Molecular Biology & Genetics:
Gene Sequencing:
Dr Muhammad Abdul Rab Faisal Sultan
PhD (Biochemistry/Molecular Biology)
Molecular Proteomics
Contents:
• Definition
• Introduction
• History
• Early methods
• New techniques
• Application
• Defects in sequencing techniques
Deoxyribonucleic acid (DNA):
• Deoxyribonucleic acid (DNA) is a nucleic acid that functions include
• Storage of genetic information
• Self-duplication & inheritance
• Expression of the genetic message
• DNA’s major function is to code for proteins. Information is encoded
in the order of the nitrogenous bases. Adenosine Cytosine Guanine
Thymine
Watson & Crick Model of DNA:
• DNA is composed of 2 chains of nucleotides
that form a double helix shape.
• The two strands are antiparallel.
• The backbone of the DNA molecule is
composed of alternating phosphate groups
and sugars.
• The complimentary nitrogenous bases form
hydrogen bonds between the strands.
• A is complimentary to T and G is
complimentary to C.
What is Sequencing:
• DNA sequencing is the process of determining
the sequence of nucleotides within a DNA
molecule.
• Every organism’s DNA consists of a unique
sequence of nucleotides.
• Determining the sequence can help scientists
compare DNA between organisms, which can
help show how the organisms are related.
• Used to determine the sequence of individual
genes, larger genetic regions, full chromosomes
or entire genomes.
• The resulting sequences may be used by
researchers in molecular biology or genetics to
further scientific progress.
Purpose:
• Understanding “code of cell life”
• Detecting mutations
• Typing microorganisms
• Identifying human halo-types
• Designating polymorphisms
History of Sequencing:
• 1972 – Earliest nucleotide sequencing – RNA sequencing of Bacteriophage
MS2 by WALTER FIESERR
• Early sequencing was performed with tRNA through a technique developed
by Richard Holley, who published the first structure of a tRNA in 1964.
• 1977 - DNA sequencing FREDRICK SANGER by Chain termination method
• Chemical degradation method by ALLAN MAXAM and WALTER GILBERT
• 1977 - First DNA genome t be sequenced of Bacteriophage ΦX174
• 1986 - LOREY and SMITH gave Semiautomated sequencing
• 1987 – Applied biosystems marketed Fully automated sequencing machines
DNA Sequencing Methods:
1)Maxam and Gilbert chemical degradation method
2) Chain termination or Dideoxy method
• Fredrick Sanger
3) Genome sequencing method
• Shotgun sequencing
• Clone contig approach
4) 2nd generation sequencing methods
• Pyrosequencing
• Nanopore sequencing
• Illumina sequencing
• Solid sequencing
Maxam Gilbert Chemical
Degradation Method:
• A. M. Maxam and [Link]-1977
• Chemical Sequencing
• Treatment of DNA with certain:-
Chemicals (formic acid, dimethyl sulfate,
hydrazine, piperidine).
• DNA cuts into Fragments
• Monitoring of sequences
Link for detail study:
[Link]
ntages-applications/
Steps Involved in Maxam-Gilbert
Method:
Labelling of DNA
• The first step involves labelling the DNA molecule with a radioactive or
fluorescent label. This allows the fragments of the DNA to be visualized on an
autoradiogram or gel.
• Chemical cleavage
• The labelled DNA is then treated with chemicals that cleave the DNA at
specific positions. There are four different chemical treatments that are used
in the Maxam-Gilbert method, each of which cleaves the DNA at different
positions:
• Hydrazine cleaves DNA at guanine (G) and adenine (A) residues.
• Dimethyl sulfate (DMS) cleaves DNA at cytosine (C) and guanine (G) residues.
• Formic acid cleaves DNA at purine residues (A and G).
• Piperidine cleaves DNA at purine residues (A and G).
Continue…:
• Fragment separation
• After the DNA has been cleaved, the resulting fragments are
separated by size using gel electrophoresis.
• Visualization and detection
• The fragments are then visualized using autoradiography or other
detection methods. The pattern of fragments generated by the
chemical cleavage is then analyzed to determine the sequence of the
DNA.
Advantages & Disadvantages:
Advantages:
• Maxam-Gilbert method can provide high accuracy in determining DNA sequence, as the
chemical cleavage is precise & predictable.
• This method can detect modifications to the DNA, such as methylated or damaged bases.
• Can be used to sequence different types of DNA, including single-stranded DNA and RNA.
• Can sequence through difficult regions of DNA, such as regions with high GC content or
secondary structures.
Disadvantages:
• Relatively complex and costly as compare to modern DNA sequencing technologies.
• Limited read length around 50-500 nucleotides
• Radioactive or fluorescent labelling can be hazardous and require special handling.
• Time-consuming process … labelling, chemical cleavage, fragment separation, and detection.
• Inconsistent yields: The yields of cleaved fragments can be variable and difficult to predict,
which can affect the accuracy of the sequencing results.
Sanger Method:
Most common approach used for
DNA sequencing .
• Invented by Frederick Sanger -
1977
• Nobel prize - 1980
• Also termed as Chain Termination
or Dideoxy method
Link for detail study:
[Link]
limitations/
Chemical Termination Method of DNA
Sequencing:
It involves following components:
a) Primer
b) DNA template
c) DNA polymerase
d) dNTPs (A,T,G,C)
e) ddNTPs
Principle:
Steps:
1. Denaturation
2. Primer attachment and extension of bases
3. Termination
4. Poly acrylamide gel electrophoresis
Other Types of Sequencing
Methods:
Pyrosequencing
• Pyrosequencing relies on light detection which occurs due to a chain
reaction initiated by the release of pyrophosphate.
• Unlike Sanger sequencing, this method does not use fluorescently
labeled nucleotides and does not rely on electrophoresis for
visualization.
• Pyrosequencing is useful for applications that require short reads and
real-time data analysis.
• This method has now been largely replaced by several new
technologies but it laid the groundwork for the development of
next-generation sequencing (NGS) technologies.
Advantages
• Accurate
• Parallel processing
• Easily automated
• Eliminates the need for nucleotides
• No need for gel electrophoresis
Disadvantages
• Only for smaller sequences
• Expensive
• Laborous
Link for detail study:
[Link]
Shotgun Sequencing:
• Shotgun sequencing, also known as shotgun cloning, is a method
used for sequencing long DNA strands or the whole genome.
• In shotgun sequencing, DNA is broken up randomly into numerous
small segments and overlapping regions are identified between all the
individual sequences that are generated.
• Multiple overlapping reads for the target DNA are obtained by
performing several rounds of this fragmentation and sequencing.
• Computer programs then use the overlapping ends of different reads
to assemble them into a continuous sequence.
• The shotgun approach was first used successfully with the bacterium
Haemophilus influenzae.
Advantages
• Faster because the mapping process was eliminated
• Uses less DNA than other methods
• Less expensive than approaches requiring a map
Disadvantages
• Requires computer processing power beyond what an ordinary
laboratory would possess
• Can introduce errors in the assembly process
• Requires a reference genome
• May not be able to assemble repetitive sequences
Link for more detail:
[Link]
Nanopore Sequencing:
• The fourth-generation DNA sequencing technology.
• Studies the interaction between DNA and protein, as well as between
protein and protein
• Nanopore sequencing uses electrophoresis to transport an unknown
sample through an orifice of 10−9 meters in diameter.
Continue…:
• The detection principle is based
on monitoring the ionic current
passing through the nanopore as
a voltage is applied across the
membrane.
• When the nanopore is of
molecular dimensions, passage
of molecules (e.g., DNA) cause
interruptions of the current level,
leading to a signal.
Types of Nanopore Sequencing:
• Biological nanopore
• Solid-state nanopore
• Hybrid nanopore
Advantages
• minimal sample preparation
• no requirement for polymerase and ligase
• potential of very long read-lengths ( > 10,000 – 50,000 nt )
• the instrument is inexpensive
Illumina Sequencing:
• Illumina dye sequencing is a technique used to determine the series
of base pairs in DNA, also known as DNA sequencing.
• This sequencing method is based on reversible dye-terminators that
enable the identification of single bases as they are introduced into
DNA strands.
• It has a similar principle to Sanger sequencing.
• Both methods use fluorescently-labelled nucleotides and identifies
each nucleotide by its fluorescent tag.
• However, while Sanger sequencing handles one DNA fragment at a
time, Illumina is massively parallel and allows millions of fragments to
be sequenced simultaneously in a single run.
Principle:
• Illumina sequencing works on the principle of sequencing by synthesis
(SBS).
• This involves identifying DNA bases as they are added to a growing
DNA strand.
• Fluorescently-labeled reversible terminator nucleotides are used and
the fluorescence emitted from each added nucleotide is captured.
• Each of the four DNA bases is labelled with a unique fluorescent dye,
allowing the sequencing system to detect which nucleotide has been
added during each cycle.
• The system captures images of these signals which are then used to
determine the exact sequence of the DNA fragment.
Continue…:
• Illumina sequencing also uses bridge amplification.
• In this process, DNA molecules with ligated adapters are used as
templates for repeated amplification on a solid surface like a glass
slide.
• The slide is coated with oligonucleotides complementary to the
adapters, which allows the DNA to form clusters of each fragment.
• This amplification process creates millions of these clusters on the
slide.
• The clusters enhance the signal and make it easier to detect and
differentiate between DNA bases by color.
Continue…:
• During sequencing, nucleotides with a unique fluorescent label are
added, incorporated, and detected in real time.
• These nucleotides also act as temporary terminators of DNA
synthesis, ensuring that only one base is added at a time which
reduces errors and provides high accuracy in reading the sequence.
• Once imaged, the terminator is cleaved and the next base is added.
• This cycle is repeated until the entire DNA fragment has been
sequenced.
Advantages
• High throughput/ cost.
• Suitable for a wide rage of applications most notably whole genome
sequencing.
Disadvantages
• Substitution error rates (recently improved).
• Lagging strand dephasing causes sequence quality deterioration
towards the end of read.
Application of Sequencing:
• DNA sequencing may be used to determine the sequence of
individual genes, larger genetic regions (i.e. clusters of genes or
operons), full chromosomes, or entire genomes of any organism.
• DNA sequencing is also the most efficient way to indirectly sequence
RNA or proteins (via their open reading frames).
• In fact, DNA sequencing has become a key technology in many areas
of biology and other sciences such as medicine, forensics, and
anthropology.
Continue…:
• Molecular biology
•Evolutionary biology
• Metagenomics
• Medicine
• Forensics
Gene Mapping
Gene Mapping:
• Gene mapping determines the specific location of genes on
chromosomes.
• Each gene occupies a fixed position, called a locus.
• It reveals which chromosome a gene is on and how close it is
to others.
• Mapping shows the order and distances between genes to
explain inheritance patterns.
Importance of Gene Mapping:
• Identifies genes responsible for diseases (e.g., cystic
fibrosis).
• Helps in medical research and development of gene-based
therapies.
• Improves crop traits in agriculture (yield, resistance).
• Supports evolutionary and genetic diversity studies.
Types of Gene Mapping:
1. Genetic (Linkage) Mapping: Based on how often genes are
inherited together.
2. Physical Mapping: Determines the actual distance between
genes using molecular techniques.
3. Sequence Mapping: Uses DNA sequencing to pinpoint exact
locations of genes.
Key Terms:
• Gene Locus: Fixed position of a gene on a chromosome.
• Recombination: Exchange of genetic material during meiosis
creating new allele combinations.
• Map Unit (Centimorgan, cM): Distance between genes with
a 1% recombination frequency.
Principle Behind Gene Mapping:
• The closer two genes are, the less likely they are to separate
during recombination.
• The farther apart they are, the more likely a crossover
occurs.
• Recombination frequency helps estimate gene distances.
• Example: 10% recombination = 10 centimorgans apart.
Example – Fruit Fly Experiment:
• Thomas Hunt Morgan’s Drosophila experiments showed
gene linkage.
• Alfred Sturtevant created the first genetic map in 1913 using
recombination data.
• Confirmed that genes are arranged linearly on
chromosomes.
Steps in Gene Mapping:
1. Cross individuals with known traits.
2. Record offspring phenotypes.
3. Calculate recombination frequency:
Recombination (%) = (Recombinants / Total Offspring) × 100
4. Construct linkage map to determine gene order and distance.
Applications of Gene Mapping:
• Medical: Identify disease-causing genes and develop gene
therapies.
• Agriculture: Improve crops through marker-assisted
selection.
• Evolutionary Biology: Compare gene maps across species.
• Forensics: Use genetic markers for identification.
Molecular Medicine
Molecular Medicine:
• Core Idea: Molecular medicine is a broad field that focuses on
understanding disease at the molecular and cellular level to develop
precise diagnostic tools and targeted therapies.
• It moves beyond treating symptoms to addressing the root cause of
illness—faulty genes, misbehaving proteins, and dysfunctional cellular
pathways.
• The Goal: To provide Personalized or Precision Medicine—treatments
tailored to an individual's unique genetic makeup and specific disease
subtype.
• Key Shift: From "What disease do you have?" to "What is the
molecular profile of your disease?"
Role in Diagnosis:
• Revolutionizing How We Detect and Monitor Disease
• Genomic Sequencing:
• Reading a patient's entire DNA code to identify genetic mutations that cause or
increase risk for diseases like cancer, cystic fibrosis, or Huntington's.
• Biomarkers:
• Measuring specific molecules (e.g., proteins, DNA fragments) in blood or tissue
that indicate the presence or progression of a disease. (e.g., PSA for prostate
cancer, viral RNA for infections).
• Molecular Imaging:
• Using advanced techniques like PET scans with molecular tracers to
visualize disease activity inside the body in real-time.
Role in Treatment:
"One-Size-Fits-All" Drugs
• Targeted Therapies:
Drugs designed to specifically block the function of a mutated protein that
drives cancer growth (e.g., Imatinib for chronic myeloid leukemia).
• Immunotherapy:
Harnessing the body's own immune system to fight disease. This includes CAR-T
cell therapy, where a patient's immune cells are engineered to recognize and
destroy cancer cells.
• Gene Therapy:
Correcting or replacing a faulty gene to cure genetic disorders. Delivers a
healthy copy of a gene to compensate for the non-functional one.
Real-World Impact & Examples:
Lab Bench to Bedside
• Oncology (Cancer):
Example: Testing for the *BRCA1/2* gene mutations to assess breast/ovarian cancer
risk and using PARP inhibitor drugs specifically for these patients.
• Rare Genetic Diseases:
Example: Spinal Muscular Atrophy (SMA) treated with Zolgensma, a one-time gene
therapy that delivers a functional copy of the SMN1 gene.
• Infectious Diseases:
Example: mRNA vaccines (like for COVID-19) are a product of molecular
medicine, providing our cells with instructions to make a harmless viral protein
and train our immune system.
Future of Molecular Medicine:
Challenges & Opportunities
• Challenges:
• High costs of development and treatment.
• Ethical considerations (genetic privacy, "designer babies").
• Ensuring equitable access for all populations.
• The Future:
• AI & Big Data: Accelerating drug discovery and diagnosis.
• CRISPR Gene Editing: Offering the potential to precisely "edit" genetic errors.
• Earlier Intervention: Predicting and preventing disease before symptoms appear.
Molecular medicine is not just a specialty—it is the future foundation of all
healthcare, making it more predictive, personalized, and powerful.
Thank You