Basic Molecular Biology

Understanding DNA, Techniques, and

Applications
 What is Molecular Biology?
• Molecular biology, the study of the biochemical and molecular processes
within cells, especially the processes of DNA replication, RNA transcription,
and protein translation, has been widely adopted in toxicology.
• Molecular biology is the study of the structure and function of molecules
and macromolecular systems associated with biological processes,
especially the molecular basis of inheritance and protein synthesis.
Introduction to DNA

DNA serves as the

molecular basis of
heredity through
replication,
expression, and
translation processes.
 What is DNA?
• DNA (Deoxyribonucleic Acid) is a molecule that carries genetic
information for the development, functioning, growth, and reproduction
of all known living organisms.
• DNA is made of two linked strands that wind around each other to
resemble a twisted ladder called “double helix”.
• Each strand has a backbone made of alternating sugar (deoxyribose) and
phosphate groups.
• Attached to each sugar is one of four bases:
1. Adenine (A)
2. Cytosine (C)
3. Guanine (G)
4. Thymine (T)
Structure of DNA

As a unit, these three

components make up
one monomer.

A monomer is an
individual unit that acts
as a building block for
large biological
molecules.
Nitrogenous Bases
DNA are Polymers of Nucleotides
Two DNA Strands – The Double Helix

‘Base Pairing’:
Cytosine + Guanine
Adenine + Thymine

The sequence of the

bases along DNA’s
backbone encodes
biological
information, such as
the instructions for
making a protein or
RNA molecule.
 DNA Replication
• DNA replication is the process by which the genome’s DNA is copied
in cells.
• Before a cell divides, it must first copy or replicate its entire genome
so that each resulting daughter cell ends up with its own complete
genome.
• Replication occurs in three major steps:
1. the opening of the double helix and separation of the DNA strands,
2. the priming of the template strand, and
3. the assembly of the new DNA segment.
DNA Replication
Transcription and Translation: DNA to mRNA to Protein
• A gene is expressed through the processes of transcription
and translation.
• In the first step, the information in DNA is transferred to a
messenger RNA (mRNA) molecule by way of a process
called transcription.
• During transcription, the DNA of a gene serves as a template
for complementary base-pairing, and an enzyme called RNA
polymerase II catalyzes the formation of a pre-mRNA
molecule, which is then processed to form mature mRNA.
• The resulting mRNA is a single-stranded copy of the gene,
which next must be translated into a protein molecule.
 Transcription &
Translation
• During transcription, the
enzyme RNA polymerase
(green) uses DNA as a template
to produce a pre-mRNA
transcript (pink).
• The pre-mRNA is processed to
form a mature mRNA molecule
that can be translated to build
the protein molecule
(polypeptide) encoded by the
original gene.
 Transcription & Translation
• During translation, which is the
second major step in gene expression,
the mRNA is "read" according to
the genetic code, which relates the
DNA sequence to the amino
acid sequence in proteins.
• Each group of three bases in mRNA
constitutes a codon, and each codon
specifies a particular amino acid
(hence, it is a triplet code).
• The mRNA sequence is thus used as a
template to assemble—in order—the
chain of amino acids that form a
protein.
 Transcription & Translation
• The amino acids specified by each
mRNA codon.
• Multiple codons can code for the
same amino acid.
• The codons are written 5' to 3', as
they appear in the mRNA.
• AUG is an initiation codon.
• UAA, UAG, and UGA are
termination (stop) codons.
 Where Translation Occurs?
• Within all cells, the translation machinery resides within a
specialized organelle called the ribosome.
• In eukaryotes, mature mRNA molecules must leave the nucleus and
travel to the cytoplasm, where the ribosomes are located.
• In prokaryotic organisms, ribosomes can attach to mRNA while it is
still being transcribed. In this situation, translation begins at the 5'
end of the mRNA while the 3' end is still attached to DNA.
• In all types of cells, the ribosome is composed of two subunits: the
large (50S) subunit and the small (30S) subunit (S, for svedberg
unit, is a measure of sedimentation velocity and, therefore, mass).
 Where Translation Occurs?
• Each subunit exists separately in the cytoplasm, but the two join together
on the mRNA molecule.
• The ribosomal subunits contain proteins and specialized RNA
molecules—specifically, ribosomal RNA (rRNA) and transfer RNA (tRNA).
• The tRNA molecules are adaptor molecules—they have one end that can
read the triplet code in the mRNA through complementary base-pairing,
and another end that attaches to a specific amino.
• Within the ribosome, the mRNA and aminoacyl-tRNA complexes are held
together closely, which facilitates base-pairing. The rRNA catalyzes the
attachment of each new amino acid to the growing chain.
• When translation begins, the small
subunit of the ribosome and an
initiator tRNA molecule assemble
on the mRNA transcript.
• The small subunit of the ribosome
has three binding sites: an amino
acid site (A), a polypeptide site (P),
and an exit site (E).
• The initiator tRNA molecule
carrying the amino acid methionine
binds to the AUG start codon of the
mRNA transcript at the ribosome’s
P site where it will become the first
amino acid incorporated into the
growing polypeptide chain.
• Here, the initiator tRNA molecule is
shown binding after the small
ribosomal subunit has assembled
on the mRNA; the order in which
this occurs is unique to prokaryotic
cells.
• In eukaryotes, the free initiator
tRNA first binds the small
ribosomal subunit to form a
complex.
• The complex then binds the mRNA
transcript, so that the tRNA and the
small ribosomal subunit bind the
mRNA simultaneously.
• Once the initiation complex is
formed on the mRNA, the large
ribosomal subunit binds to this
complex, which causes the
release of IFs (initiation factors).
• The large subunit of the ribosome
has three sites at which tRNA
molecules can bind.
• The A (amino acid) site is the
location at which the aminoacyl-
tRNA anticodon base pairs up
with the mRNA codon, ensuring
that correct amino acid is added
to the growing polypeptide chain.
 Translation

• The P (polypeptide) site is the

location at which the amino
acid is transferred from its
tRNA to the growing
polypeptide chain.
• Finally, the E (exit) site is the
location at which the "empty"
tRNA sits before being released
back into the cytoplasm to bind
another amino acid and repeat
the process.
 The Elongation Phase
• First, the ribosome moves along the mRNA in the 5'-to-3'direction,
which requires the elongation factor G, in a process
called translocation.
• The tRNA that corresponds to the second codon can then bind to
the A site, a step that requires elongation factors, as well as
guanosine triphosphate (GTP) as an energy source for the process.
• Upon binding of the tRNA-amino acid complex in the A site, GTP is
cleaved to form guanosine diphosphate (GDP).
• Next, peptide bonds between the now-adjacent first and second
amino acids are formed through a peptidyl transferase activity.
 The Elongation Phase
• After the peptide bond is formed, the ribosome shifts, or translocates,
again, thus causing the tRNA to occupy the E site.
• The tRNA is then released to the cytoplasm to pick up another amino acid.
• In addition, the A site is now empty and ready to receive the tRNA for the
next codon.
• There are three termination codons that are employed at the end of a
protein-coding sequence in mRNA: UAA, UAG, and UGA.
• No tRNAs recognize these codons. Thus, in the place of these tRNAs, one
of several proteins, called release factors, binds and facilitates release of
the mRNA from the ribosome and subsequent dissociation of the
ribosome.
Comparing Eukaryotic and Prokaryotic Translation
• The translation process is very similar in prokaryotes and
eukaryotes.
• Although different elongation, initiation, and termination factors
are used, the genetic code is generally identical.
• As previously noted, in bacteria, transcription and translation
take place simultaneously, and mRNAs are relatively short-lived.
• In eukaryotes, however, mRNAs have highly variable half-lives,
are subject to modifications, and must exit the nucleus to be
translated; these multiple steps offer additional opportunities to
regulate levels of protein production, and thereby fine-tune
gene expression.
Basic Techniques in Molecular Biology
 Basic Techniques in Molecular Biology
• Molecular Biology Techniques include DNA cloning, cut and
paste DNA, bacterial transformation , transfection,
chromosome integration, cellular screening, cellular culture,
extraction of DNA, DNA polymerase DNA dependent, reading
and writing DNA, DNA sequencing, DNA synthesis,
molecular hybridization, rewriting DNA: mutations, random
mutagenesis, point mutation, chromosome mutation.
• Most important techniques are Polymerase Chain Reaction
(PCR), Expression cloning, Gel electrophoresis, Macromolecule
blotting and probing, Arrays (DNA array and protein array).
 Polymerase Chain Reaction (PCR)
• PCR is a laboratory technique for rapidly producing (amplifying)
millions to billions of copies of a specific segment of DNA,
which can then be studied in greater detail.
• PCR involves using short synthetic DNA fragments called
primers to select a segment of the genome to be amplified, and
then multiple rounds of DNA synthesis to amplify that segment.
• PCR is so sensitive that the DNA present in an individual cell can
be isolated and amplified. This process is faster and less tedious
than the traditional methods of gene cloning.

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 PCR Steps
The PCR involves three major cyclic reactions:
1. Denaturation
2. Annealing
3. Elongation
 PCR Step 1: Denaturation
• Denaturation occurs when the reaction
mixture is heated to 94℃ for about 0.5
to 2 minutes. This breaks the hydrogen
bonds between the two strands of DNA
and converts it into a single-stranded
DNA.
• The single strands now act as a template
for the production of new strands of
DNA.
• The temperature should be provided for
a longer time to ensure the separation of
the two strands.
 PCR Step 2: Annealing
• The reaction temperature is lowered to
54-60℃ for around 20-40 seconds.
Here, the primers bind to their
complementary sequences on the
template DNA.
• Primers are single-strand sequences of
DNA or RNA around 20 to 30 bases in
length.
• They serve as the starting point for the
synthesis of DNA.
• The two separated strands run in the
opposite direction and consequently
there are two primers- a forward primer
and a reverse primer.
 PCR Step 3: Elongation
• At this step, the temperature is raised to 72-
80℃. The bases are added to the 3’ end of
the primer by the Taq polymerase enzyme.
• This elongates the DNA in the 5’ to 3’
direction.
• Taq Polymerase can tolerate very high
temperatures. It attaches to the primer and
adds DNA bases to the single strand. As a
result, a double-stranded DNA molecule is
obtained.
• These 3 steps are repeated 20-40 times in
order to obtain a number of sequences of
DNA of interest in a very short time period.
 Applications of PCR
1. Medicine 3. Research and Genetics
• Testing of genetic disease mutations. • Compare the genome of two
• Monitoring the gene in gene therapy. organisms in genomic studies.
• Detecting disease-causing genes in • In the phylogenetic analysis of
the parents. DNA from any source such as
fossils.
2. Forensic Science
• Analysis of gene expression.
• Used as a tool in genetic
fingerprinting. • Gene Mapping
• Identifying the criminal from millions
of people.
• Paternity tests
 Gel Electrophoresis
• Gel electrophoresis is a technique used to separate DNA
fragments (or other macromolecules, such as RNA and
proteins) based on their size and charge.
• Electrophoresis involves running a current through a gel
containing the molecules of interest.
• Based on their size and charge, the molecules will travel
through the gel in different directions or at different speeds,
allowing them to be separated from one another.
 Gel Electrophoresis

• All DNA molecules have the same amount of charge per mass.
• Because of this, gel electrophoresis of DNA fragments separates
them based on size only.
• Using electrophoresis, we can see how many different DNA
fragments are present in a sample and how large they are relative
to one another.
• Gels for DNA separation are often made out of a polysaccharide
called agarose, which comes as dry, powdered flakes.
• When the agarose is heated in a buffer (water with some salts in
it) and allowed to cool, it will form a solid, slightly squishy gel.
• In gel electrophoresis, the molecules to be separated are pushed by
an electrical field through a gel that contains small pores.
• Because DNA and RNA are negatively charged molecules, they will be
pulled toward the positively charged end of the gel.
• The molecules travel through the pores in the gel at a speed that is
inversely related to their lengths.
• This means that a small DNA molecule will travel a greater distance
through the gel than a larger DNA molecule.
 DNA Cloning
• DNA cloning is a molecular biology technique which is used for the
creation of exact copies or clones of a particular gene or DNA.
• During this technique, the selected DNA fragment is inserted into a
plasmid (the circular piece of DNA) using enzymes. Restriction
enzymes and DNA ligase are used in the process.
• The restriction enzymes are used to cut the DNA fragments at specific
sequences and DNA ligase enzymes are used to join the nicks.
• The recombinant DNA is introduced into bacteria.
• These bacteria reproduce and produce an exact copy of the plasmid.
• These copies are known as clones.
Importance of DNA Cloning

1. DNA cloning can be used to

make proteins such as insulin
with biomedical techniques.
2. It is used to develop
recombinant versions of the
non-functional gene to
understand the functioning of
the normal gene. This is applied
in gene therapies also.
3. It helps to analyse the effect of
mutation on a particular gene.
 DNA Sequencing
• DNA Sequencing refers to the general laboratory techniques for
determining the exact sequence of nucleotides, or bases, in a
DNA molecule.
• The sequence of the bases, often referred to by the first letters
of their chemical names (A, T, C and G), encodes the biological
information that cells use to develop and operate.
• Establishing the sequence of DNA is key to understanding the
function of genes and other parts of the genome.
DNA
Sequencing:
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 Application of Molecular Biology
• Medical Applications • Forensic Applications
• Genetic Testing • DNA Fingerprinting
• Gene Therapy • Crime Scene Analysis DNA
• Disease Diagnosis Profiling
• Drug Development • Crime Scene Investigation
• Personalized Medicine
• Agricultural Applications
• Genetically Modified Organisms (GMOs)
• Crop Improvement
• Crop Pest Resistance
• Improved Nutritional Content