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NUCLEIC ACIDS
Sem I, 2011/2012
Khadijah Hanim bt Abdul Rahman
School of Bioprocess Eng, UniMAP
Week 12: 30/11 & 1/12/2011
[email protected]
Learning Outcomes
 DISCUSS
basic structures, properties and
functions of nucleic acids.
 DISCUSS DNA isolation methods
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

Two polynucleotide chains wind
around a common axis to form a
double helix.

The two strands of DNA are
antiparallel, but each forms a
right-handed helix.

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

Consists of 2 polynucleotide strands wound around each
other to form a right-handed double helix
 Each nucleotide monomer in DNA is composed of:
- Nitrogenous base (purine @ pyrimidine)
- Deoxyribose sugar (pentose, 5C)
- Phosphate
 Mononucleotides are linked to each other by 3’,5’phosphodiester bonds
 These bonds join the 5’-hydroxyl group of the
deoxyribose of 1 nucleotide to the 3’-OH group of the
sugar unit of another nucleotide thru a phosphate group.
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
Nucleic acid structure
 The
antiparallel
orientation of the 2
polynucleotide strands
allows H bond to form
between nitrogenous
bases that are
oriented toward the
helix interior.

There are 2 types of base pairs (bp) in DNA:
- Adenine (A- purine) pairs with thymine (Tpyrimidine)- 2 hydrogen bonds
- Guanine (G- purine) pairs with cytosine (Ccytosine)- 3 hydrogen bonds
 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.


The overall structure of DNA
resembles a twisted
staircase.
The Dimension of crystalline
DNA have been precisely
measured :
1) one turn of double helix
span 3.4nm and consist 10.4
base pairs.
2) diameter of double helix is
2.4nm- interior space of
double helix- suitable for
base-pairing purinepyrimidine.
3) distance between adjacent
base pairs is 0.34nm.

Noncovalent bonding that contribute to the
stability of DNA helical structure :
1) Hydrophobic interactions. The base ring π
cloud of electrons 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-between nucleotides.Base
pairs, on close approach form hydrogen bond,
three between GC pairs and two between ATkeeps the strands in correct complementary
orientation.
3) Base stacking. Stacking interactions are a form
of van der waals interaction. Base stacking
interactions are among the aromatic
nucleobases. 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. Repulsion between
nearby phosphate groups- potentially
destabilizing force- minimized by shielding
effects of divalent cations ie. Mg2+.
The DNA helix
The geometry of DNA

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 its base pair sequence and/or
isolation conditions. Each molecular form possesses the
same no. of base pairs.

DNA can assume different conformations becoz
deoxyribose is flexible and the C1-N- glycosidic linkages
rotates.
A-DNA








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 instead of 2.4 nm
The A form of DNA is observed when it is extracted with
solvents such as ethanol.
Significance of A-DNA under cellular conditionsstructure of RNA duplexes and RNA/DNA duplexes
formed during transcription.
Z-DNA

Named for it zigzag conformation
 Diameter = 1.8nm, slimmer than B-DNA= 2.4 nm.
 Twisted into left-handed spiral with 12bp per turn, B
DNA= 10.4 bp
 Each turn occur in 4.5nm compared with 3.4 nm for BDNA.
 DNA segments with alternating purine-pyrimidines bases
(CGCGCG) are most likely to adopt a Z configuration.
 Regions of DNA rich in GC repeats are often regulatory,
binding specific proteins that initiate/block transcription.
Genome structure


The genome of each living organism- full inherited
instructions required to sustain living processes
Genome size: the no of base-paired nucleotides, varies
over an enormous range from less than 1 million bp in
Mycoplasma to greater than 1010 bp in certain plants.
Prokaryotic Genomes

Genome size
- The genomes are relatively small
- Considerably fewer genes than eukaryotes. Eg: the E.
coli chromosome contains about 4.6 Mb that code for
4300 genes.
 Coding capacity
- Genes are compact and continuous- that is they contain
little, if any, concoding DNA either between/ within gene
sequences.
 Gene expression
- The regulation of many functionally related genes is
enhanced by organizing them into operons. An operon is
a set of linked genes that are regulated as a unit.

Prokaryotes possess additional small pieces of
DNA- plasmids.
 Plasmids- have genes that are not present on
the main chromosome.
 Genes that are not essential for growth and
survival but genes that provide growth/survival
advantage: antibiotic resistance genes, unique
metabolic capacities (N2 fixation, degradation of
aromatic compounds) and virulence (toxins)
Eukaryotic genomes


-

-
-
Organization of genetic information in eukaryotic
chromosomes- more complex.
Genome size:
Larger than prokaryotes but size does not necessarily a
measure of the complexity of the organism. Some
species accumulated vast amounts of non-coding DNA.
Coding capacity
Although there is enormous coding capacity- majority of
DNA sequences in eukaryotes do not have coding
functions- do not possess intact regulatory regions to
initiate transcription.
The function is unknown- some may have
regulatory/structural roles. Not more than 1.5% of human
genome codes for protein.
 Coding
-
-
-
continuity
Eukaryotic genes are discontinuous. Noncoding sequences (introns) are
interspersed between sequences called
exons.
Exons- code for a gene product.
Intron sequences are removed from premRNA transcript by splicing mechanism to
produce functional mRNA molecules.
RNA







Ribonucleic acid is a class of polynucleotides, involved in
protein synthesis.
RNA molecules are synthesized in a process referred as
transcription.
During transcription- RNA is synthesized thru
complementary base pair formation.
The sequence of bases in RNA is therefore specified by
the base sequence in one of 2 strands in DNA.
Only 1 DNA strand that acts as template for synthesis of
RNA molecule- referred as antisense (non-coding
strand).
The nontranscribed DNA strand is called sense strand
(coding).
The base sequence of the sense strand is the DNA
version of the mRNA used to synthesize the polypeptide
product of gene.
 For
example, the antisense DNA
sequence
5’- CCGATTACG-3’ is transcribed into the
RNA sequence 3’- GGCUAAUGC-5’.
RNA molecules differ from DNA:
 The
sugar moiety of RNA is ribose.
DNA=deoxyribose.
 The nitrogenous bases in RNA differ from
those observed in DNA. Instead of
thymine, RNA molecules use uracil
(Adenine base pairing with uracil).
 In contrast to double helix DNA, RNA
exists as a single strand.
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
 The
most prominent types of RNA:
 Transfer RNA (tRNA)
 Ribosomal RNA (rRNA)
 Messenger RNA (mRNA)
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
Exercise
 Consider
the following antisense DNA:
5’-CGCTATAGCGTTTCAT-3’
- Determine the sequence of its
complementary strand
- Determine the mRNA transcript
- Determine the antisense mRNA
sequences.
QUIZ
 When
DNA is heated, it denatures, that is
the strands separate. Determine which of
the following molecules will denature first
as the temperature is raised. Why?
a) 5’- GCATTTCGGCGCGTTA-3’
3’- CGTAAAGCCGCGCAAT-5’
b) 5’- ATTGCGCTTATATGCT-3’
3’- TAACGCGAATATACGA-5’
QUIZ
 Consider
the following sense DNA
sequence:
5’-GCATTCGAATTGCAGACTCCT-3’
a)
b)
Determine the sequence of its
complementary strand
Determine the mRNA and antisense RNA
sequences
Transfer RNA (tRNA)
Transfer RNA
 tRNA transport amino acids to ribosomes
for assembly into protein
 Comprising about 15% of cellular RNA,
 Average length of tRNA = 75 nucleotides
 tRNA molecules bound to a specific amino
acid- cells possess at least 1 type of tRNA
for each of the 20 amino acids commonly
found in protein.
 tRNA-
cloverleaf structure.
 The structure allows it to perform 2
important functions:
- The 3’-terminus- forms a covalent bond to
a specific amino acid
- Anticodon loop- contains 3-base-pair
sequence that is complementary to the
DNA triplet code for the specific amino
acid.
tRNA structure
Ribosomal RNA
 rRNA is the most abundant RNA in living cells
 rRNA is the component of ribosomes
 Ribosomes = cytoplasmic structures that
synthesized proteins
 Ribosomes of prokaryotes and eukaryotes are
similar in shape and function- differ in size and
chemical composition.
 Both types of ribosome consist of 2 subunits of
unequal size.
 Prokaryotic ribosome: 50 S and 30 S subunit.
 Eukaryotic ribosome: 60 S and 40 S subunit.
Ribosomal RNA
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
Prokaryotic mRNA;
polycistronic- contain coding information for several
polypeptide chains
Are translated into proteins by ribosomes
during/immediately after they are synthesized
 Eukaryotic
mRNA:
 Typically codes for a single polypeptidemonocistronic.
 Are modified extensively- capping at the
5’-residue, splicing (removing of introns),
attachment of poly A tails.
Nucleic acid extraction protocol

Ruptured bacterial cells or isolate eukaryotic nucleus
- to expose the nucleic acid
 Bacterial nucleic acid can be precipitated by treating cells
with alkali and lysozyme (an enzyme that degrades
bacterial cell walls by breaking glycosidic bonds)
 Partially degraded protein is extracted using certain
solvents (phenol & chloroform)
 Eukaryotic nuclei can be treated with detergents/
solvents to release their nucleic acid.
 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
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
- The temp at which one-half of a DNA sample is denatured
referred as Tm- varies among DNA molecules according to
base composition.
- Renaturation DNA can be prepared by maintain the temp.
~ 25oC 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:

Chromatography
 Electrophoresis
 Ultracentrifugation
Chromatography

Many of the chromatographic techniques that are
used to separate proteins also apply to nucleic
acids
 Several types of chromatography: ion-exchange,
gel filtration and affinity.
 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 doublestranded nucleic acid than single-stranded
nucleic acid molecules
 So dsDNA can be effectively separate
from ssDNA, RNA or other protein
contaminants by this method

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

Affinity chromatography is used to isolate
specific nucleic acids.

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

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

Nucleic 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
 The
unique properties of nucleic acid:
under certain conditions DNA duplexes
reversibly melt and reanneal.
 Enable researcher to detect and analyze
particular DNA sequence- to locate
specific nucleic acid sequences.
 The basis of detecting specific sequence :
nucleic acids hybridization
 Single-stranded DNA from different
sources hybridize if there is a significant
sequence homology.
 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 blot technique
Probe labelling
 Sequences with known identities- DNA or
RNA probe is radioactively/fluorescent
labeled.
2) restriction fragment preparation
 DNA samples to be tested are treated with
restriction enzymes that cut at specific
nucleotides sequences to produce a
restriction fragments
1)
Southern blot technique
3) 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





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
 Eg: mRNA that codes for B-globin binds
specifically to the B-globin gene, even
though B-globin mRNA lacks the intron
present in the gene- sufficient base
pairing.
5) Autoradiography
 Rinse away unattached probe
 Autoradiograph showing hybrid DNA
fragment
Ultracentrifugation


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.
DNA Sequencing
 To
determine the DNA nucleotide
sequences.
 The classical chain-termination method
requires a single-stranded DNA template,
a DNA primer, a DNA polymerase, normal
deoxynucleotidetriphosphates (dNTPs),
and modified nucleotides (dideoxyNTPs)
that terminate DNA strand elongation.

The DNA sample is divided into four separate
sequencing reactions, containing all four of the
standard deoxynucleotides (dATP, dGTP, dCTP
and dTTP) and the DNA polymerase.
 To each reaction is added only one of the four
dideoxynucleotides (ddATP, ddGTP, ddCTP, or
ddTTP) which are the chain-terminating
nucleotides, lacking a 3'-OH group required for
the formation of a phosphodiester bond between
two nucleotides, thus terminating DNA strand
extension and resulting in DNA fragments of
varying length.
 The
newly synthesized and labelled DNA
fragments are heat denatured, and
separated by size (with a resolution of just
one nucleotide) by gel electrophoresis on
a denaturing polyacrylamide gel.