Molecular Biology and

Genetics
Rabia Sultan
PhD Scholar

1
Molecular Biology and Genetics

Basic Molecular Biology

Advanced Molecular
Biology

2
PART 1 (Basics of Molecular Biology)
 Structure of DNA
 DNA Replication and Recombination
 Mutation and DNA repair
 Transcription and Translation
 Cell-based DNA cloning
 Nucleic acid hybridization assays
 PCR, DNA sequencing and in-vitro mutagenesis
 Organization of human genome
 Human gene expression
 Instability of Human Genome
 Physical and Transcript mapping

3
 PART 2 (Advanced Molecular Biology)
 Introduction to Molecular Medicine and Gene
Therapy.
 Gene therapy for neurological disorders
 Gene therapy for musculoskeletal disorders
– Bones
– Ligaments and Tendons
– Cartilage
– Intervertebral disc
– Muscles

4
Outlines:
• Overview of Genome
• Central Dogma of Life
– Chemical structure of DNA
– Forms of DNA
• DNA Replication
• References

5
Cells come in a variety of shapes and sizes

6
 How big is genome?
WHY ARE WE INTERESTED?

7
8
Where does this diversity come from?

9
Conversion of biological information from a
one- to a three- and finally a four
dimensional
state.

10
Central Dogma of Biology!

11
 DNA (deoxyribonucleic acid)
• Made up of :
– Sugar (de-oxy ribose)
– Bases (ATGC)
• Adenine
• Thymine
• Guanine
• Cytosine
– Phosphate group

• DNA is the polymer made up

of small subunits nucleotides.

12
 Purine and Pyrimidine

BASES

13
14
15
 DNA
(deoxyribonucleic
acid)

16
Phosphodiester Bonds
Link Successive
Nucleotides in
Nucleic Acids

• Phosphodiester bond
is formed between two
nucleotides.
• 3’ of the previous
nucleotides, 5’ of the
next join together.

17
Under high temperature conditions, hydrogen bonds are broken
and DNA strands are denatured. When the temperature is
optimal, the strands reanneal. 18
Nucleotide vs Nucleoside

19
WATSON
and
CRICK
model of
DNA

20
 Comparison of A, B, and Z
forms of DNA.

• The Watson-Crick structure

is also referred to as B-
form DNA, or B-DNA.
The B form is the most
stable structure for a
random-sequence DNA
molecule under
physiological conditions.

21
 Chargaff rule
1. The base composition of DNA generally varies from one species to
another.
2. DNA specimens isolated from different tissues of the same species
have the same base composition.
3. The base composition of DNA in a given species does not change
with an organism’s age, nutritional state, or changing environment.
4. In all cellular DNAs, regardless of the species, the number of
adenosine residues is equal to the number of thymidine residues
(that is, A = T), and the number of guanosine residues is equal to the
number of cystidine residues (G = C).
From these relationships it follows that the sum of the purine
residues equals the sum of the pyrimidine residues; that is, A and G
= T and C.

22
 Question: 1
• The nucleotide sequence of one DNA strand of
a DNA double helix is

5’-GGATTTTTGTCCACAATCA-3’
3’-CCTAAAAACAGGTGTTAGT-5’
• What is the sequence of the complementary
strand?

23
 Question: 2
• In the DNA of certain bacterial cells, 13% of
the nucleotides are adenine. What are the
percentages of the other nucleotides?
ATGC= (100%)
A= 13% T=13% = 26 A=13%
AT (26) =CG (100-26 = 74) T=13%
C= 37%
74/2 = 37 G=37%
C= 37% G=37%
24
 Question: 3
The two strands of a DNA double helix can be separated by
heating. If you raised the temperature of a solution containing
the following three DNA molecules, in what order do you
suppose they would “melt”? Explain your answer.

A) 5’-GCGGGCCAGCCCGAGTGGGTAGCCCAGG-3’
3’-CGCCCGGTCGGGCTCACCCATCGGGTCC-5’
B. 5’-ATTATAAAATATTTAGATACTATATTTACAA-3’
3’-TAATATTTTATAAATCTATGATATAAATGTT-5’
C. 5’-AGAGCTAGATCGAT-3’
3’-TCTCGATCTAGCTA-5’

25
 Question: 3 (ANSWER)
The two strands of a DNA double helix can be separated by heating. If you
raised the temperature of a solution containing the following three DNA
molecules, in what order do you suppose they would “melt”? Explain
your answer.

helix C (containing a total of 34 hydrogen bonds) would melt at the lowest

temperature,
helix B (containing a total of 65 hydrogen bonds) would melt next, and
helix A (containing a total of 78 hydrogen bonds) would melt last.

Helix A is the most stable, largely owing to its high GC content. Indeed, the
DNA of organisms that grow in extreme temperature environments, such as
certain prokaryotes that grow in geothermal vents, has an unusually high GC
content.
26
 Question: 4
Base Pairing in DNA In samples of DNA isolated from
two unidentified species of bacteria, X and Y, adenine
makes up 32% and 17%, respectively, of the total bases.

1. What relative proportions of adenine, guanine, thymine,

and cytosine would you expect to find in the two DNA
samples?

2. What assumptions have you made? One of these species

was isolated from a hot spring (64 8C). Which species is
most likely the thermophilic bacterium, and why?
27
 Question: 4 (ANSWER)
Answer
• X Specie • Y Specie
Specie X has 36% GC
• A= 32% • A= 17% Content
• T= 32% • T= 17% Specie Y has 66% GC
• G= 18% • G= 33% Content
• C= 18% • C=33%
Specie Y can tolerate
high temperature so it is
thermophilic

28
 DNA Replication

29
CELL CYCLE

30
 DNA Replication

• The process in which DNA makes multiple

copies of it.

31
 Models of DNA Replication

32
 DNA Replication
The direction of DNA replication is 5` to 3`

33
 Players of DNA replication

• Helicase (unwinding enzyme)

• DNA Polymerase III (Catalytic enzyme)
• DNA Primase
• Single-stranded binding proteins (SSBP)
• Ligase

34
 HELICASE
• Unwinds the double stranded DNA by
breaking the hydrogen bonds between bases.
• Y-Shaped structure is formed---
REPLICATION FORK

35
 DNA POLYMERASE III
• A) DNA polymerase catalyzes the stepwise addition
of a deoxyribonucleotide to the 3ʹ-OH end of a
polynucleotide chain, the growing primer strand that
is paired to an existing template strand.
• The newly synthesized DNA strand therefore
polymerizes in the 5ʹ-to-3ʹ direction.

36
 Proofreading
mechanism of
DNA
Polymerase III

37
DNA Polymerase III

38
Sliding clamp holds DNA polymerase on
the DNA.

39
Limitations of DNA polymerase III
• DNA polymerase is limited by the fact that it
lacks exonuclease activity in a 5‘ to 3'
direction.
• It cannot initiate synthesis on its own. It also is
prone to making errors.

40
DNA Polymerase cant start replication!
• DNA Primase is the
enzyme that make
RNA primer and
facilitate DNA
Polymerase in the
replication

41
Single-Strand DNA-binding proteins (SSB
proteins)
• Bind to the single strand of DNA and prevent
the annealing of the strand.

42
 Leading and Lagging Strand
• The synthesis of each Okazaki fragment ends when
this DNA polymerase runs into the RNA primer
attached to the 5ʹ end of the previous fragment.

43
 LIGASE
• The RNA primers are removed by DNA
polymerase I.
• The enzyme Ligase seals the two strands
together.

44
45
 TOPOISOMERASES
• The over winding, in turn, is continually
relieved by proteins known as DNA
Topoisomerases.
• A DNA

46
 Summary
• DNA replication takes place at a Y-shaped structure called a replication
fork.
• A self-correcting DNA polymerase enzyme catalyzes nucleotide
polymerization in a 5ʹ-to-3ʹ direction, copying a DNA template strand
with remarkable fidelity.
• Since the two strands of a DNA double helix are antiparallel, this 5ʹ-to-
3ʹ DNA synthesis can take place continuously on only one of the
strands at a replication fork (the leading strand).
• On the lagging strand, short DNA fragments must be made by a
“backstitching” process.
• Because the self-correcting DNA polymerase cannot start a new chain,
these lagging-strand DNA fragments are primed by short RNA primer
molecules that are subsequently erased and replaced with DNA.

47
• DNA replication requires the cooperation of many proteins. These
include
• (1) DNA polymerase and DNA primase to catalyze nucleoside
triphosphate polymerization;
• (2) DNA helicases and single-strand DNA-binding (SSB)
proteins to help in opening up the DNA helix so that it can be
copied;
• (3) DNA ligase and an enzyme that degrades RNA primers to
seal together the discontinuously synthesized laggingstrand DNA
fragments; and
• (4) DNA topoisomerases to help to relieve helical winding and
DNA tangling problems.
Many of these proteins associate with each other at a replication
fork to form a highly efficient “replication machine,” through
which the activities and spatial movements of the individual
components are coordinated.
48
 References
• Molecular Biology of the cell- Albert
• Essential cell biology- Albert
• Genetics from Gene and Genomes
• Albert Lehninger- David L. Nelson

49
• https://www.youtube.com/watch?v=2_-jSoSa
aTA

50