Plant Biotechnology

BO-6223
 Content of Theory Course
Unit I
Chapter Introduction to Biotechnology:
No. 1 Definitions, History, Development, Scope and Importance of biotechnology
(Self-study).
Chapter
No. 2 Plant Biotechnology:
Plant Tissue Culture:
Definition, history, principle, scope and Importance of plant tissue culture.
Totipotency, Sterilization methods, Culture media composition, types of
medium and role of hormones in tissue culture.
Inoculation, Incubation and Acclimatization.
Chapter
No. 3 Applications of Plant Tissue Culture in plant improvement and conservation:
Callus, single cell and suspension culture and its significance.
Organ culture: Anther, Embryo and Meristem culture.
Organogenesis, somatic embryogenesis and artificial seeds.
 Biotechnology

Use of Biology to develop new products, methods and organisms

intended to improve human health and society.

It existed from the beginning of the civilization.

 Recombinant DNA technology
• It comprises of altering genetic material to obtain enhanced and
desired characteristics in living organisms or their products.
• It involves splicing or joining DNA fragments from different biological
sources.
Milestones
• 1967 – DNA Ligase
• 1970 – Restriction endonuclease – Hind III (from Haemophilus
influnzae)
• 1971 – Paul Berg – spliced DNA from virus SV40 and lambda
bacteriophage
• 1973 – Stanley Cohen and Herbert Boyer – spliced gene for antibiotic
resistance from Salmonella into E. coli.
Restriction enzyme/
molecular scissors
 Definition
• A restriction enzyme is a protein isolated from bacteria that cleaves
DNA sequences at sequence-specific sites, producing DNA fragments
with a known sequence at each end.
• Arber discovered restriction enzymes while studying a phenomenon
known as host-controlled restriction of bacteriophages.
 Discovery
• 1960 - Werner Arber
• Arber discovered restriction enzymes
while studying a phenomenon known
as host-controlled restriction of
bacteriophages.
• He named it endonuclease R, which
was later changed to EcoB
Arber proposed that
• Bacterial cells were able to protect themselves against foreign DNA.
• Only those bacteriophages that had previously been in contact with
the same bacterial strain could successfully infect new host cells, and
that the previous exposure somehow modified the phage DNA in a
way that protected it from restriction.
• There were specific sites in the genome at which restriction activities
occurred.
 Isolation
• Hamilton Smith and Kent Wilcox isolated and characterized the first
restriction enzyme from Haemophilus influenzae.

• They confirmed that

• Restriction endonuclease degrades foreign phage DNA but not the bacterial
host's DNA
 Isolation
• Hamilton Smith and Kent Wilcox isolated and characterized the first
restriction enzyme from Haemophilus influenzae.

• They confirmed that

• Restriction endonuclease degrades foreign phage DNA but not the bacterial
host's DNA
• Restriction enzymes are extremely selective with regard to where they make
their cuts
 First application of RE
• By Daniel Nathans and Kathleen Danna.
• Digested, the DNA of the eukaryotic virus SV40 into 11 unique
linearfragments.
• Separated the fragments using gel electrophoresis.
 Explanation
• Bacteria protect their DNA by modifying their own recognition
sequences, usually by adding methyl (CH3) molecules to nucleotides
in the recognition sequences
• Bacteriophages that have previously replicated in a particular host
bacterial strain and survived, are similarly modified with methyl-
labelled nucleotides and thereby protected from cleavage within that
same strain.
• The restriction enzymes catalyze the hydrolysis of the bond between
the 3’-oxygen atom and the phosphorus atom in the phosphodiester
backbone of DNA. The enzymes require Mg2+ or other divalent ions
for their activity.
 Current scenario
• More than 800 different restriction enzymes isolated from bacteria.
• They recognize and cut more than 100 different restriction sites.
• Most restriction sites are 4 to 6 bases long,
• Most sites are palindromic
• HindIII
• Isolated from Haemophilus influenzae
• Recognizes the sequence 5'AAGCTT-3' (upper strand)/3'TTCGAA-5' (lower
strand) and cleaves between the two A's on both strands.
 Nomenclature of RE
• Names begin with an italicized three-letter acronym.
• The first letter indicates the first letter of the bacterial genus from
which the enzyme has been isolated and the next two letters are
derived from the specific epithet.
• These may be followed by extra letters or numbers to indicate the
serotype or strain.
• This is followed by a space and a Roman numeral to indicate the
chronology of identification.
• Hind III was the third of four enzymes isolated from Haemophilus
influenza serotype d.
 Categories of restriction enzymes

Cleavage Site
Types Co-factors Example Enzymes
•Cleave at sites away from the
recognition site
Type I ATP, Mg2+ EcoB, EcoK
•Possess both restriction and
methylase activities
2+ •Cleave within or at short specific EcoR I,
Type II Mg
distances from the recognition site BamH I
2+ •Cleave at sites 25 – 27 bp from the
Type III ATP, Mg EcoP I, Hinf III
recognition site
 ISOSCHIZOMERS AND NEOSCHIZOMERS

• Isoschizomers are restriction enzymes with

the same recognition sequence and
cleavage sites. Example: Sph I (CGTAC/G)
and Bbu I (CGTAC/G)

• Neoschizomers are restriction enzymes with

the same recognition sequence but cleave
the DNA at a different site within that
sequence. Example: Tai I (ACGT/) and Mae II
(A/CGT)
PRODUCTS OF RESTRICTION DIGESTION
• Blunt ends possess a 5’-phosphate group that
promotes ligation. They are universally
compatible with other blunt-ended DNA.
• Blunt ends generated by EcoR V

• Sticky ends are small stretches of single-stranded

DNA capable of self-ligation or ligation with a
complementary region from another DNA
molecule. The sticky ends possess 3’- or 5’-
overhangs of 1–4 nucleotides.
• 5’ Cohesive end generated by Bln I
• 3’ Cohesive end generated by Kpn I
 Applications
• Molecular cloning
• Restriction mapping of DNA
• Gene sequencing
• RFLP
 DNA ligase
• DNA ligase is a type of enzyme that facilitates the joining
of DNA strands together by catalyzing the formation of
a phosphodiester bond.
• It plays a role in replication (DNA Ligase I), repair (DNA Ligase III) and
recombination of DNA (DNA Ligase IV).
• They generally repair single-strand breaks in duplex DNA in living
organisms, but some forms (such as DNA ligase IV) they repair
double-strand breaks
 Role of DNA ligase in recombination DNA
technology
• DNA ligation is commonly used in molecular cloning projects to
physically join a DNA vector to a gene of interest. The ends of the
DNA fragments can be blunt or cohesive and must contain
monophosphate groups on the 5' ends.
• This recombinant DNA molecule, can replicate autonomously within a
microbial host as an extra-chromosomal circular DNA (Molecular
cloning).
Building a recombinant plasmid
You must remove or destroy the restriction enzymes (REs)
before you ligate. Otherwise the REs will just recut your
newly ligated DNA.

This is often done by purifying the cut DNA — usually by

running the digest (cut DNA) on an agarose gel and then
cutting out the band of interest.
Bad plasmids
 Alkaline phosphatases
• The enzyme alkaline phosphatase (ALP) has the physiological role
of dephosphorylating compounds.
• They are found across prokaryotes and eukaryotes alike, with the
same general function, but in different structural forms suitable to
the environment they function in.
• Alkaline phosphatase is found in the periplasmic space of E.
coli bacteria. This enzyme is heat stable and has its maximum activity
at high pH.
Application of ALP
in research

Removing these phosphates

prevents the DNA
from ligating (the 5' end
attaching to the 3' end),
thereby keeping DNA
molecules linear until the
next step of the process for
which they are being
prepared.
 T4 Polynucleotide Kinase
• Polynucleotide kinase is a T4 bacteriophage enzyme that catalyzes the
transfer of a gamma-phosphate from ATP to the free hydroxyl end of
the 5' DNA or RNA.

• Transfers the γ-Phosphate from ATP to the 5´ End of DNA for

subsequent ligation
• Plays a critical role in the repair of DNA strand breaks.
Reverse transcriptase (RT)

• Reverse transcriptase is an enzyme

encoded from the genetic material
of retroviruses that catalyzes the
transcription of retrovirus RNA
into DNA.
• Discovered in 1970 – by Temin &
Satoshi, and David Baltimore,
working independently.
• It is also known as RNA-
dependent DNA polymerase
which generates
complementary DNA (cDNA)
from an RNA template, a
process termed reverse
transcription.
• This enzyme is able to
synthesize a double helix DNA
once the single-strand DNA is Double helix
synthesised.
 Two Enzymes in One
• C-DNA synthesis on an RNA template -
performed in the polymerase active
site.
• After building the c-DNA strand, the
enzyme then removes the original RNA
strand by cleaving it into pieces. This is
performed by a nuclease active site.
• It builds a second DNA strand matched
to the one that was just created to
form the final DNA double helix - also
performed by the polymerase site.
 Application of RT in research
• Reverse transcriptase is also a fundamental component of reverse
transcription-polymerase chain reaction (RT-PCR), a powerful tool
used in research.
• RT-PCR is commonly used to quantify the amount of messenger
RNA (mRNA) transcribed from a gene.
• Because RNA is fragile and difficult to study, a strand of
complementary DNA (cDNA) is synthesized from RNA, using reverse
transcriptase during the RT-PCR procedure.
• The cDNA can then be amplified by polymerase chain reaction and
used for subsequent experiments.
 Taq DNA polymerase
• It is a thermostable DNA polymerase I
• Named after the thermophilic bacteria Thermus aquaticus that lives
in hot springs and hydrothermal vents.
• Originally isolated by Chien et al. in 1976
• often abbreviated to Taq or Taq pol
 Taq DNA polymerase
• Optimal temperature - 72°C, nucleotides are incorporated at a rate of
2–4 kilobases per minute.
• Extremely heat resistant with a half-life of 40 minutes at 95°C.
• At temperatures above 90 °C, Taq demonstrates very little or no
activity at all, but the enzyme itself does not denature and remains
intact.
• Due to it’s heat resistant nature, it has replaced the DNA polymerase
from E. coli originally used in PCR.
• Taq DNA polymerase is the most common enzyme used for PCR
amplification.
 Terminal Deoxynucleotidyl Transferase
• Terminal deoxynucleotidyl transferase (TdT) / DNA
nucleotidylexotransferase (DNTT) / terminal transferase
• It is a unique DNA polymerase which catalyzes the polymerization
of deoxyribonucleotides in the absence of a template
• It catalyzes the repetitive addition of deoxyribonucleotides to the 3'-
OH of oligo-deoxyribonucleotides and single-stranded and double-
stranded DNA.
• It requires an oligonucleotide of at least three nucleotides to
serve as a primer
 Steps involved in recombinant DNA technology

3. Isolation of
1. Isolation of 4. Amplification
2. Restriction desired DNA
genetic of desired DNA
enzyme digestion fragment from
material using PCR
agarose gel

5. Ligation of
6. Insertion of
7. Isolation of desired DNA with
recombinant DNA
recombinant cells plasmid DNA –
into host
recombinant DNA
1. Isolation of genetic material
Step 2 and 3
4. Amplification of desired DNA using PCR
5. Ligation of desired
DNA with plasmid DNA –
recombinant DNA
6. Insertion of recombinant DNA into host
• by Transformation - uptake and expression of foreign DNA by a cell,
It was observed that plasmid DNA were taken up easily and expressed
better in certain bacterial cell, hence they were used as vecttors)
7. Isolation of recombinant cells