capable of self-replication inside the
host cell.
Cloning vector is used for replicating donor DNA fragment within host cell.
Concept of vectors
Characteristics of cloning vectors
it must be small in size.
It must be self-replicating inside host cell.
It must possess restriction site for Restriction Endonuclease enzymes.
Introduction of donor DNA fragment must not interfere with replication property
of the vector.
It must possess some marker gene such that it can be used for later
identification of recombinant cell.
It must possess multiple cloning site.

Concept of vectors
Types of Cloning Vectors
There are the following different types of cloning vectors:
1. Plasmids
 These were the first vectors used in gene cloning.
 These are found in bacteria, eukaryotes and archaea.
 These are natural, extrachromosomal, self-replicating DNA molecules.
 They have a high copy number and possess antibiotic-resistant genes.
 They encode proteins which are necessary for their own replication.
 pBR322, pUC18, F-plasmid are some of the examples of plasmid vectors

Concept of vectors
Concept of vectors
2. Bacteriophage
The viruses that infect bacteria are called bacteriophage. These are intracellular
obligate parasites that multiply inside bacterial cell by making use of some or all
of the host enzymes.
Bacteriophages have a very high significant mechanism for delivering its
genome into bacterial cell. Hence it can be used as a cloning vector to deliver
larger DNA segments.
Most of the bacteriophage genome is non-essential and can be replaced with
foreign DNA.
Using bacteriophage as a vector, a DNA fragment of size up to 20 kb can be
transformed.
Concept of vectors
2. Bacteriophage
These are more efficient than plasmids for cloning large DNA inserts.
Phage λ and M13 phage are commonly used bacteriophages in gene cloning.
The screening of phage plaques is much easier than the screening of

recombinant bacterial colonies.
Plaque assay is a well established method for measuring virus concentration as it relates to infectious dose.
Concept of vectors
3. Bacterial artificial chromosomes (BACs)
Bacterial artificial chromosomes (BACs) are simple plasmid which is designed to
clone very large DNA fragments ranging in size from 75 to 300 kb.
BACs basically have marker like sights such as antibiotic resistance genes and
a very stable origin of replication (ori) that promotes the distribution of plasmid
after bacterial cell division and maintaining the plasmid copy number to one or
two per cell.
BACs are basically used in sequencing the genome of organisms in genome
projects (example: BACs were used in human genome project).
Several hundred thousand base pair DNA fragments can be cloned using BACs.
Concept of vectors
Fig: Bacterial artificial chromosomes (BACs)

Concept of vectors
4. Yeast artificial chromosomes (YACs)
YACs are yeast expression vectors.
A very large DNA fragments whose sizes ranging from 100 kb to 3000 kb can be
cloned using YACs.
Mostly YACs are used for cloning very large DNA fragments and for the physical
mapping of complex genomes.
YACs have an advantage over BACs in expressing eukaryotic proteins that
require post translational modifications.
But, YACs are known to produce chimeric effects which make them less stable
compared to BACs.
Concept of vectors
Concept of vectors
Human artificial chromosomes (HACs)
Human artificial chromosomes (HACs) or mammalian artificial chromosomes
(MACs) are still under development.
HACs are microchromosomes that can act as a new chromosome in a
population of human cells.
HACs range in size from 6 to 10 Mb that carry new genes introduced by human
researchers.
HACs can be used as vectors in transfer of new genes, studying their
expression and mammalian chromosomal function can also be elucidated
using these microchrosomes in mammalian system.

Concept of vectors
Concept of vectors
Uses of Vectors
Vectors have been developed and adapted for a wide range of uses. Two primary
uses are:
To isolate, identify and archive fragments of a larger genome
To selectively express proteins encoded by specific genes.
Vectors were the first DNA tools used in genetic engineering, and continue to be
cornerstones of the technology.

Marker genes
Marker genes
Marker systems are tools for studying the transfer of genes into an experimental
organism.
In gene transfer studies, a foreign gene, called a transgene, is introduced into an
organism, in a process called transformation.
A common problem for researchers is to determine quickly and easily if the target
cells of the organism have actually taken up the transgene.
 A marker allows the researcher to determine whether the transgene has been
transferred, where it is located, and when it is expressed.
.
Marker genes
Marker genes
Marker genes exist in two broad categories:
I. Selectable marker genes
II. Reporter genes

1. Selectable Marker Genes
The selectable marker genes are usually an integral part of plant transformation
system. They are present in the vector along with the target gene. In a majority of
cases, the selection is based on the survival of the transformed cells when grown
on a medium containing a toxic substance (antibiotic, herbicide, antimetabolite).
This is due to the fact that the selectable marker gene confers resistance to
toxicity in the transformed cells, while the non- transformed cells get killed.
A large number of selectable marker genes are available and they are grouped
into three categories— antibiotic resistance genes, antimetabolite marker
genes and herbicide resistance genes.
Marker genes
(a) Antibiotic Resistance Genes
In many plant transformation systems, antibiotic resistance genes (particularly of E.
coli) are used as selectable markers. Despite the plants being eukaryotic in nature,
antibiotics can effectively inhibit the protein biosynthesis in the cellular organelles,
particularly in chloroplasts.
Eg: Neomycin phosphotransferase II (npt II gene):
The most widely used selectable marker is npt II gene encoding the enzyme
neomycin phosphotransferase II (NPT II). This marker gene confers resistance to
the antibiotic kanamycin. The trans-formants and the plants derived from them can
be checked by applying kanamycin solution and the resistant progeny can be
selected.
Marker genes
(b) Antimetabolite Marker Genes
Eg: Dihydrofolate reductase (dhfr gene)
The enzyme dihydrofolate reductase, produced by dhfr gene is inhibited by the
antimetabolite methotrexate.
A mutant dhfr gene in mouse that codes for this enzyme which has a low affinity to
methotrexate has been identified.
This dhfr gene fused with CaMV promoter results in a methotrexate resistant marker
which can be used for the selection of transformed plants.

Marker genes
(c) Herbicide Resistance Markers
Eg: Enolpyruvylshikimate phosphate synthase (epsps/aroA genes):
The herbicide glyphosate inhibits photosynthesis. It blocks the activity of
enolpyruvylshikimate phosphate (EPSP) synthase, a key enzyme involved in
the biosynthesis of phenylalanine, tyrosine and tryptophan.
Mutant strains of Agrobacterium and Petunia hybrida that are resistant to
glyphosate have been identified. The genes epsps/aroA confer resistance to
transgenic plants which can be selected.

Marker genes
Marker genes
Reporter genes
Gene whose products are easily detected or monitered.
A gene that is used to `tag' another gene or DNA sequence of interest.
Identifying whether a certain gene has been taken up by cell.
Marker genes
Measurement of gene expression
Easily quantifiable
Relatively rapid degradation of the enzyme
Lack of endogenous activity in the concerned cell
Should not be toxic to cells
Assay should be sensitive and reliable

Marker genes
Types of Reporter gene
Scorable reporter genes
Expression of this results in quantifiable phenotype
Easily detected through highly sensitive enzyme analyzes
Selectable reporter genes
Expression of resistance to a toxin
Selection of tranformants from nontransformants in growth media containing
selective agent.
Marker genes
Scorable reporter gene
Green Fluorescent Protein
Derived from jellyfish Aequorea victoria
Formed by nucleophilic reaction between C-ter of S with N-ter of G, formed
imidazoline heterocyclic ring which oxidise with Y to yield floroscence .
Variants of GFP
Yellow Fluorescent Protein
Formed by mutation of Thr 203 residue to tyrosine
Blue Fluorescent Protein
Modification of tyr66 to his
Cyan Fluorescent Protein
Modification of tyr66 to tryptophan
Green Fluorescent Protein
Derived from Discosoma striata (Ds Red)
Alternation of >30 amino acid to yield RFP-1
Marker genes
Selectable reporter gene
Antibiotic Resistance Genes
1. Neomycin phosphotransferase II (npt II gene)
Derived from the transposon Tn5 code foraminoglycoside 3` phosphotransferase
Resistance to the antibiotic kanamycin neomycin by phosphorylation
2. Hygromycin phosphotransferase (hpt gene)
Derives from E. coli
Resistant against hygromycin by phosphorylation

Marker genes
Herbicide Resistance Markers
1.Phosphinothricin acetytransferase (pat/bar gene)
Derived from Streptomyces hygroscopicus
Converts herbicides into acetylated forms
Resistant against Bialophos, phosphinothricin and glufosinate
2. Enolpyruvylshikimate phosphate synthase (epsps/aroA genes)
Derived from Agrobacterium sp CP4
Resistance against glyphosate which blocks the activity of EPSP synthase, a key
enzyme involved in the biosynthesis of aromatic amino acid
3. Bromoxynil nitrilase (bxn gene)
Derived from Klebsiella pneumoniae
The herbicide bromoxynil inhibits photosynthesis (photosystem II)
 Encode a specific nitrilase that converts bromoxynil to its primary metabolite 3,5-
dibromo-4-hydroxybenzoic acid
Marker genes
Reporter gene for functional genomics
Identify a promoter, to study the expression pattern and strength of the promoter
Reporter gene is simply placed under the control of the target promoter
Gene expression assays
Reporter is directly attached to the gene of interest to create a gene fusion
The two genes are under the same promoter elements and are transcribed and
then translated into protein
Transformation and transfection assays
Reporter genes expressed under their own promoter independent from that of the
introduced gene of interest Reporter gene can be expressed constitutively or
inducibly
Identification and analysis of cloned genes
After successful introduction of recombinant DNA molecule into appropriate host
cells, the transformants (i.e. the host cells that contain the desired genes) are
selected and separated from the colony.
More efficient methods have been designed to identify and select the recombinant
containing the desired DNA fragment. These are:
1. Colony hybridization
2. Immunological detection
3. PCR
4. Blotting
1. Colony hybridization
Colony hybridization is a method of selecting bacterial colonies with desired
genes. This method was discovered by Michael Grunstein and David S.
Hogness.
Colony hybridization can define as the method for the isolation of the specific
DNA sequences or genes from the bacterial cells containing hybrid DNA, by the
means of a nitrocellulose membrane filter.
The transferring medium then goes through several chemical and physical
treatments.
Method
Colony hybridization begins with culturing thinly populated bacterial colonies on
a nutrient agar plate.
These colonies are symmetrically replicated on a nitrocellulose filter by direct
contact, after which the cells on the filter membrane are lysed (break down of a
cell caused by damage to its plasma membrane) and their DNA is denatured,
allowing it to bind to the filter.
These DNA clusters are then hybridized to a desired radioactively-labelled RNA
or DNA probe (DNA probes are stretches of single-stranded DNA used to detect
the presence of complementary nucleic acid sequences (target sequences) by
hybridization) and screened by autoradiography.

DNA clusters that exhibit a desired gene are then matched up to the
corresponding (living) bacterial colonies, which can be isolated for further
growth and experimentation.
Compare the developed autoradiogram with the master plate to identify the
colonies containing a gene of interest.
The cells which contain the desired gene can grow in the liquid medium and
can further process for the isolation of recombinant plasmid DNA.
Transferring medium of Colony Hybridization
The nitrocellulose filter paper is the transferring medium of the colony
hybridization which forms replicas of the master plate.

The nitrocellulose acts as a membrane which contains the exact copies of the
gene to that of the master plate. Nitrocellulose filter paper acts as the ―Blotting
pad‖ (is a pad of blotting paper).
Therefore, the colony hybridization method is the ―Screening technique‖ which
makes the use of the radioactive probe.
The radioactively labelled probe then screen or isolate the particular gene from
the number of bacterial colonies.
..\Study_Videos\Colony hybridization method _ screening genomic or

cDNA libraries.mp4
https://www.youtube.com/watch?v=FtNreXY7poA
2. Immunological detection
Developed in 1970 when plasmid vectors are used to construct genomic libraries.
Immunological screening involves the use of antibodies that specifically
recognize antigenic determinants on the polypeptide.
This technique can be applied to any protein for which an antibody is available.
The molecular target for recognition is generally an Epitope (the part of an
antigen molecule to which an antibody attaches itself).
Earlier immune screening methods employed radio-labeled primary antibodies to
detect antibody binding to the nitrocellulose sheet.
It is now superseded or updated by antibody sandwiches resulting in highly
amplified signals.
The secondary antibody recognizes the constant region of the primary antibody
and is, additionally, conjugated to an easily assayable enzyme (e.g. horseradish
peroxidase or alkaline phosphatase) which can be analyzed using colorimetric
change or emission of light using X-ray film.
Procedure
In this technique, the cells are grown as colonies on master plates and transferred
to a solid matrix.
These colonies are subjected to lysis releasing the proteins which bind to the
matrix.
These proteins are treated with a primary antibody which specifically binds to the
protein (acts as antigen). The unbound antibodies are removed by washing.

 A secondary antibody is added which specifically binds to the primary antibody
removing the unbound antibodies by washing.
The secondary antibody carries an enzyme label (e.g., horse radish peroxidase
or alkaline phosphatase) bound to it which converts colorless substrate to
colored product.
The colonies with positive results (i.e. colored spots) are identified and sub
cultured from the master plate).
..\Study_Videos\IMMUNOLOGICAL SCREENING FOR RECOMBINANT CLONES.mp4

Polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) is a laboratory technique used to amplify
DNA sequences.
The method involves using short DNA sequences called primers to select the
portion of the genome to be amplified.
The temperature of the sample is repeatedly raised and lowered to help a DNA
replication enzyme copy the target DNA sequence.
The technique can produce a billion copies of the target sequence in just a few
hours.
..\Study_Videos\Polymerase Chain Reaction (PCR).mp4

PCR was developed in 1983 by Kary B. Mullis, an American biochemist who
won the Nobel Prize for Chemistry in 1993 for his invention.
Before the development of PCR, the methods used to amplify or generate copies
of recombinant DNA fragments were time-consuming and labour-intensive.
In contrast, a machine designed to carry out PCR reactions can complete many
rounds of replication, producing billions of copies of a DNA fragment, in only a
few hours.
What is PCR?
PCR is a method of copying DNA molecules.
DNA replication is common in life; for example it takes place inside your own
cells every time they divide.
An enzyme known as polymerase uses, one strand of DNA as a template to
create a complementary strand. The result is that one double stranded DNA
molecule is converted into two, both identical to the first.
PCR, or the polymerase chain reaction, adds two components to this process.
The initial reaction yields twice the number of starting molecules, but then is
immediately followed by a subsequent reaction, which yields twice the molecules
as the first reaction. This is why PCR is known as a chain reaction.

Commonly 25-40 reactions are chained together, theoretically resulting in 225 –
240 more molecules of DNA then were initially present.
Additionally, the goal of a PCR reaction is commonly to replicate only a portion
of the genome of interest.
For example, somewhere between 75- 1000 bases, instead of the entire human
genome of 3 billion bases. As PCR produces billions of copies of only the
DNA of interest, this process is known as “amplification”.

Why is PCR important?

PCR and related techniques have many applications. Here are just a few.
 Human Diagnostics
Detecting viral infections (HIV, COVID-19 etc.)
Detecting bacterial infections (Tuberculosis, etc.)
Genotyping (detecting genetic variants, which can indicate susceptibility to
disease)
Environmental Monitoring
Water quality monitoring
Food safety testing
Scientific Research
Preparing DNA to sequence
Monitoring gene expression levels
Manipulating DNA in genetic engineering and synthetic biology
How does PCR work?
The principles behind every PCR, whatever the sample of DNA, are the same.
Five core ‗ ingredients‘ are required to set up a PCR. These are:
The DNA template to be copied.
Primers, short stretches of DNA that initiate the PCR reaction, designed to bind
to either side of the section of DNA you want to copy.
DNA nucleotide bases (also known as dNTPs). DNA bases (A, C, G and T) are
the building blocks of DNA and are needed to construct the new strand of DNA.
Taq polymerase enzyme, to add in the new DNA bases
Buffer to ensure the right conditions for the reaction.
PCR involves a process of heating and cooling called thermal cycling which is
carried out by machine.
Three main stages

There are three main stages:
1. Denaturing
When the double-stranded template DNA is heated to separate it into two single
strands.
2. Annealing
When the temperature is lowered to enable the DNA primers to attach to the template
DNA.
3. Extending
When the temperature is raised and the new strand of DNA is made by the Taq
polymerase enzyme.
These three stages are repeated 20-40 times, doubling the number of DNA
copies each time.
A complete PCR reaction can be
performed in a few hours, or even less
than an hour with certain high-speed
machines.
After PCR has been completed, a
method called electrophoresis can be
used to check the quantity and size of
the DNA fragments produced.

What happens at each stage of PCR?

1. Denaturing stage
During this stage the cocktail containing the template DNA and all the other core
ingredients is heated to 94- 95⁰C.
The high temperature causes the hydrogen bonds between the bases in two
strands of template DNA to break and the two strands to separate.
This results in two single strands of DNA, which will act as templates for the
production of the new strands of DNA.
It is important that the temperature is maintained at this stage for long enough to
ensure that the DNA strands have separated completely.
This usually takes between 15-30 seconds.

2. Annealing stage
During this stage the reaction is cooled to 50-65⁰C. This enables the primers to
attach to a specific location on the single-stranded template DNA by way of
hydrogen bonding (the exact temperature depends on the melting temperature of
the primers you are using).
Primers are single strands of DNA or RNA sequence that are around 20 to 30
bases in length.
The primers are designed to be complementary in sequence to short sections of

DNA on each end of the sequence to be copied.
Primers serve as the starting point for DNA synthesis. The polymerase enzyme
can only add DNA bases to a double strand of DNA. Only once the primer has
bound can the polymerase enzyme attach and start making the new
complementary strand of DNA from the loose DNA bases.
The two separated strands of DNA are complementary and run in opposite
directions (from one end - the 5‘ end – to the other - the 3‘ end); as a result,
there are two primers – a forward primer and a reverse primer.
This step usually takes about 10-30 seconds.
3. Extending stage
During this final step, the heat is increased to 72⁰C to enable the new DNA to be
made by a special Taq DNA polymerase enzyme which adds DNA bases.
Taq DNA polymerase is an enzyme taken from the heat-loving bacteria Thermus
aquaticus.
This bacteria normally lives in hot springs so can tolerate temperatures above
80⁰C. The bacteria's DNA polymerase is very stable at high temperatures, which
means it can withstand the temperatures needed to break the strands of DNA
apart in the denaturing stage of PCR.
DNA polymerase from most other organisms would not be able to withstand
these high temperatures, for example, human polymerase works ideally at 37˚C
(body temperature).
72⁰C is the optimum temperature for the Taq polymerase to build the
complementary strand. It attaches to the primer and then adds DNA bases to the
single strand one-by-one in the 5‘ to 3‘ direction.
The result is a brand new strand of DNA and a double-stranded molecule of
DNA.
The duration of this step depends on the length of DNA sequence being amplified
but usually takes around one minute to copy 1,000 DNA bases (1Kb).
These three processes of thermal cycling are repeated 20-40 times to produce
lots of copies of the DNA sequence of interest.
The new fragments of DNA that are made during PCR also serve as templates to
which the DNA polymerase enzyme can attach and start making DNA.
The result is a huge number of copies of the specific DNA segment produced in a
relatively short period of time.
Blotting
Blotting is a method in which a macromolecule (DNA, RNA and protein) is

immobilized on a solid medium and then investigated with a detectable ligand
(molecule that binds to another (usually larger) molecule) to determine whether
the macromolecule binds specifically to its ligand.
Depending on whether the immobilized macromolecule is DNA, RNA or protein,

one generates DNA blots (Southern blots), RNA blots (Northern blots) or
protein blots (Western blots).
The macromolecule can be applied to the blotting matrix directly (dot blot), or it
can be derived and eluted(remove (an adsorbed substance) by washing with a
solvent) from an electrophoretic gel (gel blot).
Southern blotting
 Southern blotting is an example of RFLP (restriction fragment length
polymorphism). It was developed by Edward M. Southern (1975).
 Southern blotting is a hybridization technique for identification of particular size
of DNA from the mixture of other similar molecules. This technique is based on
the principle of separation of DNA fragments by gel electrophoresis and
identified by labelled probe hybridization.
 Basically, the DNA fragments are separated on the basis of size and charge
during electrophoresis. Separated DNA fragments after transferring on nylon
membrane, the desired DNA is detected using specific DNA probe that is
complementary to the desired DNA.
 A hybridization probe is a short (100-500bp), single stranded DNA. The probes

are labeled with a marker so that they can be detected after hybridization.
Procedure/ Steps
1. Restriction digest: by RE enzyme and amplification by PCR
2. Gel electrophoresis: SDS gel electrophoresis
3. Denaturation: Treating with HCl and NaOH
4. Blotting
5. Baking and Blocking with casein in BSA
6. Hybridization using labelled probes
7. Visualization by autoradiogram
Work flow for Southern Blot

Step 1: DNA digestion
The DNA is fragmentized by using suitable restriction enzyme. RE cuts the DNA
at specific site generating fragments.
The number of fragments of DNA obtained by restriction digest is amplified by
PCR.
Step 2: Gel electrophoresis
Fragmented DNA is typically electrophoresed on an agarose gel to separate the
fragments according to their molecular weights. Acrylamide gels can alternatively
be used for good resolution of smaller DNA fragments (<800 bp).
Step 3: Denaturation
The SDS gel after electrophoresis is then soaked in alkali (NaOH) or acid (HCl) to
denature the double stranded DNA fragments.
DNA strands get separated.
Step 4: Blotting
The separated strands of DNA is then transferred to positively charged

membrane nylon membrane (Nitrocellulose paper) by the process of blotting.
Traditional transfer of DNA is done overnight using an upward-transfer method.

For reliable and consistent transfer with minimal background, Nylon Membranes
are highly recommended.
Step 5: Baking and blocking
After the DNA of interest bound on the membrane, it is baked on autoclave to fix
in the membrane.
The membrane is then treated with casein or Bovine serum albumin (BSA) which
saturates all the binding site of membrane.
Step 6: Hybridization with labelled probes
The DNA bound to membrane is then treated with labeled probe.
The labeled probe contains the complementary sequences to the gene of

interest.
The probe bind with complementary DNA on the membrane since all other non-
specific binding site, on the membrane has been blocked by BSA or casein.
Step 7: Visualization by Autoradiogram
The membrane bound DNA labeled with probe can be visualized under
autoradiogram which give pattern of bands.
Identification and analysis of cloned gen

Gene Transfer in Plants

Uploaded by

Copyright:

Available Formats

Gene Transfer in Plants

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Gene Transfer in Plants

Uploaded by

Copyright:

Available Formats

Plant Biochemistry and Plant Biotechnology

3rd Year Courses Course Code: Bot. 301

Unit B: Plant Biotechnology

Gene cloning (DNA cloning)

The production of exact copies (clones) of a particular gene or DNA sequence

using genetic engineering techniques is called gene cloning.

The term gene cloningDNA cloningmolecular cloningrecombinant

(DNA) cloning a particular gene is copied forming ―clones.

DNA cloning can be achieved by two different methods:

1. Cell based DNA cloning

2. Cell-free DNA cloning (PCR)

fragment of interest from one organism to a self-replicating genetic element,

such as a bacterial plasmid.

and protein expression.

amplifying a gene of interest.

and sequencing before PCR can be performed for further analysis.

Requirements for Gene Cloning (Cell-based)

Principle of Gene Cloning

A fragment of DNA, containing the gene to be cloned, is inserted into a suitable

vector, to produce a recombinant DNA molecule.

bacterium, although other types of living cell can be used.

only of itself but also of the gene that it carries.

to the progeny and further vector replication takes place.

molecule; the gene carried by the recombinant molecule is now said to be

Steps in Gene Cloning

Isolation of DNA [gene of interest] fragments to be cloned.

Insertion of isolated DNA into a suitable vector to form recombinant DNA.

Introduction of recombinant DNA into a suitable organism known as host.

Multiplication/Expression of the introduced Gene in the host.

Isolation of multiple gene copies/Protein expressed by the gene.

Purification of the isolated gene copy/protein.

A. Isolation of the DNA fragment or gene

 The target DNA or gene to be cloned must be first isolated.

 A gene of interest is a fragment of gene whose product (a protein, enzyme or a

 The desired gene may be isolated by using restriction endonuclease (RE)

enzyme, which cut DNA at specific recognition nucleotide sequences known as

 Sometimes, reverse transcriptase enzyme may also be used which

 The different types of vectors available for cloning are plasmids,

bacteriophages, bacterial artificial chromosomes (BACs), yeast artificial

chromosomes (YACs) and mammalian artificial chromosomes (MACs).

bacteriophages (phage λ) beside all the other available vectors.

have few common features in general such as:

 It must be self-replicating inside host cell.

 It must possess a unique restriction site for RE enzymes.

identification of recombinant cell (usually an antibiotic resistance gene that is

absent in the host cell).

 They should be easily isolated from host cell.

D. Formation of Recombinant DNA

E. Transformation of recombinant vector into suitable host

methods such as Ca ++ ion treatment, electroporation, etc.

F. Isolation of Recombinant Cells

The transformation process generates a mixed population of transformed and

non-trans- formed host cells.

For isolation of recombinant cell from non-recombinant cell, marker gene of

plasmid vector is employed.

become sensitive to Ampicillin.

G. Multiplication of Selected Host Cells

necessary to provide them optimum parameters to grow and multiply.

recombinant DNA carried by them.

H. Isolation and Purification of the Product