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Nucleic acids
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Nucleic acids are polymers of monomers called
nucleotides
2 types of nucleic acid – DNA & RNA
Each nucleotides composed of 3 parts :
- A nitrogenous base
pyrimidines
purines
- A pentose (5-C sugar)
- Phosphate group
Nitrogenous base

A, G, C found as both RNA and DNA
 U is found as RNA
 T is found as DNA
PENTOSE SUGAR
 In
ribonucleotides, the
pentose is ribose
 In
deoxyribonucleotide (or
deoxynucleotides) the
sugar is 2’-deoxyribose –
the carbon at position 2’
lacks a hydroxyl group
 In
a nucleic acid polymer/polynucleotide,
nucleotides are joined by covalent bonds =
phosphodiester linkage (between
phosphate of one nucleotides and the
sugar of the next)
Nucleic acid structure

Nucleotides can be joined to each other to form RNA and
DNA.
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The nucleic acids are chains of nucleotides whose
phosphates bridge the 3' and 5' positions of neighboring
ribose units
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The phosphates of these polynucleotides are acidic, so
at physiological pH, nucleic acids are polyanions.

The linkage between individual nucleotides is known as a
phosphodiester bond.
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Each nucleotide that has been incorporated into
the polynucleotide is known as a nucleotide
residue.
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The terminal residue whose C5' is not linked to
another nucleotide is called the 5' end
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The terminal residue whose C3' is not linked to
another nucleotide is called the 3' end.
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By convention, the sequence of nucleotide
residues in a nucleic acid is written, left to right,
from the 5' end to the 3' end.
Nucleic acid structure
Definitions
 DNA stands
for deoxyribonucleic acid. It is
the genetic code molecule for most
organisms.
 RNA stands
for ribonucleic acid. RNA
molecules are involved in converting the
genetic information in DNA into proteins.
In retroviruses, RNA is the genetic
material.
DNA structure : Watson and Crick

Watson and Crick 1953 1st
proposed the double helix as 3-D
structure of DNA
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Two polynucleotide chains wind
around a common axis to form a
double helix.
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The two strands of DNA are
antiparallel, but each forms a
right-handed helix.
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The bases occupy the core of the
helix and sugar-phosphate chains
run along the periphery, thereby
minimizing the repulsions
between charged phosphate
groups.
 DNA consist
of 2 polynucleotide strands
wound around each other to form a righthanded double helix
Nucleotides are linked each other by 3’-5’
phosphodiester bonds (join 5’-hydroxyl group of
deoxyribose sugar of one nucleotide to the 3’hydroxyl group of deoxyribose of another
nucleotide)
 Hydrogen bond form between the nitrogenous
base of 2 antiparallel polynucleotide strands
 TWO types of base pairs in DNA :
1) adenine (purine) pairs with thyamine
(pyrimidine)
2) Guanine (purine) pairs with cytosine
(pyrimidine)

 If
1 strand has the base sequence
AGGTCCG, so the other strand must have
sequence TCCAGGC
 These hydrogenbonding interactions, a
phenomenon known as complementary
base pairing, result in the specific
association of the two chains of the double
helix.
 Dimension
of DNA :
1) one turn of double helix span 3.4nm
consist 10.4 base pairs.
2) diameter of double helix is 2.4nm
3) distance between adjacent base pairs is
0.34nm.

Noncovalent bonding in DNA structure :
1) Hydrophobic interactions. Electron between
stacked purine & pyrimidine bases is nonpolar.
the clustering of bases component of nucleotide
within double helix stabilize structure, because it
minimize their interaction with water.
2) Hydrogen bond. Base pairs, on close
approach form hydrogen bond, three between
GC pairs and two between AT.
3) Base stacking. Stacking interactions are a form
of van der waals interaction. Interaction between
stacked G and C bases are greater than those
between stacked A and T bases, which largely
accounts for the greater thermal stability of
DNAs with a high G+C content
4) Electrostatic interaction. DNA external surface,
sugar-phosphate backbone possesses –ve
charged phosphate group.
The DNA helix
The geometry of DNA
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The biologically most common form of DNA is known as
B-DNA, - structural features first noted by Watson and
Crick together with Rosalind Franklin and other.

DNA is flexible molecule. It can assume several distinct
structural depending on the solvent composition and base
sequence. The major structural variants of DNA are ADNA and Z-DNA.
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Under dehydrating conditions, B-DNA undergoes a
reversible conformational change to A-DNA which forms a
wider and flatter right-handed helix than does B-DNA.
A-DNA
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When DNA become partially dehydrated, it
assumes the A form.
The base pairs no longer at right angle
They tilt 20° away from the horizontal
Distance between adjacent base pairs slightly
reduced (11bp helical turn instead or 10.4bp
found in B form)
Each turn of double helix occur in 2.5nm, instead
of 3.4nm
Diameter swell to 2.6nm
Z-DNA
 Named
for it zigzag conformation
 Diameter = 1.8nm, slimmer than B-DNA
 Twisted into left-handed spiral with 12bp
per turn
 Each turn occur in 4.5nm
RNA
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Ribonucleic acid is a class of polynucleotides
In contrast, RNA occurs primarily as single
strands, which usually form compact structures
rather than loose extended chains
 Nearly all involve in some aspect of protein
synthesis
 RNA molecules are synthesized in a process
called TRANSCRIPTION
 New RNA mol. are produced by mechanism
similar to DNA, through complementary base
pair formation
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New RNA mol. Are produced by mechanism
similar to DNA, through complementary base
pair formation (A=U G=C)
 Eg. DNA sequence 5’-CCGATTACG-3’ is
transcribe into RNA sequence
3’-GGCUAAUGC-5’
Differences between DNA & RNA
RNA
DNA
Sugar moiety is ribose
Sugar moiety is
deoxyribose
Nitrogenous base
Adenine, Urasil,
Guanine, Cytosine
Exist in single strand
Nitrogenous base
Adenine, Thyamine,
Guanine, Cytosine
Exist in double helix
Content of A and U, as
well as G and C are
equal
Content of A and T, as
well as G and C are
equal
Secondary structure of RNA

RNA exist as single strand.
 RNA can coil back on itself and form a unique
secondary structure
 The shape of these structures determined by
complementary base pairing by specific RNA
sequence, as well as base stacking
Types or RNA
 Types
of RNA = transfer RNA, ribosomal
RNA, messenger RNA
Transfer RNA
 tRNA transport amino acids to ribosomes
for assembly into protein
 Average length of tRNA = 75 nucleotides
Ribosomal RNA
 rRNA is the most abundant RNA in living cells
 rRNA is the component of ribosomes
 Ribosomes = cytoplasmic structures that
synthesized proteins
Messenger RNA
 mRNA is the carrier of genetic information from
DNA for the synthesis of protein
 mRNA is transcribed from a DNA template, and
carries coding information to the sites of protein
synthesis: the ribosomes
Denaturation and renaturation of
DNA

Unique properties of nucleic acids- under certain
conditions DNA duplexes reversibly melt (separate)
and reanneal (base pair to form duplex again)
 Binding forces that hold the DNA double helix can
be disrupted
 This process = denaturation, promoted by :
- heat (most common denaturing method)
- low salt concentrations
- extremes in pH
- Renaturation DNA can be prepared by maintain the temp.
below denaturing temp.
- requires some time because the strands
explore various configurations until they achieve the most
stable one
Nucleic acid methods

Most of technique used in nucleic acid research
are based on differences in molecular weight or
shape, base sequences, or complementary base
pairing
 Some of the most useful nucleic acid fractionation
procedure are:
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Chromatography
 Electrophoresis
 Ultracentrifugation
Nucleic acid extraction protocol

Ruptured bacterial cells or isolate eukaryotic nucleus
- to expose the nucleic acid
- done by grinding or sonicating the sample
 Removing membrane lipids by adding a detergent or
enzyme lysozyme
 Removing proteins by adding a protease
 Precipitating the DNA with an alcohol
- usually ice-cold ethanol or isopropanol. Since DNA is
insoluble in these alcohols, it will aggregate together,
giving a pellet upon centrifugation. This step also
removes alcohol-soluble salt
Chromatography
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Many of the chromatographic techniques that are
used to separate proteins also apply to nucleic
acids
Objectives : purify nucleic acid of interest or
isolation of individual nucleic acid sequences
A type of column chromatography that uses a
calcium phosphate gel called hydroxyapatite been
used in nucleic acid research
Hydroxyapatite bind tightly to double-stranded
nucleic acid than single-stranded nucleic acid
molecules
So dsDNA can be effectively separate from
ssDNA, RNA or other protein contaminants by this
method
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dsDNA can be rapidly isolated by passing a cell
lysate through a hydroxyapatite column
 wash the column with a low concentration of
phosphate buffer to release only the ssDNA, RNA
and protein
 Elute the column with a concentrated phosphate
buffer tp collect dsDNA
hydroxyapatite
RNA +
protein
dsDNA
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Affinity chromatography is used to isolate
specific nucleic acids.
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For example, most eukaryotic messenger RNAs
(mRNAs) have a poly (A) sequences or cellulose
to which poly (U) is covalently attached. The
poly(A) sequences specifically bind to the
complementary poly(U) in high salt and low
temperature and can later be released by
altering these condition.
Electrophoresis
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Gel electrophoresis separate nucleic acids on
the basis of molecular weight and 3-D structure
in an electric field
 The technique involves drawing DNA molecules,
which have an overall negative charge, through
a semisolid gel by an electric current toward the
positive electrode within an electrophoresis
chamber.
 The used gel is typically composed of a purified
sugar component of agar called agarose.
Electrophoresis

Nucleuic acids mixture
placed in well
 Nucleic acids are -ve
charge (phosphate group)
 Nucleic acid migrate to
anode
 Rate of migration are
proportional to molecular
size
 In
genetic engineering, scientists use the
technique to isolate fragments of DNA
molecules that can then be inserted into
vectors, multiplied by PCR, or preserved in
a gene library.
Southern blotting
 Enable
researcher to detect and analyze
particular DNA sequence
 The basis of detecting specific sequence :
nucleic acids hybridization
 Hybridization can be used to locate and/ or
identify specific genes or other sequence
 Eg. ssDNA from two diff sources (tumor
cell and normal cell) can be screened for
sequence differences
Southern blott technique
1) restriction fragment preparation
 DNA samples to be tested are treated with
restriction enzymes that cut at specific
nucleotides sequences to produce a restriction
fragments
2) electrophoresis
 The mixture of restriction fragments from each
sample are separated by electrophoresis
according to their size
 Each sample forms a characteristic patterns of
band
 The gel soaked with 0.5M NaOH to convert
dsDNA to ssDNA
Southern
blot
technique
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3) Blotting
The DNA fragments are transferred to
nitrocellulose filter paper by placing them on a wet
sponge in a tray with a high salt buffer
(nitrocellulose bind strongly to ssDNA)
As buffer is drawn through the gel and filter paper
by capillary action, the DNA is transferred and
become permanently bound to nitrocellulose filter
4) hybridization with radioactive probe
Nitrocellulose filter is exposed to radioactively
labeled probe, which bind to ssDNA with a
complementary sequence
4) hybridization with radioactive probe
 Nitrocellulose filter is exposed to a solution
containing radioactively labeled probe.
 The probe is ssDNA complementary to
DNA sequence of interest, and it attaches
by base pairing to restriction fragment of
complementary sequence
5) Autoradiography
 Rinse away unattached probe
 Autoradiograph showing hybrid DNA
fragment
Ultracentrifugation
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Equilibrium density gradient ultracentrifugation in
CsCl is one of the most commonly used DNA
separation procedures.
At high speeds, a linear gradient of CsCl is
established.

Mixture of DNA, RNA and protein migrating
through this gradient separate into discrete
bands at position where their densities are
equal to density of CsCl.
 DNA mol. with high Guanine and Cytosine
content are more dense than those with a
higher proportion of adenine and thyamine.
 The difference helps separate heterogenous
mixtures of DNA fragments
 Single stranded DNA denser than the double
stranded DNA, so the two can be separated by
equilibrium density gradient ultracentrifugation.