1962 Nobel Prize
The double helix duo
James D. Watson and
Francis H. C. Crick
Nature 171, 737-738 (1953)
“A Structure for Deoxyribose Nucleic Acid”
Phoebus Levene DNA was first isolated from WBC
DNA (phosphate, by Johannes Friedrich Miescher,
sugar, base), 1919 1869
Erwin Chargaff
No. of A = No. of T
No. of G = No. of C
Rosalind Franklin Maurice Wilkins
Linus Pauling
Nobel Prize 1954, 1962 X-ray diffraction of DNA
The Structure and constituents of nucleic acids
DNA: genetic storage unit
RNA: genetic storage in viruses,
Acts as catalysts and gene-regulator
Most naturally occurring DNA is
dsDNA, locked in a helical structure.
Consists of: deoxy-ribose/ribose
sugar, phosphates and heterocyclic
nucleobases (A, T, G, C).
Alignment: Sugar and phosphates
along the helical axis, nucleobase
Perpendicular to helical axis.
Direction: from 5’→3’. 3’-OH is free,
5’-OH is phosphorylated.
Watson-Crick Double Helix
5
Nature 171, 737-738 (1953)
Eukaryotic Cell Nucleus
Human chromosomes
46 chromosomes: 22 pairs,
Plus X and Y (male) or two X (female)
3 billion base-pairs
packed in Chromatin
Total length: 1.8 m or about 6 ft
Total length of DNA in a human
Body: 100 trillion cells x 6 feet
It would reach the sun and back
600 times
Prokaryotes do not have cell nucleus
All materials are stored in cytoplasm
6
Helix Diameter
Major Groove:
Hosts large biomolecules
e.g., Proteins, Nucleic Acids
Minor Groove:
Binding domain for small molecules
e.g., ions, organic molecules
Helix pitch
7
DNA B-form (right-handed helix)
H
N N H O CH3
N A N H N T
R
N N
R
O
Building Blocks of DNA and RNA
Nucleic acids Nucleobase + D-ribofuranose + Phosphate
Nucleobase + sugar: Nucleoside
Nucleobase + sugar + phosphate: Nucleotide
RNA bases: A, U, G, C
DNA bases: A, T, G, C
H
N N H O CH3
N A N H N T
R
N N
R
O
9
Forces that stabilize DNA
1. Hydrogen bonding (Watson-Crick, Hoogsteen, Wobble)
2. pi-stacking between adjacent base pairs
All nucleobases are
Neutral at pH=7
pKa values of the nucleobases at 20°C in water
10
syn anti syn anti
Rotation of the bases around C-1’ is highly restricted due to steric interaction with C-2’
hydrogen
A ═ T base pair G Ξ C base pair
11
Secondary structure of DNA/RNA
D-deoxy-ribose (DNA) D-ribose (RNA)
D-deoxy-ribose (DNA) conformations:
HO
HO Base HO Base
O O O
OH
OH OH
HO
C2’-endo twist C3’-endo twist
Envelope form This puckered form exists in This puckered form exists in
DNA RNA
Twisted form: C1’-O4’-C4’ are in plane, others out of plane in an endo twist. 12
Out of the two twisted forms the preferred conformation will be dependent on
the substituent at C-2’. Presence of EW group e.g. –OH, will lead to C3’-endo twist
as in RNA. Deoxyribose is more flexible than ribose and can adopt both conformations
Secondary structure of DNA
A ═ T base pair
G Ξ C base pair
13
DNA melting, concentration and hybridization
14
15
16
DNA Replication
DNA Replication
Major enzymes involved
Helicase: The protein(s) that unwound a portion of the double helix to form the replication
fork. The enzyme targets A-T rich sequence in the gene.
SSB Protein (single strand DNA binding): The proteins prevent the replication fork from re-
annealing.
DNA polymerase: Two polymerases are involved. One DNA polymerase (called
polymerase δ) binds to the parent DNA strand which is 5’-3’, uses it as a template and
begins replication (i.e synthesizing new strand) along the new 5' to 3' direction. This is
known as leading strand.
Because DNA synthesis can only occur 5' to 3‘ direction, a molecule of a second type of
DNA polymerase (epsilon, ε, in eukaryotes) binds to the other template strand as the double
helix opens. This molecule uses short RNA sequences as primers and continue strand
elonogation incorporating DNA nucleotides. (called Okazaki fragments). Another enzyme,
DNA ligase I then stitches these together into the lagging strand
RNA primase: Type of RNA polymerase that provides RNA primers required to initiate
replication process.
RNAse: This enzyme destroys the RNA primers once the synthesis is over.
20
DNA ligase: Stitches (ligates) the synthesized DNA fragments (Okazaki fragments).
3’
5’
5’
5’
Replication fork
3’
Helicase
Single strand binding protein
Primase
RNA primer
5’
DNA polymerase δ
DNA polymerase ε
dNTP
Newly
Synthesized
strand
3’
5’
3’
5’
5’
3’
3’
5’
3’
5’
Leading strand
Okazaki fragment
Lagging strand
Newly
Newlysynthesized
synthesizedDNA
DNAmolecules
fragment
with RNA
with Impurity
nick
Exonuclease
DNA polymerase
DNA ligase joins the newly synthesized DNA
strands and fills up the nick
DNA Replication: Steps
5’- P
3’ -OH
3’ -OH P-5’
Helicase
5’- P
Replication fork is prevented from
Replication fork
re-hybridizing by SSB proteins
3’ -OH
Helicase
Polymerase δ 3’ (Leading strand)
5’
5’- P
Polymerase always recognizes
5’ RNA primer 3’-5’ strand and controlled DNA synthesis
always occurs from 5’-3’ direction
3’ -OH
Polymerase ε
5’ (Lagging strand)
27
dNTPs 3’ (Leading strand)
5’
5’- P
Leading strand: continuous synthesis
5’
Lagging strand: Discrete synthesis
3’ -OH Okazaki fragments
RNA
3’
DNA
5’ (Lagging strand)
RNAse/
exonuclease
5’- P 3’ -OH
New DNA from Leading strand
3’ -OH P-5’
5’ 5’ 5’
3’ -OH P-5’
DNA Ligase
5’- P 3’ -OH
New DNA from Lagging strand
28
3’ -OH P-5’
Polymerase Chain Reaction (PCR)
PCR is a technique to amplify genomic DNA, extracted from organisms. It’s one of the most important
discoveries that has revolutionized biochemical/biological research.
Applications: Identifying cDNAs or any genomic DNA, detection of genomic mutations, diagnosis of genetic
diseases.
Principles: Complementary to DNA replication, requires thermal cycles.
The basic process of PCR requires mainly 5 components:
The whole setup and PCR Mixture
Figure 1: PCR Machine
1. DNA template: isolated DNA
2. dNTPs: Deoxynucleotide Triphosphate is the building block of DNA. Which helps to make the entire
DNA Strand. Two types of dNTPs are there, Purines and Pyrimidines.
Example: Purines: Adenine and Guanine. Pyrimidines: Thymine and Cytosine.
3. DNA Polymerase: DNA Polymerase catalyzes the synthesis of DNA during genomic DNA
replication and Repair. Taq DNA Polymerase, used in the Polymerase Chain Reaction is a thermostable
DNA polymerase, isolated from the thermophillic bacterium Thermus aquaticus (Taq).
Figure 3: Structure of Taq DNA Polymerase.
Source: DNA polymerases engineered by directed evolution to
incorporate non-standard nucleotides Roberto Laos*, J.
Michael Thomson and Steven A. Benner
4. Oligonucleotide Primers: Primer is a short nucleic acid sequence that provides a starting point for DNA
synthesis. Two primers used in the PCR reaction are Forward Primer and Reverse Primer.
5. Mgcl2 Buffer System: Magnesium ion acts as an cofactor of the enzyme Taq Polymerase, which binds in
the catalytic sides of Taq and increases the efficiency of the enzyme.
Denaturation Cycle 1 95°C
5’ 3’
5’ 3’
3’ 5’
3’ 5’
Annealing Cycle 1 60°C
Extension 72°C
Two Copies of Target DNA
5’ 3’
3’ 5’
Hydrogen Bonds
5’ 3’
3’ 5’
Taq Polymerase
DNA Primer
dNTP
Denaturation Cycle 2 95°C
5’ 3’
5’ 3’
3’ 5’
3’ 5’
5’ 3’
5’ 3’
3’ 5’
3’ 5’
Cycle 2
Annealing 60°C
Four Copies of Target DNA
Extension
5’ 3’ 72°C
3’ 5’
5’ 3’
3’ 5’
5’ 3’
3’ 5’
5’ 3’
3’
DNA Primer Taq Polymerase dNTP
Cycle 3
8 copies of target DNA
DNA sequencing methods
DNA sequencing methods
DNA sequencing methods
DNA sequencing methods
Sanger’s di-deoxy method
Sanger’s Method
•To know the exact sequence of given DNA strand
•Usually done by controlled termination/interruption
of replication
•Single stranded DNA is used for sequencing
3’ -OH 5’- P
dATP
5’- P dTTP DNA Polymerase I
dCTP
dGTP
ddTTP ddGTP
ddCTP
ddATP
O O O
O P O P O P O Base
O O O O
ddNTP H H 51
DNA sequencing methods
DNA sequencing methods
DNA sequencing methods
DNA sequencing methods
DNA sequencing methods
DNA sequencing methods
DNA synthesis: Solid phase phosphoramidite chemistry
58
59
60
61
Solid support is CPG (controlled pore glass)
62
1. Detritylation of the support-bound 3′-nucleoside by 3% TFA or TCA
63
2. Activation and coupling: by tetrazole as catalyst
64
3. Capping: by acetic anhydride and N-methyl imidazole
65
4. Oxidation: by iodine H2O/pyridine
66
5. Cleavage from the CPG: by NH3/MeOH, 55C
67
68
Operation Reagent/solvent Time
Wash acetonitrile 30 s
3% trichloroacetic acid in
Detritylate 50 s
dichloromethane
Monitor trityl - -
Wash acetonitrile 30 s
Flush argon 10 s
0.1 M phosphoramidite
Couple monomer and 0.5 M 30 s
tetrazole in acetonitrile
Wash acetonitrile 30 s
10 s
Flush argon
acetic
anhydride/pyridine/THF
Cap 1/1/8 and 17.6% w/v N- 30 s
methyl imidazole in
acetonitrile
Wash acetonitrile 30 s
Flush argon 10 s
0.015 M iodine in
Oxidize water/pyridine/THF 45 s
2/20/78
Wash acetonitrile 30 s
Flush argon 10 s 69
