Download 4-Biochemical Properties of DNA and The Technology involve them

FOURTH WEEK
Gihan E-H Gawish, MSc, PhD
Ass. Professor
Molecular Genetics and Clinical
Biochemistry
KSU
Biochemical Properties of Genetic Material
(DNA & RNA)
Tropp, B; Molecular Biology genes to proteins. Jones and Bartlett Publisher. 2008
 1-
1 Absorption Spectra
Absorb light in ultraviolet range, most strongly
in the 254-260 nm range
Due to the purine and pyrimidine bases
Useful for localization, characterization and
quantification of samples
 1-2
Sedimentation
and density
• Can be characterized by
sedimentation velocity
(Svedberg coefficient, S)
 Sedimentation velocity
centrifugation
 Related to MW and shape
• Or by buoyant density
 CsCl (DNA) or CsSO4 for
RNA
 Sedimentation
equilibrium centrifugation
 G-C
base pairs are more dense than
A-T pairs
 Agarose
or polyacrylamide gels
 DNA is negatively charged and migrates
toward positive pole when placed in an
electric field
 Smaller fragments move through the gel
matrix more quickly and therefore migrate
faster per unit of time
 Extremely
common
method
for
characterizing and purifying DNA fragments
• Including DNA sequencing procedures
Gel
Electrophoresis
Animation
Denaturation
Reassociation
Nucleic Acid
Properties
Fragile
Supercoiling
Chemical Modification
Degradation
Mutation
 Denaturation
involves the breaking of
hydrogen bonds
• Disrupts the base stacking in the helix and lead to
increased absorbance at 260 nm
 Hyperchomic shift
 By
increasing temperature slowly and
measuring absorbance at 260 nm as melting
profile can be generated
• Temperature for midpoint of denaturation is called
the Tm
 Increased
G+C gives
increased Tm
• 3 vs. 2
hydrogen
bonds
 Increased
ionic
strength also
increases Tm
 Denatured
DNA duplexes can reassociate
complementary strands to reform duplex
with
• Chemical reaction, rate depends upon conditions
 including substrate concentration
Co (starting concentration)
t1/2 (time taken for half the DNA to reassociate).
 DNA
concentration is routinely measured
in micrograms per ml (mass/volume)
• But here the relevant concentration is copies of
complementary DNA (not mass) per unit volume
• And this depends upon both the mass per volume
and the size of the genome being studied
 Previous
curves were for genomes
generally lacking repetitive sequence
regions
• Al or nearly all sequences present at one copy per
genome
 What
happens to the C0t analyses when
genomes have repetitive sequences?
• Single copy, middle and highly repetitive
2-2 C0t Analyses
When the nucleotide sequence of
one strand is known,
The sequence of the
complementary strand can be
predicted

After nucleic acids are denatured they can be allowed
to reform base pairs with complementary molecules
• Molecular hybridization
• Close but not perfect match required
 stringency
• Can involve DNA:DNA or DNA:RNA
• FISH, Southern transfer (blotting) and DNA microarray
analyses involve hybridization
 FISH
 Use
DNA or RNA
probes for
hybridization
• Originally radioactive
• Now biotin and
fluorescent dyes
 Cells/chromosomes
fixed to slide before
hybridization
 Can detect single copy
genes
Objective: Identify Specific Parts of a Chromosome
Procedure
http://FISH

This image shows chromosomes
with fluorescent R-bands.

The bright green dots are probes
complementary
to
olfactory
receptor homologues.

In most chromosomes these areas
are subtelomeric, i.e. near the end
of the chromosomes, but in
chromosome 2 (bottom, left) we
see that the probe has hybridized
to the middle of the chromosome.
•A comparison with ape chromosomes shows that the human chromosome 2
is the result of an end to end fusion of two ancestral chromosomes.
•As a result the two subtelomeric ends became the middle of chromosome 2,
which is why we get hybridization of the probe there
 The
southern blot is
used to verify the
presence or absence of
a specific nucleotide
sequence in the DNA
from different sources
 And
to identify the
size of the restriction
fragment
that
contains the sequence
(2) Southern Blot
(Animation) , (Procedure)
The
great lengths of DNA molecules make them
extremely susceptible to breakage by the
hydrodynamic forces
Pipetting
Pouring
Mixing
Great care is taken when larger DNA molecule are isolated
Nature of Supercoiling

It is a physical rearrangement of
the DNA double helix that allows
it to conform more closely.
 DNA can adopt a more compact
configuration due to supercoiling.
 Linking number (Lk) is the
number of times the two
phosphodiester backbones wrap
around one another in a given
distance.
 It depend on the solution
conditions
Enzymes
 DNA gyrase adds negative
supercoils
DNA topoisomerases remove
negative super coils
DNA replication
introduce
Supercoiling
tends to
positive
OXIDATION
OXYGEN SPECIES
METHYLATION
METHYLTRANSFERASES

Cytosine:

8-oxo-7,8-dihydroguanine
(8-oxoG)
HYDROLYSIS
4
N - methylcytosine

Adenine: N 4- methyladenine
Deamin
-ation
Depuri
-nation
Remodeling
Block
Replication
Mutation
DNA Repair
Chemical Modification
•Alkylation (Methylation)
•Oxidation
•Hydrolysis
Nucleases

They are hydrolytically
cleave the phosphodiester
backbone of DNA
1Endo-
2ExoCleave
Middle of Chain The End of the Chain
3- Restriction Endonucleases
 Mutations
in DNA sequences generally occur
through one of two processes:
 DNA
damage from environmental agents such as
ultraviolet light (sunshine), nuclear radiation or
certain chemicals
 Mistakes
that occur when a cell copies its DNA in
preparation for cell division.
Addition
Deletion
Translocation
Mutation