4. Ribose and phosphate
• β-furanose
pentoses in both
• carbons
numbered:
• 1’,2’,3’,4’,5’
• DNA:
• 2’ Deoxyribose
• or just
deoxyribose
Phosphate
• Gives negative
charge
2’
3’
4’
5’
1’
5. •Cells can store information for long term & if alterations happens, they
sometimes become significant like mutations
•Carcinogenesis and aging are also slow irreversible accumulation of
alterations in DNA
•Among these alterations some are non-destructive and important for
function strand separation for replication & transcription
8. Ribose and phosphate
• β-furanose
pentoses in both
• carbons
numbered:
• 1’,2’,3’,4’,5’
• DNA:
• 2’ Deoxyribose
• or just
deoxyribose
Phosphate
• Gives negative
charge
2’
3’
4’
5’
1’
9. •Cells can store information for long term & if alterations
happens, they sometimes become significant like mutations
•Carcinogenesis and aging are also slow irreversible accumulation
of alterations in DNA
•Among these alterations some are non-destructive and
important for function strand separation for replication &
transcription
Nucleic Acid Chemistry a powerful array of
technologies that have applications in molecular
biology, medicine and forensics
10. Solubility
• DNA is polar in nature and thus soluble in water. Its highly charged
phosphate-sugar backbone gives it its polarity. However, in the presence of
salt and alcohol, it is insoluble.
• Dielectric constant of water and alcohol 78.4, 24.5, high dielectric constant of
water decreases the attraction between charged species.
•Salting in and Salting out
11. Absorption
• At 260 nanometers, the DNA bases can absorb ultraviolet light. A
spectrophotometer can measure this absorption. The amount of ultraviolet
light absorbed increases with the order of the bases. For example, at 260 nm,
a single-stranded DNA absorbs 1.37 units, whereas a double-stranded DNA
absorbs 1.00 unit at 260 nm
12. • All molecules absorb radiant energy at a
specific wavelength, from which it is possible to
extrapolate the concentration of a solute within a
solution.
According to the Beer-Lambert law there is a linear
relationship between the absorbance A (also called
optical density, OD) and the concentration of the
macromolecule given by the following equation:
A=OD=εlc
Where ε is the molar extinction coefficient, c is the
concentration; and l is the pathlength of the cuvette.
Proteins and nucleic acids absorb light in the ultraviolet
range within wavelengths of between 210 and 300 nm. As
previously explained, the maximum absorbance of DNA
and RNA solutions is at 260 nm whereas the maximum
absorbance of protein solutions is at 280 nm.
13. Denaturation and Renaturation
• On heating, both strands denature, and on cooling, they can renature. The
melting temperature, which varies depending on the precise DNA sequence,
is the temperature at which these strands are permanently separable.
• In contrast to the region of higher concentration A-T, which is only bonded with
two hydrogen bonds, the region of higher concentration of C-G has a higher
melting temperature because these bases are bonded with three hydrogen
bonds, which require more energy to break.
14. DNA/ RNA denaturation
DNA/ RNA denaturation: (Reversible, Change in 3-D structure that is
due cleavage of all weak cross linkages inside the molecule except
covalent backbone and can be renatured)
E.g. at pH=7, 25oC DNA solutions are viscous but extreme pH +
temperature above 80 oC decreases viscocity (physical change, solubility)
similar to denaturation of proteins denaturation of DNA
15. DNA/ RNA denaturation
Disruption of the hydrogen bonds between
paired bases and of base stacking cause
unwinding of the double helix to form two
single strand , no covalent bond broken on
attaining same pH or temp renaturation or
annealing to intact duplex partial
denaturation
If completely strands are separated
complete denaturation both strands first
find by random collisions each other short
complementary segments and then zipper
themselves fastly to duplex
Hypochromic effect absorption of UV
light decreases as free strands or nucleotides
are again in duplex form Hyperchromic
effect when denatured
16.
17. When DNA is denatured, the melting point is when half of the DNA is
present as separated single strands melting temperature tm different
for different DNA
The greater the G≡C content, the higher the melting point (tm) tm indicates base
compositions
18. Electron micrograph showing partially denatured DNA
Red arrows indicate regions of high A-T content, which denature first on careful
increase of temperature
Bubbles
The same happens during replication and transcription which starts from AT rich
sequences
DNA duplexes ↓ stable as compared to DNA-RNA hybrid duplex which is itself ↓
stable to RNA-RNA or RNA duplexes
19. Hybridization (DNA or RNA)
2 complementary DNA strands can pair with one another to detect
similar DNA sequences in 2 different species or within the same specie
genome.
E.G., human and mouse DNA below tm Duplex hybrids
DNA annealing temperature, length & conc. of DNA fragments
annealed, concentration of salts of
reaction mixture, and properties of the sequence (GC%)
Low temperature important too low can anneal even distant
matching parts
20.
21. Common evolutionary heritage related functional and
structural proteins have same DNAs
applications in
modern genetic techniques (Blotting) to nitrocellulose
membrane and checking gene sequences by probing
unknown genes with known functions can be isolated,
individuals can be identified, diseases before onset can be
detected.
