Deoxyribonucleic acid

(DNA)
There are two types of nucleic acids:
• DNA (Deoxyribonucleic acids)
• RNA (Ribonucleic acids)
Molecular Scissors
Molecular scissors also called the restriction
enzyme is a DNA cutting enzyme that recognizes
a specific target sequence and cut DNA into two
pieces at near that site
Types of restriction enzymes
Types Co-factor Cleavage site Example enzyme
Type I ATP, Ado MET, Mg2+ • Cleave at site away EcoB I (Catalog No.
from the recognition R0260)
site
• Possess both
restriction and
mythylase activities

Type II Mg2+ • Cleave within or at EcoR I (Catalog No.

short specific distances R6265)
from the recognition Bam H I (Catalog No.
site R0260)

Type III ATP, Mg2+ • Cleave at sites 25-27 Eco I

BP from the Hinf III
recognition site
Type IIII Mg2+ • Cleave close to or
within the recognition
sequence
• Target modified DNA,
such as methylated,
hydroxymethylated
DNA
Factors affecting the activity of
restriction enzymes
• Star activity – they cleave at non-specific sites.
Some factors that induce start activity are high
salt and glycerol concentration etc.
• Methylated DNA – methylated DNA are resistant
to cleavage by certain restriction enzymes.
• Temperature – endonucleases optimally digest
the target DNA at 37°C
Molecular glue
• In DNA replication, ligase’s job is to join together
fragments of newly synthesized DNA to form a
seamless strand.
• If two pieces of DNA have matching ends, DNA
ligase can join them together to make an
unbroken molecule
• DNA ligase do this by using ATP as an energy
source
 T4 DNA Ligase
APPLICATIONS • Joining DNA with cohesive or
blunt termini
• Repairing nicks in duplex DNA,
RNA or DNA-RNA hybrids
• Ligase-mediated RNA detection
• Site-directed mutagenesis
• Amplify fragment length
polymorphisms
SUBSTRATE • dsDNA
• dsRNA
• Phosphorylated proteins
T4 RNA ligase
• RNA 3’-end labeling with
cytidine 3’,5’-bis [alpha—P]
Phosphate
• Joining RNA to RNA
• Circularization of synthetic
oligonucleotides
• Site-specific generation of
composite primers for PCR
• ssDNA
• ssRNA
• Poly and oligonucleotides
Reaction catalyzed ATP-dependent intra and
intermolecular formation of a
phosphodiester bond between
juxtaposed
ATP-dependent intra and
intermolecular formation of
phosphodiester bonds between 5’-
phosphate and 3’-hydroxyl termini
termini of poly and oligonucleotides,
ssRNA AND ssDNA.
CRISPR/Cas9

• CRISPR/Cas9 is a simple and efficient genome

editing tool.
• OriGene offers genome-wide CRISPR gene
knockout/knockin kits containing 2 gRNA vectors
and donor DNA.
Types of CRISPR knockout kits
There are two types:
• Non-homology mediated CRISPR gene knockout
kits
• HDR mediated CRISPR gene knockout kits
 Recombinant DNA and RNA
technology
Recombinant DNA Technology is defined by
“the joining together of DNA molecules from
different organisms and inserting it into a host
organism to produce new genetic combinations
that are of value to science, medicine, agriculture
and industry. of value to science, medicine,
agriculture and industry.
From: An Introduction to Ethical, Safety and Intellectual Property Rights Issues in Biotechnology, 2017
• joining together of DNA molecules from different organisms and
Tissue plasminogen activator
• When wounds start to bleed, a blood clot forms to stop the
blood flow and later the blood clot is dissolved.
• Plasmin is found in the blood in an inactive called
plasminogen
• (TPA) activates the conversion of plasminogen to plasmin
• Recombinant DNA technology was useful in isolating the
gene that codes for TPA in humans and then using this gene
to manufacture the protein in large amounts for medicinal
purposes
Gene therapy
Human gene therapy (HGT) is defined as the transfer of DNA
encoding a therapeutic gene to the somatic cells of a patient in
order to treat a disease
It correcting genetic defects or by expressing proteins that are
therapeutically useful.
Patients with cystic fibrosis lacks a gene that codes for a
protein trans membrane carrier of chloride ions
Using recombinant technology the gene for the trans
membrane was isolated and placed in ribosomes.
 Genetically modified organisms
(GMOs)
Genetically modified organisms (GMOs) are
living organisms whose genetic material has
been artificially manipulated in a laboratory
through genetic engineering. Most GMOs have
been engineered to withstand the direct
application of herbicide and/or to produce an
insecticide.
Implications of using GMO’s
• GMO’s may become invasive species
• The transfer of gene’s through cross pollination and
fertilization may occur between GMOs and closely
related species
• Foods containing ingredients derived from GMO’s may
cause allergies in susceptible persons
• GMOs engineered for virus resistance may generate
new diseases
Natural
Selection
Environmental factors as factors of
natural selection
Environmental factors can change the
genetic make up of organisms and the
evolutionary agent is called gene flow,
occurs when migrating individuals breed
in their new location
Mechanism of natural selection
Types of selection are as follows:
Natural selection
Stabilising selection
Directional selection
Darwin’s theory
This is the formation of new species because of
some members are isolated in new environments
 The Biological species concept
This is the concept which states that is a
organism wonders into a new isolated
environment that supplies a foreign food
source, the organism will adapt to the
changes by developing new features that
can better withstand the new
environment.
Types of speciation
•Allopathic speciation
•Sympatric speciation
Allopathic speciation
• When organisms of an ancestral species evolve
into two or more descendant species after a
period of physical separation caused by a
geographic barrier, such as a mountain range,
rockslide, or river.
Sympathic speciation
•Occurs when a geographic barrier does not
separate the members of the population
but the gene pool is divided. Sympathic
speciation occurs within the same habitat
Isolating mechanism

•Reproductive
•Behavioral
•Temporal
Reproductive

Different species are unable to interbreed

and produce healthy, fertile offspring due to
barriers called mechanisms of reproductive
isolation.
Behavioral isolation
•Behavioral isolation is based on how
members of a species find and choose
mates. Many species have very specific
behaviors and rituals that they carry out
before reproducing.
Temporal isolation
•Temporal isolation occurs when two
populations reproduce at different times of
the day or in different seasons.