Chap IV: Nucleic Acids

• Role of nucleic acids in living organisms

• Nomenclature

• Chemical composition and structures of

nucleosides and nucleotides

• Structure of DNA double helix

• Packaging of DNA

• Replication

• Mutations and repair mechanisms

• Transcription
• Genetic code
• Translation
• DNA sequencing
 Historical background
• Nuclein were discovered by Friedrich Miescher in 1869.
• In 1889 Richard Altmann discovered that nuclein has
acidic properties, and it became called nucleic acid
• In 1938 Astbury and Bell published the first X-ray
diffraction pattern of DNA.
• 1950s: Rosalind Franklin and Maurice Wilkins obtained X-
ray diffraction pattern of DNA AND deduced that DNA
was helical.
• In 1953 Watson and Crick determined the structure of
DNA.
Friedrich Miescher Astbury Richard Altmann
 Nucleic Acids
• They are molecules that store information for
cellular growth and reproduction.
• They are two types : Deoxyribonucleic Acid
(DNA) and Ribonucleic Acid (RNA).
• Large molecules consisting of long chains of
monomers called nucleotides.
 The distribution of nucleic acids in the eukaryotic
cell
• DNA is found in the nucleus with small amounts
in mitochondria and chloroplasts
• RNA is found throughout the cell.
 Ribonucleic Acid (RNA)
• Transmits information from DNA to make proteins.
• RNA is much more abundant than DNA
• There are several important differences between RNA and
DNA:
 Structure:
• DNA: Typically exists as a double-stranded helix, with two
long strands of nucleotides twisted around each other. The
backbone is made of deoxyribose sugar and phosphate
groups.
• RNA: Usually single-stranded and consists of a ribose sugar
backbone and phosphate groups.
Nucleotides:
• DNA: Contains four nitrogenous bases: adenine
(A), thymine (T), cytosine (C), and guanine (G).
• RNA: Contains the same bases as DNA, except
thymine (T) is replaced by uracil (U), so the bases
are adenine (A), uracil (U), cytosine (C), and
guanine (G).
• Function:
• DNA: Serves as the long-term storage of genetic
information; it holds the instructions for building
and maintaining an organism.
• RNA: Plays several roles, including serving as a messenger
between DNA and the protein synthesis machinery
(mRNA), forming the core of ribosomes (rRNA),and
transferring amino acids during protein synthesis (tRNA).
 Stability :
• DNA: More stable than RNA due to its double-stranded
structure and lack of a hydroxyl group on the 2' carbon of
the sugar, making it less prone to hydrolysis.
• RNA: Less stable than DNA, as the presence of the hydroxyl
group on the ribose sugar makes it more reactive and
prone to degradation.
• DNA synthesis needs a primer, RNA synthesis does not
need a primer
Location:
• DNA: Primarily found in the nucleus of eukaryotic
cells, with some DNA located in mitochondria and
chloroplasts.
• RNA: Found throughout the cell, including the
nucleus and cytoplasm, where it is involved in
various cellular processes.
There are three main types of RNA:
• Messenger RNA (mRNA) carries genetic
information from DNA to the ribosomes. where
proteins are synthesized. It serves as a template for
the translation process
• Transfer RNA (tRNA):Brings amino acids to the
ribosome to make the protein. During protein
synthesis. Each tRNA molecule has an anticodon that pairs
with a corresponding codon on the mRNA, ensuring that
the correct amino acid is added to the growing polypeptide
chain
• Ribosomal RNA (rRNA): rRNA is a key component
of ribosomes, which are the cellular machinery
that synthesizes proteins. It helps to stabilize the
structure of the ribosome and plays a role in
catalyzing the formation of peptide bonds between
amino acids.
Types of RNA
Ribosomal RNA and Messenger RNA
• Ribosomes are the sites of protein synthesis
 They consist of ribosomal RNA (65%) and
proteins (35%)
-They have two subunits, a large one and a small
one
 Messenger RNA carries the genetic code to the
ribosomes
- They are strands of RNA that are complementary
to the DNA of the gene for the protein to be
synthesized.
 Transfer RNA
• Transfer RNA (tRNA) is a type of RNA molecule
that plays a crucial role in the process of
translating genetic information from messenger
RNA (mRNA) into proteins. Here are some key
points about tRNA:
 Structure
• Shape: tRNA molecules have a distinctive
cloverleaf structure, which is essential for their
function. This structure allows tRNA to interact
with both mRNA and amino acids.
• Anticodon: Each tRNA has a specific sequence of
three nucleotides called an anticodon that is
complementary to the corresponding codon on
the mRNA. This ensures that the correct amino
acid is added to the growing polypeptide chain
during protein synthesis.
• Amino Acid Attachment: At the opposite end of
the tRNA molecule, there is an acceptor stem
where a specific amino acid is attached. Each
tRNA is linked to its corresponding amino acid by
a specific enzyme called aminoacyl-tRNA
synthetase.
 Function
• Translation: During translation, ribosomes read
the sequence of codons in mRNA. tRNA
molecules bring the appropriate amino acids to
the ribosome according to the sequence of
codons.
• Peptide Bond Formation: As each tRNA
molecule delivers its amino acid, the ribosome
catalyzes the formation of peptide bonds
between the amino acids, creating a
polypeptide chain that will eventually fold into
a functional protein.
• Types
• There are many different tRNA molecules, each
specific to one of the 20 amino acids used in protein
synthesis. This specificity ensures that the correct
amino acid is incorporated into the protein being
synthesized.
• Importance
• tRNA is essential for translating the genetic code
into functional proteins. Without tRNA, the
translation of mRNA into proteins would not occur,
making it vital for the expression of genes and the
overall functioning of living organism.
• One end attaches to the amino acid and the other binds
to the mRNA by a 3-bases complementary sequence.
 Deoxyribonucleic acid (DNA)
• is a molecule that carries the instructions used in
the growth, development, functioning
and reproduction of all known living organism and
many viruses. DNA stores biological information.
• Most DNA molecules consist of
two biopolymer strands coiled around each other to
form a double helix.
• The two DNA strands are polynucleotides since they
are composed of simpler monomer units
called nucleotides.
• contain nucleobases (cytosine [C], guanine [G], ade
nine [A] or thymine [T])
• They contain also a sugar called deoxyribose, and
a phosphate group.
• The nucleotides are joined to one another in a chain
by covalent bonds between the sugar of one
nucleotide and the phosphate of the next, resulting
in an alternating sugar-phosphate backbone.
• The nitrogenous bases of the two separate
polynucleotide strands are bound together,
according to base pairing rules (A with T and C with
G), with hydrogen bonds to make double-stranded
DNA.
• The nitrogenous base pairs are divided into two
main groups, pyrimidines and purines.
Chemical composition and structures of
nucleosides and nucleotides

The nucleic acids DNA and

RNA consist of monomers
called nucleotides that
consist of :
• Pentose sugar.
• Nitrogen-containing
base.
• Phosphate group.
Nucleotide structure
Nucleotide structure…

PHOSPHATE SUGAR BASE

PURINES PYRIMIDINES
Ribose or

Deoxyribose Adenine (A) Cytosine (C)

Guanine(G) Thymine (T)

Uracil (U)

NUCLEOTIDE
Nitrogen Bases
The nitrogen bases in
DNA and RNA are
•Pyrimidines: C, T,
and U.
•Purines: A and G.
Nitrogen Bases in DNA and RNA
DNA contains the nitrogen bases
•Cytosine (C)
•Guanine (G)same in both DNA and RNA
•Adenine (A)
•Thymine (T): Only in DNA
RNA contains the nitrogen bases
•Cytosine (C)
•Guanine (G)same in both DNA and RNA
•Adenine (A)
•Uracil (U): Only in RNA
Pentose Sugars

The pentose (five-carbon) sugar

•in RNA is ribose.
•in DNA is deoxyribose with no O atom on carbon 2’.
•The carbon atoms are numbered with primes to
distinguish them from the atoms in nitrogen bases.
• Nucleoside:
• Is composed of pentose sugar
• consists of a nitrogenous base covalently
attached to a sugar (ribose or deoxyribose)
but without the phosphate group. The general
structure consist of Nitrogen base + Sugar
Nomenclature
Nucleosides
A nucleoside
•has a nitrogen base linked by
a glycosidic bond to C1’ of a
sugar (ribose or deoxyribose).
•Nucleoside are named by HO
changing the nitrogen base
ending to:-osine for purines
and -idine for pyrimidines.
Nucleotides

A nucleotide
•is a nucleoside that forms a
phosphate ester with the C5’ –OH
group of a sugar (ribose or
deoxyribose).
•is named using the name of the
nucleoside followed by
5’-monophosphate.
Nucleosides and Nucleotides with Purines
Nucleosides and Nucleotides with Pyrimidines
Names of Nucleosides and
Nucleotides
 Watson and Crick model for the structure of DNA
• In 1953, Watson and Crick postulated that,
– It consists of two antiparallel strands of
nucleotides twisted around each other form a
right-handed double helix.
– The strands of DNA comprising the double helix
run in opposite direction
 the key features of their model include:
 Base Pairing: A-T, C-G. This is done through the
H- bond.
Anti -parallel strand: The two strand run in
opposite directions , which is essential for
replication and function.
The two DNA strands are not IDENTICAL, but they
are COMPLEMENTARY
Sugar - Phosphate backbone: is formed by
alternating sugar and phosphate groups, providing
structural stability.
Major and minor Grooves: the twisting of the helix
creates grooves that are important for protein
binding. Both grooves are essential for the
recognition of DNA sequences and the regulation
of biological processes like replication,
transcription and repair.
 This model laid the foundation for understanding
DNA replication, transcription and the molecular
basis of heredity
 Hydrophilic backbone of alternating
sugar/phosphate units of the nucleotides are on
outside of DNA molecule exposed to water
 Hydrophobic bases are stacked neatly inside the
molecule. The hydrogen bond between the base
pairs hold the double helix together.
Hydrogen bonding pattern in the base pairs
defined by Watson and Crick
Schematic drawings of complementary anti
parallel strand of DNA following the pairing rules
proposed by Watson and Crick
Watson-Crick model for the structure of DNA
(Physical Dimensions)
Double Helix of DNA

In the double helix of DNA

•two strands of nucleotides
form a double helix structure
like a spiral stair case.
•hydrogen bonds link bases
A–T and G–C.
•the bases along one strand
complement the bases along
the other.
•is held in place by of two H
bonds that form between the
base pairs A-T and three
 Packaging of DNA
DNA Packaging refer to the way DNA is organized and
compacted within the cell. The packaging allows DNA to fit
within the nucleus while still being accessible for
replication and transcription.
In eukaryotes: DNA is wrapped around Histone proteins, to
form nucleosomes.
-The nucleosomes coil and fold again into high ordered
structures which result in formation of chromatin.
- In cell division, the chromatin condenses into
chromosomes, allowing for efficient segregation of genetic
material.
In Prokaryotes: DNA is circular and not associated with
histones. Other proteins help organize and stabilize this
DNA
• Effective DNA Packaging is crucial for regulating
gene expression , protecting DNA integrity and
ensuring proper cell division.
How DNA is Packaged ?
The following are key steps involved in the process
• Nucleosomes: DNA wraps around histone protein,
forming nucleosome.
• Chromatin structure: Nucleosomes coil and fold to
form a more condensed structure called
chromatin.
• Two type of chromatins:
Euchromatin ( less condensed, active in
transcription)
Heterochromatin ( More condensed and inactive)
• Higher-order folding: chromatin further folds into
loops and domains , organizing itself into a high-
order structure that enables efficient packaging
and regulation of gene expression.
• Chromosomes: During cell division, chromatin
condenses even more to form visible
chromosomes ensuring the accurate distribution
of DNA to daughter cells.
 DNA Replication
DNA replication : is the process by which a cell
makes an identical copy of its DNA. This is essential
for cell division, ensuring that each new cell receives
the same genetic information.
 How DNA replication a role in cell division
process :
DNA replication: is absolutely necessary in cell
division, it ensure that each new cell has the
complete set of genetic information needed for
proper function and development
Bellow shows the way it plays a role in cell
Division process:
Preparation for Division: Before a cell divides, it
must duplicate its DNA so that each daughter cell
receives an identical set of genetic instructions. This
occurs during the S phase of the cell cycle.
Mechanism: The double helix structure of DNA
unwinds during replication and each strand serves as
a template for the synthesis of a new complementary
strand. DNA polymerase plays an important role in
adding nucleotides to form new strands.
Accuracy: DNA replication includes proofreading
mechanisms to minimize errors, ensuring that the
genetic information remains stable and consistent.
This accuracy is essential for maintaining the
integrity of the organism’s genome.
Cell Cycle Coordination: when DNA replication is
done successively, triggers the cell to proceed to
mitosis (in somatic cells) or meiosis (in germ cells),
where the cell physically divides. Any issues in
replication can stop the cell cycle and lead to cell
death or diseases like cancer.
 Genetic Variation: In meiosis, the process of DNA
replication is coupled with genetic recombination, which
contributes to genetic diversity in offspring
• Watson and Crick model during DNA
replication: They suggested that DNA is
replicated on a DNA template.
- Each strand of DNA in the double helix acts as a
template or a pattern for the synthesis of its
complement. Since DNA is double-stranded,
complementary replication would produce two
double-helical DNA molecules, each containing a
strand of the original DNA and a new strand
complementary to it.
This type of replication is called semiconservative
replication, which was confirmed by M.S.
Meselson and F.W. Stahl in 1957.
- In 1955, Arthur Kornberg and his associates
discovered DNA polymerase — a large protein
which catalyses the formation of DNA.
- DNA strand elongates. Elongation or the chain
growth is always from the 5 end to the 3 end of
the elongating DNA molecules.
Replication of DNA as suggested by Watson and
Crick
 DNA Replication Process
• DNA replication involves the following processes:
Initiation:
- Origin of Replication: Replication begins at specific
locations called origins of replication. In eukaryotic
cells, there are multiple origins of replication on
each chromosome.
- Helicase Enzyme: This enzyme unwinds and
separates the two strands of the DNA double helix,
creating a replication fork.
Elongation:
– Primase: This enzyme synthesizes a short RNA primer
complementary to the DNA template strand, providing a
starting point for DNA synthesis.
– DNA Polymerase: This enzyme adds nucleotides to the
growing DNA strand, synthesizing new DNA in a 5’ to
3’ direction. It requires the RNA primer to initiate
synthesis.
 Leading and Lagging Strands:
– The leading strand is synthesized continuously in the
direction of the replication fork.
– The lagging strand is synthesized in short segments
called Okazaki fragments, which are later joined
together.
Termination:
- Once the entire DNA molecule has been replicated, the
RNA primers are removed and replaced with DNA
nucleotides.
- The enzyme DNA ligase seals any gaps between the newly
synthesized DNA fragments.
Proofreading and Repair:
-DNA polymerases have proofreading abilities to
correct any errors that occur during replication.
-Additional repair mechanisms can fix any remaining
errors to ensure genetic fidelity.
-The process is called semi-conservative replication
because one strand of each daughter DNA comes
from the parent DNA and one strand is new
-The energy for the synthesis comes from hydrolysis
of phosphate groups as the phosphodiester bonds
form between the bases
 The result of DNA replication is two identical DNA
molecules, each consisting of one old (template)
strand and one newly synthesized strand, a process
known as semi-conservative replication.
- This mechanism is crucial for growth, development,
and repair in living organisms.
Semi-Conservative DNA Replication
 Direction of Replication

The enzyme helicase unwinds several sections of

parent DNA
At each open DNA section, called a replication fork,
DNA polymerase catalyzes the formation of 5’-3’ester
bonds of the leading strand
The lagging strand, which grows in the 3’-5’
direction, is synthesized in short sections called
Okazaki fragments
The Okazaki fragments are joined by DNA ligase to
 There are several enzymes involved.
1. Topoisomerase: Is responsible for initiation of the
unwinding of the DNA by eliminating supercoiling.
The tension holding the helix in its coiled and
supercoiled structure can be broken by nicking a
single strand of DNA
2. Helicase : Accomplishes unwinding of the original
double strand, once supercoiling has been eliminated
by the topoisomerase. The two strands bind together
by hydrogen bonding, so the helicase activity
requires energy in the form of ATP to break the
strands apart.
3. DNA polymerase proceeds along a
single- stranded molecule of DNA catalyze DNA
formation.
o There are different forms of DNA polymerase , but
it is DNA polymerase III that is responsible for the
processive synthesis of new DNA strands.
o DNA polymerase cannot start synthesizing de novo
on a bare single strand.
o It needs a primer with a 3'OH group onto which it
can attach a dNTP.
4. Primase: is an enzyme that creates a primer on a
DNA strand by adding RNA nucleotides to the
strand according to the DNA template sequence.
This process occurs during DNA replication.
This RNA primer is eventually removed before
DNA replication is complete
[Link]-Strand Binding Proteins (SSBs): Stabilize
the unwound single strands of DNA to prevent
them from re-annealing or forming secondary
structures.
6. Ligase :The Okazaki fragments are joined by DNA
ligase to give a single 3’-5’ DNA strand
 Some Terminology
• Primers : Are short sequences of nucleotides that
provide a starting point for DNA synthesis
Primers are essential in various molecular biology
techniques, particularly in polymerase chain reaction
(PCR), where they help amplify specific DNA
segments.
Key Points about Primers:
Composition: Primers are typically 18-25
nucleotides long and are designed to be
complementary to the target DNA sequence.
Function: They bind to the template DNA strand
and provide a free 3' hydroxyl group, which is
necessary for DNA polymerase to initiate
replication.
Types:
– Forward Primers: Complementary to the start of
the target sequence.
– Reverse Primers: Complementary to the end of
the target sequence, usually on the opposite
strand.
 The main difference between DNA primers and
RNA Primers lie in their chemical structure,
stability, and specific roles in nucleic acid processes.
• DNA Primers: Used in DNA replication and techniques
like PCR (Polymerase Chain Reaction) to initiate DNA
synthesis.
• RNA Primers: Typically used during DNA replication in
cells, where they are synthesized by the enzyme primase
to provide a starting point for DNA polymerase
 Mutations
• A mutation in DNA: Refers to a change in the
nucleotide sequence of an organism's genetic
material. DNA polymerase can make mistakes
while adding nucleotides.
• Most mistakes are corrected, but if they are not,
they may result in a mutation. Mutation can alter
the nucleotide sequence in DNA as result from
mutagens such as radiation and chemicals.
• Produce one or more incorrect codons in mRNA.
 Produce a protein containing one or more incorrect
amino acids.
 Produce defective proteins and enzymes. Cause genetic
diseases.
Types of mutations
-Chromosomal mutations: affecting xmes
 -Gene mutations:
Gene mutation: is a permanent alteration in the DNA
sequence that makes up a gene. A gene codes for
the formation of a particular
protein. If the sequence of bases is changed a different
protein is produced. The new protein may have
different properties.
Types of gene mutation: Point Mutation: affect single
nucleotide of nucleic acid
• Frame shift mutation: are due to either insertions or
deletions of nucleotides. It can be Deletion,
insertion, substitution.
 Protein Synthesis
The two main processes involved in protein
synthesis are
- the formation of mRNA from DNA
(transcription)
- the conversion by tRNA to protein at the ribosome
(translation)
Transcription takes place in the nucleus, while
translation takes place in the cytoplasm
Genetic information is transcribed to form mRNA
much the same way it is replicated during cell
division
 The central dogma
The central dogma outlines the plan for the storage
and transmission of hereditary information.
The central dogma describe the Two-step process,
transcription and translation, by which the
information in genes flows into proteins: DNA →
RNA → protein.
Now we know that DNA is the bearer of genetic
information in all living organisms.
The entire basis of information storage and
transmission in the cell is embodied in three steps:
REPLICATION,TRANSCRIPTION
TRANSLATION
 Transcription
- Transcription is the production of aRNA strand
that is complementary in base sequence to a DNA
strand
- It Occurs in nucleus
- The DNA template is copied starting at the
initiation point called TATAAA
- The produced RNA strand = messenger RNA
(mRNA)
-RNA is synthesized from template DNA following
strand separation of the double helix
• During transcription, RNA polymerase moves along
the DNA template in the 3’-5’direction to synthesize
the mRNA
• The mRNA is released at the termination point
• mRNA moves out of the nucleus to ribosomes in the
cytoplasm and the DNA rewinds
Transcription is done in three steps:
• Initiation - binding of RNA polymerase to double-
stranded DNA at the Promoter (binding is catalyzed
by transcription initiation factors)
• Elongation : RNA polymerase catalyzes the covalent
addition of nucleotides (complementary to the
template DNA strand) to the 3' end of the growing
RNA strand.
• Termination : of transcription at the transcription
termination sequence in the DNA (terminator)
 Processing of mRNA
• DNA in nucleus contains exons (that code)and
introns (that do not code) for protein( in
Eukaryotes)
• The initial mRNA(pre-RNA) has introns, it must be
processed before it can be read by the tRNA
• So, while the mRNA is still in the nucleus, the
introns are removed from pre-mRNA
• The exons remained are joined to form the mRNA
that leaves the nucleus to ribosome for protein
synthesis
Removing Introns from mRNA
 Genetic code
• This relationship between bases and AA is known
as the genetic code
Characteristics of genetic code:
• It represents a triplet code, each codon is presented
by 3 successive nucleotides in the sequence 5 prime
to 3 prime on mRNA, so they are 64 different
triplets
• From the 64 codes, three: UAA,UAG and UGA do
not code for any AA. They are called non-sense
codons or terminator codons
• The same AA may be coded by more than one codon for
example: UUU and UUC. This property is known as
degeneracy or redundancy of genetic code
• The two codons AUG and GUG are initiator codons when
they occupy the first position on mRNA. So, AUG is
universal initiator, GUG acts as initiator codon only in
eukaryotes.
• The genetic code is universal: the same triplet
codons specify the same amino acids in all species.
• The sequence of AA in polypeptide chain
corresponds to codons in mRNA which in turn are
complementary to anticodons of sense strand of
DNA
The Genetic Code: mRNA Codons
Translation
• Translation: results in the synthesis of a
polypeptide chain composed of a linear chain
of amino acids whose order is specified by the
sequence of codons in mRNA
• Translation occurs in the cell cytoplasm, at the
ribosomes
• Ribosomes consist of ribosomal RNA (rRNA)
and ribosomal proteins
• Direction of Synthesis: mRNA read from 5’-
to-3’
• Messenger RNA (mRNA) contains genetic code
in triplet base codons that specify the
sequence of amino acids in proteins
Translation start:
• AUG (ATG in DNA) codes for methionine
Translation stop:
• stop codon (UAG, UAA or UGA) causes release
of the polypeptide
ORF: from start codon to stop codon (open
reading flame)
DNA 5’ 3’
3’ 5’
Synthesis of RNA
Transcription
mRN A
5’ 3’
synthesis of polypeptide
(Translation)

NH2 cooH
• Transfer RNAs (tRNA): contain triplet base
sequences called anticodon which are
complementary to codons in mRNA and position
amino acids during translation.

• Each transfer RNA (tRNA) is covalently attached

to a specific amino acid (by aminoacyl-
tRNAsynthetases) and contains an anti-codon
complementary to an mRNA codon
Phases of translation:
• Initiation: The ribosome assembles around the
target mRNA. The first tRNA is attached at the
start codon.
• Elongation: The tRNA transfers an amino acid to
the tRNA corresponding to the next codon. The
ribosome then moves (translocates) to the next
mRNA codon to continue the process, creating an
amino acid chain.
• Termination: When a stop codon is reached, the
ribosome releases the polypeptide
 steps
• Binding of mRNA to ribosome
• AA activation and attachment to tRNA
• Initiation and Translocation
• Polypeptide chain elongation
• Chain termination
 AA activation and attachment to tRNA
• The first step in translation is
activation of the tRNA
• Each tRNA has an anticodon
that complements codon of
mRNA
• An aminoacyl tRNA
synthetase uses ATP
hydrolysis to attach an amino
acid to a specific tRNA
 Initiation and Translocation
• mRNA attaches to a ribosome
• On the mRNA, the start codon (AUG) binds to a
tRNA with methionine
• The second codon attaches to a tRNA with the
next amino acid
• A peptide bond forms between the adjacent
amino acids at the first and second codons
• The first tRNA detaches from the ribosome and
the ribosome shifts to the adjacent codon on the
mRNA (translocation)
• A third codon can now attach where the second
one was before translocation …..etc
Scheme of Translation
Polypeptide chain elongation and
Termination
• After a polypeptide with all the amino acids for a
protein is synthesized, the ribosome reaches the
“stop” codon: UGA, UAA, or UAG
• There is no tRNA with an anticodon for the “stop”
codons
• Therefore, protein synthesis ends (termination)
• The polypeptide is released from the ribosome and
the protein can take on it’s 3-D structure(some
proteins begin folding while still being synthesized,
while others do not fold up until after being released
Summary of Protein Synthesis
• A mRNA attaches to a ribosome.
• tRNA molecules bonded to specific amino
acids attach to the codons on mRNA.
• Peptide bonds form between an amino acid
and the peptide chain.
• The ribosome shifts to each codon on the
mRNA until it reach the STOP codon.
• The polypeptide chain detaches to function as
an active protein
DNA Sequencing
• DNA sequencing is the process of determining the
precise order of nucleotides within a DNA molecule.
• It includes any method or technology that is used to
determine the order of the four bases:
adenine, guanine, cytosine, and thymine in a strand
of DNA.
• The development of rapid DNA sequencing methods
has greatly accelerated biological and medical
research and discovery.
• Knowledge of DNA sequences has become
indispensable for basic biological research, and in
numerous applied fields such as medical diagnosis.
• biotechnology, forensic biology, virology and
biological systematics.
• The rapid speed of sequencing attained with modern
DNA sequencing technology has been instrumental in
the sequencing of complete DNA sequences,
or genomes of numerous types and species of life,
including the human genome and other complete DNA
sequences of many animal, plant, and microbial species
• There are Three main types of DNA sequencing.
1. Maxam/Gilbert chemical sequencing
[Link] classical chain termination method or Sanger
method: it is the older one
High-Throughput Sequencing (HTS) techniques or
Next-Generation Sequencing (NGS)methods :
Newer methods that can process a large number
of DNA molecules quickly.
3. Third-Generation Sequencing: Useful for complex
genomes and structural variant detection.
 Maxam/Gilbert chemical sequencing

Walter Gilbert
Allan Maxam Walter Gilbert
• Maxam–Gilbert sequencing is a method of DNA
sequencing developed by Allan
Maxam and Walter Gilbert in 1976–1977.
• This method is based on chemical modification
of DNA and subsequent cleavage at specific
bases.
• Maxam–Gilbert sequencing was the first widely
adopted method for DNA sequencing
• and, along with the Sanger dideoxy method,
represents the first generation of DNA sequencing
methods.
• Maxam–Gilbert sequencing is no longer in
widespread use, having been supplanted by next-
generation sequencing methods
• Maxam-Gilbert sequencing requires radioactive
labeling at one 5' end of the DNA and
purification of the DNA fragment to be
sequenced.
• How is it done: The DNA sample is prepared
and is sequenced using chemical termination
process.
- From the DNA sample which is double stranded
a homogeneous single- stranded DNA is
prepared.
- When a DMS (dimethyl sulfate) is added on
single strand it will clean and show the
nucleotides out.
 Sanger chain termination sequencing

Frederick Sanger
• Sanger sequencing is a method based on the
selective incorporation of chain-
terminating dideoxynucleotides by DNA
polymerase during in vitro DNA replication.
• Developed by Frederick Sanger and colleagues in
1977, it was the most widely used sequencing
method for approximately 40 years.
• When invented, the chain-terminator method used
fewer toxic chemicals and lower amounts of
radioactivity than the Maxam and Gilbert method.
• Because of its comparative ease, the Sanger
method was soon automated and was the method
used in the first generation of DNA sequencers.
• The human genome was sequenced using Sanger
method in 2001.
• The classical chain-termination method requires a
single-stranded DNA template, a DNA primer,
a DNA polymerase, normal
deoxynucleosidetriphosphates (dNTPs), and
modified di-deoxynucleotidetriphosphates
(ddNTPs), the latter of which terminate DNA strand
elongation.
• These chain-terminating nucleotides lack a 3'-
OH group required for the formation of
a phosphodiester bond between two nucleotides.
• causing DNA polymerase to cease extension of
DNA when a modified ddNTP is incorporated.
• The ddNTPs may be radioactively
or fluorescently labeled for detection in automated
sequencing machines.
• The DNA sample is divided into four separate
sequencing reactions, containing all four of the
standard deoxynucleotides (dATP, dGTP, dCTP and
dTTP) and the DNA polymerase.
• More recently, higher volume Sanger sequencing
has been supplanted by "Next-Gen" sequencing
methods, especially for large-scale,
automated genome analyses.
Next-generation sequencing
-This method is different from the earlier methods by
being highly scalable, allowing the entire genome to
be sequenced at once.
-This was accomplished by fragmenting the genome
into small pieces,
- multiple fragments are sequenced at once in an
automated process.
• Third-Generation Sequencing:
• Techniques like PacBio and Oxford Nanopore that can read
longer DNA sequences in real-time.
• Useful for complex genomes and structural variant
detection
Applications of DNA sequencing:
• Medical Diagnostics: Identifying genetic
disorders or mutations that may lead to disease.
• Genomic Research: Understanding genetic
variation within populations or species.
• Forensic Analysis: Analyzing DNA samples for
criminal investigations.
• Agricultural Biotechnology: Developing
genetically modified organisms with desirable
traits