Dr.

Ramith Ramu
Department of Biotechnology & Bioinformatics,
JSS AHER, Mysuru-570015
 What is Nucleic Acid ?
• Deoxyribonucleic acid (DNA)
• Ribonucleic acid (RNA)
• The chemical basis of hereditary
• They organized into genes, the fundamental units of genetic information.

• Nucleoproteins:
• Conjugated proteins, presence of non-protein prosthetic group, nucleic acid
& attached to one or more molecules of a simple protein, a basic protein
histone or protamine is called nucleoprotein.
Structure of DNA was discovered by James Watson & Francis Crick
DNA double helix with two strands as Proposed by Watson & Crick
 Nucleic Acid

• First discovered in the nuclei of cells.

• Huge organic molecules that contains C, H, O, N, & P.

• Nucleic acid is a polymer of NUCLEOTIDE held by 3’ and 5’ phosphate

bridges.

• These are of two types:

• Deoxyribonucleic acid (DNA)

• Ribonucleic acid (RNA)

 Functions of nucleic acids

• DNA is the chemical basis of heredity

• Reserve bank of genetic information

• Responsible for maintaining the identity of different species of organisms over

millions of years

• Cellular function is under the control of DNA

• The basic information pathway

• DNA directs the synthesis of RNA, which in turn directs protein synthesis
 Central dogma of life

DNA RNA Protein

• DNA to DNA = Replication

• DNA to RNA = Transcription

• RNA to Protein = Translation

 Composition of nucleic acids

Nucleic acids

Polymer of

Nucleotides

Nucleosides Phosphate

Bases Sugar
Purines Ribose
Pyrimidines deoxyribose
 Composition of Nucleoside

• Nucleosides are composed of nitrogenous base and sugar

• The pentose sugar is either ribose in ribonucleosides or 2-deoxyribose in
deoxyribonucleosides
• Glycosidic bond:
• The linkage of base & sugar (N-glycosidic bond) for nucleosides involves
distinct nitrogen atoms in purine & pyrimidine ring
• In purine nucleosides, nitrogen 9 of purine ring is linked to carbon 1 of
pentose sugar.
• In pyrimidine nucleosides, nitrogen 1 of pyrimidine ring is linked to carbon
1 of pentose sugar.
• Nucleosides with purine bases have the suffix - sine, while pyrimidine
nucleosides end with - dine.

10
 Nucleoside

5’
OH-CH2

4’ 1’
SUGAR
3’ 2’

11
 Composition of Nucleotides

• Phosphate esters of nucleosides.

• Nucleotides are composed of nitrogenous base, a pentose sugar and
phosphate
• Nucleotide = nucleoside + phosphate
• The esterification occurs at 5th or 3rd hydroxyl group of the pentose sugar
• Most of the nucleoside phosphates involved in biological function are 5’
phosphates.
• 5’ AMP is abbreviated as AMP, but 3’ variety is written as 3’-AMP.
 Purines
• Purine bases are nine membered ring structures consisting of pyrimidine ring
fused to imidazole ring.
• The atoms of purine ring are numbered in the anticlockwise manner.
• Major bases in nucleic acids: Adenine & Guanine
• Adenine (6-amino purine):
• It contains amino group at 6th position
• Guanine (2-amino 6-oxypurine):
• It contains amino group at 2nd position & oxygen at 6th position.
 Purine Nucleotides

2-amino-6-oxypurine 6-aminopurine
 Minor purines present nucleic acids
• Several minor & unusual bases are often found in DNA & RNA

• These include 5-methylcytosine, N4-acetylcytosine, N6 methyl adenine, N6

dimethyl adenine & N7 methylguanine

• Importance:

• The unusual bases in nucleic acids help in the recognition of specific

enzymes.

15
 Purine bases of plants

• Plants contain certain methylated purines.

• Caffeine (1,3,7-trimethylxanthine):

• It is found in coffee.

• It acts as a stimulant.

• Theophylline (1,3-dimethylxanthine):

• Present in tea leaves.

• It acts as a bronchial smooth muscle relaxant.

16
 Purine analogs
• These have structural similarities to purines.
• They inhibit the enzymes involved in the metabolism of purine
nucleotides.
• Allopurinol:
• Inhibits xanthine oxidase & used in the treatment of hyperuricemia
(gout).
• 6-mercaptopurine:
• It inhibits purine nucleotide synthesis & used as an anticancer drug.
• Metabolic intermediates:
• These are formed during metabolism of nucleotides
• E.g. hypoxanthine, xanthine & uric acid.
 Pyrimidines

• Pyrimidines contain six membered nitrogenous ring.

• The atoms in pyrimidine ring are numbered in clockwise direction.

• Major pyrimidines found in nucleic acids:

• Cytosine

• Uracil

• Thymine
 Pyrimidines

• Cytosine: Cytosine is found in both RNA & DNA.

• Cytosine (2-oxy,4-amino pyrimidine) has oxygen at position 2 & amino group
at position 4.
• Uracil: Uracil is found only in RNA.
• Uracil (2,6-dioxy-pyrimidine) has oxygens at position 2 & 4
• Thymine:
• Thymine found in DNA and thymine (methyluracil) has oxygen at position 2
& 4, methyl group at position 5
Pyrimidines: Cytosine, Uracil & thymine (CUT)

2-oxy-4-aminopyrimidine
Cytosine (C)

2,4-dioxy
pyrimidine 2,4-dioxy-5-
methylpyrimidine
Uracil (U)
Thymine (T)
Minor (unusual) pyrimidines found in nucleic acids
• Methylcytosine present in DNA & dihydrouracil present in tRNA.
• Pyrimidine analogs:
• These have structural similarities to pyrimidines.
• They act either as inhibitors of enzymes in the metabolism of pyrimidines
or interact with nucleic acids.
• 5-fluorouracil:
• It inhibits the enzyme thymidylate synthase.
• It is used in the treatment of cancer.

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 Minor/Unusual bases

• Specific DNA and RNA contains small quantities of Minor/modified

bases also.

• These modifications includes-

• Methylation

• Hydroxymethylation

• Glycosylation

• Alteration of atoms.
 Minor/Unusual base

• Modification of Adenine:

N-methyladenine,

N6N6- dimethyladenine

• Modification of Guanine:

7-methylguanine

• Modification of Cytosine:

5-methylcytosine

5-hydroxymethylcytosine
• Modification of Uracil:

Dihydroxyuracil

• Special Bases:

Hypoxanthine (6-oxopurine)

Xanthine (2,6-dioxopurine)

Uric acid (2,6,8-trioxopurine)

 Special Bases

Uric Acid (2,6,8-trioxypurine)

 Sugars Present in DNA & RNA

• DNA & RNA are distinguished on the basis of the pentose sugar present.
• DNA contains β-D-2-deoxyribose
• RNA contains β-D-ribose.
 Principal Nucleotides
• Ribonucleotides are named as
• Adenine = Adenosine monophosphate (AMP)
• Guanine = Guanosine monophosphate (GMP)
• Cytosine = Cytidine monophosphate (CMP)
• Thymine = Thymidine monophosphate (TMP)
• Uracil = Uridine monophosphate (UMP)
• Deoxyribonucleotides are named as
• Adenine = Deoxyadenosine monophosphate (dAMP)
• Guanine = Deoxyguanosine monophosphate (dGMP)
• Cytosine = Deoxycytidine monophosphate (dCMP)
• Thymine = Deoxythymidine monophosphate (dTMP)
• Uracil = Deoxyuridine monophosphate (dUMP)
 Functions of Nucleotides
• Activated precursors of DNA & RNA.

• ATP – Universal currency of energy.

• Required for activation of intermediates in many biosynthetic pathway.

• GTP-involved in protein biosynthesis as source of energy.

• Components of coenzymes: NAD, FAD & CoA.

• Metabolic regulators, e.g. cAMP, cGMP.

 Physiological Important Nucleotides

• Nucleotides of Adenine

• Adenosine triphosphate (ATP)

• Adenosine diphosphate (ADP)

• Adenosine monophosphate (AMP)

• Cyclic adenosine monophosphate (cAMP)

• Phospho adenosine phospho sulfate (PAPS)

• S-adenosyl methionine (SAM)

 ATP and ADP

• In oxidative phosphorylation,

• ADP is substrate,

• ATP is product

• ATP is universal currency of energy in biological systems.

 Nucleotides of Guanine

• Guanosine triphosphate (GTP)

• Guanosine diphosphate (GDP)

• Cyclic guanosine monophosphate (cGMP)

• GTP and GDP:

• In Substrate level phosphorylation, GDP & GTP is used

• GTP is required for activation of adenylate cyclase.

• Energy source for protein synthesis (GTP)

• Acts as allosteric regulator

 Nucleotide of Uracil

• Uridine diphosphate - sugar derivatives (UDP-sugar) acts as sugar donar

• UDP-glucose in synthesis of glycogen

• Other UDP-sugar in glycoproteins

• UDP-glucuronate in conjugation reaction

 Nucleotide of Cytosine

• CTP is required for synthesis of phosphoglycerides.

• CMP-N-acetylneuraminic acid required for synthesis of glycoproteins.

• CDP-choline involved in the synthesis of sphinogomyelin

 Modification of bases in DNA

• About 5% of cytosine in eukaryotic DNA is methylated.

• Methylation is catalysed by methylase (DNA methyl transferase)
• Methylation suppresses migration of segment of DNA (which are called
as transposons) & increase the tendency of DNA to assume Z form.
• Hypomethylation of DNA is associated with development of cancer.

34
 Structure of DNA

• DNA is a polymer of deoxyribonucleotides

• Composed of monomeric units namely

• Deoxyadenylate (dAMP)

• Deoxyguanylate (dGMP)

• Deoxycytidylate (dCMP)

• Deoxythymidylate (dTMP)

• The monomeric units held together by 3’5’-phosphodiester bonds as back

bone.
DNA structure
 Base pairing rules: Chargaff’s rule
• DNA has equal numbers of adenine & thymine residues (A=T) and equal
number of guanine & cytosine residues(G=C).

• This is called as Chargaff’s rule of molar equivalence of between purines &

pyrimidine's in DNA structure.

• RNAs which are usually single stranded, do not obey Chargaff’s rule.
 Base pairing rules: Chargaff’s rule
The sum of purines is equal to sum of pyrimidine's---

A+G=T+C

Ratio of Adenine to thymine is 1--- A/T=1

Ratio of guanine to cytosine is 1--- G/C=1

Bases with 6-amino groups are equal to bases with 6-keto groups--- A+C=G+T
 DNA double helix
• Double helical structure was proposed by Watson & Crick in 1953.

• The DNA is a right handed double helix.

• It consists of two polydeoxyribonucleotide chains twisted around each other

on a common axis of symmetry.

• The chains are paired in an antiparallel manner, that is, the 5'-end of one
strand is paired with the 3'-end of the other strand
• The two strands are antiparallel, i.e., one strand runs in the 5 ' to 3 ' direction
while the other runs in 3' to 5 ' direction.

• The width (or diameter) of a double helix is 20A0 (2nm)

• Each turn of helix is 34 A0 (3.4nm) with 10 pairs of nucleotides, each pair

placed at a distance of about 3.4 A0

• The DNA helix, the hydrophilic deoxyribose-phosphate backbone of each

chain is on the outside of the molecule, whereas the hydrophobic bases are
stacked inside.
• The polynucleotide chains are not identical but complementary to each other
due to base pairing.

• The two strands are held together by hydrogen bonds.

A = T, G = C

• The hydrogen bonds are formed between a purine & pyrimidine.

• The spatial relationship between the two strands in the helix creates a major
(wide) groove and a minor (narrow) groove.
• These grooves provide access for the binding of regulatory proteins to their
specific recognition sequences along the DNA chain.

• DNA helix proves Chargaff’s rule.

• The genetic information resides on one of the two strands known as template
strand or sense strand.

• The opposite strand is antisense strand.

Types of DNA
 Conformations of DNA double helix

• The double helical structure of DNA exists in 6 forms A,B,C,D,E and Z form.
• Among these, B, A & Z forms are important.
• B-form is most predominant form under physiological conditions.
• A-from is also right-handed helix.
• Contains 11 base pairs.
• There is a tilting of the base pairs by 200 away from the central axis.
• Z-form is a left –handed helix and contains 12 base pairs per turn.
• The polynucleotide strands of DNA move in a somewhat zig-zag fashion,
hence called as Z-DNA.

• Other types of DNA:

• DNA also exists in certain unusual structures.

• These structure are important for molecular recognition of DNA by proteins

& enzymes.
 Bent DNA

• Adenine base containing DNA tracts are rigid & straight.

• Bent conformation of DNA occurs when A-tracts are replaced by other bases or
a collapse of the helix into minor groove of A-tract.

• Bending in DNA structure is due to photochemical damage or mispairing bases.

• Certain antitumor drugs (e.g.,cisplastin) produce bent structure in DNA.

• Such changed structure can take up proteins that damage the DNA.
Bent DNA
 Triple-stranded DNA
• Triple stranded DNA formation may occur due to additional hydrogen bonds
between the bases

• Thymine can selectively form two Hoogsteen hydrogen bonds to the adenine
of A-T pair to form T-A-T.

• Cytosine can also form two hydrogen bonds with guanine of G-C pairs that
results in C-G-C.

• Triple helical structure is less stable than double helix.

• Due to three negatively charged backbone strands in triple helix results in an

increased electrostatic repulsion.
Triple-stranded DNA
 Four-stranded DNA

• Polynucleotides with very high content of guanine can form a

tetrameric structure called G-quartets.

• These structures are planar & are connected by Hoogsteen

hydrogen bonds.

• Antiparallel four stranded DNA structures referred to as G-

tetraplexes.

• The ends of eukaryotic chromosomes namely telomeres are rich

in guanine,& forms G-tetraplexes.
Four-stranded DNA
 Size of the DNA molecule

• Huge in size
• B-DNA with a thickness of 0.34nm
• Molecular weight of 660 daltons
• Length is expressed in base pairs(bp)
• A kilobase pair 103bp, a megabase pair(Mb) is 106bp & gigabase
pair (Gp) is109
• 1kb=1000bp
• 1Mb=1000kb=1,000,000bp
• 1Gb=1000Mb=1,000,000,000bp
• Length varies from species to species
• Length of Human DNA is 2 meters &10µ diameter
 Erwin Chargaff
• Chargaff, an Austrian refugee biochemist, was born in 1905 in
Czernowitz, Austria.

• Using the simple but sensitive technique of paper

chromatography, he along with his collaborators at Columbia
University analyzed the base composition of DNA from various
sources.

• He described Watson and Crick as two pitchmen in the search of

a helix and explains his objection to molecular biology as ‘by its
claim to be able to explain everything, is acutally inpedes the
flow of scientific explanation’. His autobiography is Heraclitean
Fire: Sketches From a Life Before Nature, Rockefeller University
Press, New York, 1978
a characteristic regularity of pattern as first noted by Erwin Chargaff and his
coworkers in 1950.

The important conclusions drawn by him are :

1. The sum of purines (Pu) is equal to the sum of pyrimidines (Py), i.e., Pu/Py =
1. In other words, A + G = T + C (+ MC) where MC stands for
methylcytosine, where it occurs.

2. The ratio of adenine to thymine is also one, i.e., A/T = 1.

3. The ratio of guanine to cytosine (plus methylcytosine where it occurs) is also

one, i.e., G/C(+ MC) = 1.
4. Bases with 6-amino groups are equal to bases with 6-keto (hydroxyl) groups, i.e., A + C
(+ MC) = G + T.

5. The ratio of A + T/G + C(+ MC), known as dissymmetry ratio, varies greatly from one

species of DNA to the other and is characteristic of that species. When the dissymmetry

ratio exceeds one, such a DNA is called AT type ; when the value is less than one, such

a DNA is designated as GC type. In bacteria, both AT and GC types of DNA are found.

However, in the higher organisms, the range of dissymmetry ratio is more limited : in

most animals the value ranges from 1.3 to 2.2 and in higher plants from 1.1 to 1.7. The

value of dissymmetry ratio in human beings is 1.4 and that in Mycobacterium tuberculosis

is 0.60.
• Erwin Chargaff, in late 1940s, independently had provided the crucial
observation that, in DNA obtained from a wide variety of organisms, the
molar ratio of adenine to thymine and that of guanine to cytosine were very
close to unity. These results indicated that a specific relationship must exist
between the two bases within each of the ratios.

• These and some other generalizations were collectively termed as

“Chargaff’s equivalence rules”.

• Later, the results of titration studies (= analytical studies) also suggested that
the long polynucleotide chains were held together by hydrogen bonding
between the base residues.
• Chargaff’s data suggest that A is always paired with T and G is
always paired with C.

• A-paired bases occur in any order :

• A...T

• T...A

• C...G

• G…C
 Base pairing rules: Chargaff’s rule

The sum of purines is equal to sum of pyrimidine's---

A+G=T+C

Ratio of Adenine to thymine is 1--- A/T=1

Ratio of guanine to cytosine is 1--- G/C=1

Bases with 6-amino groups are equal to bases with 6-keto groups---
A+C=G+T
• The structure of DNA derives its strength from Chargaff’s rule. Single-stranded DNA,
and RNAs which are usually single-stranded, do not obey Chargaff’s rule. However,
double-stranded RNA which is the genetic material in certain viruses satisfies
Chargaff’s rule.

Complementary base pairing in DNA

(A) Thymine pairs with adenine by 2 H-bonds (B) Cytosine pairs with guanine by 3 H-
bonds.
 Denaturation and Renaturation of DNA

• DNA denaturation is the separation of double strand into two

single strands, which occurs when the hydrogen bonds between
the strands are broken.

• Denaturation of DNA is a loss of biologic activity and is

accompanied by cleavage of hydrogen bonds holding the
complementary sequences of nucleotides together.
 Denaturation of DNA

• Denaturation of DNA double helix takes place by the

following denaturating agents:

(i) Denaturation by Temperature

(ii) Denaturation by Chemical Agents

(iii) Effect of pH on Denaturation

 (i) Denaturation by Temperature

• If a DNA solution is heated to approximately 90°C or above

there will be enough kinetic energy to denature the DNA
completely causing it to separate into single strands.

• This denaturation is very abrupt and is accelerated by chemical

reagents like urea and formamide.
• The chemicals enhance the aqueous solubility of the purine and
pyrimidine groups.

• This separation of double helix is called melting as it occurs abruptly

at a certain characteristic temperature called denaturation
temperature or melting temperature (Tm).

• It is defined as temperature at which 50% of the DNA is melted. The

abruptness of the transition indicates that the DNA double helix is
highly cooperative structure, held together by many reinforcing bonds.
• The melting of DNA can be followed spectrophotometrically by
monitoring the absorbance of DNA at 260 nm. Tm is analogous
to the melting point of crystal. The Tm value depends on the
nature of the DNA.
• If several samples of DNA are melted, it is found that the Tm is
highest for those DNAs that contain the highest proportion of
G—C.
• Actually the value is used to estimate the percentage of G—C in
a DNA sample. In fact, the Tm of DNA from many species varies
linearly with G—C content.
• This relationship between Tm and G—C content arises due to
guanine and cytosine form three hydrogen bonds when base
paired, whereas adenine and thymine form only two.
Melting Temperature (Tm)

• Primer melting temperature (Tm) - All primers in the reaction must have
similar melting temperature (Tm) so they anneal to and dissociate from
complementary DNA sequences at approximately the same temperatures,
allowing each amplification to precede at the selected temperature.
• Primer melting temperature (Tm) by definition is the temperature at which
one half of the DNA duplex will dissociate to become single stranded and
indicates the duplex stability.
• Primers with melting temperatures in the range of 52-58°C generally produce
the best results. Primers with melting temperatures above 65°C have a
tendency for secondary annealing.
• The guanine-cytosine (GC) content of the sequence gives a fair indication of
the primer Tm. The formula for primer Tm calculation: Tm = 4(G + C) + 2(A
+ T)=°C
• GC content - Primers should optimally contain 40-60% GC
content.
• The GC content is the number of G's and C's in the primer as a
percentage of the total bases.
• The presence of G or C bases within the last five bases from the
3' end of primers, known as the GC clamp, helps promote
specific binding at the 3' end due to the stronger bonding of G
and C bases. More than 3 G's or C's should be avoided in the last
5 bases at the 3' end of the primer.
• Try to have uniform distribution of G and C nucleotides, as
clusters of G’s or C’s can cause non-specific priming.
 Denaturation involves the following changes of the
properties of DNA:
• (a) Increase in Absorption of UV-Light:
• If denaturation is followed spectrophotometrically by monitoring
the absorbance of light at 260 nm, it is observed that the
absorbance at 260 nm increases as the DNA become denatured, a
phenomenon known as the hyperchromatic effect or
hyperchromacity or hyperchromism. This is due to un-stacking
of base pairs.
• A plot of the absorbance at 260 nm against the temperature of a
DNA solution indicates that little denaturation occurs below
approximately 70°C, but further increases in temperature result
in a marked increase in the extent of denaturation.
• (b) Decrease in Specific Optical Rotation:
• Double-stranded DNA shows a strong positive rotation which
highly decreases with denaturation. This change is analogous to
the change in rotation observed when the proteins are denatured.
• (c) Decrease in Viscosity:
• The solutions of native DNA exhibit high viscosity because of
the relatively rigid double helical, long and rod like character of
DNA molecule. Denaturation causes a marked decrease in
viscosity.
DNA melting curves
• If melted DNA is cooled it is possible to reassociate the
separated strands, a process known as renaturation. However, a
stable double-stranded molecule may be formed only if the
complementary strands collide in such a way that their bases are
paired precisely. But renaturation may not be precise if the DNA
is very long and complex.
• Thus the rate of renaturation (renaturation kinetics) can give
information about the complexity of a DNA molecule. Complete
denaturation is not a readily reversible process. If a heat-
denatured DNA solution is cooled slowly (anneling) and hold the
solution at about 25°C below Tm and above a concentration of
0.4M Na+ for several hours, some amount of
• DNA (50-60%) is renatured. Rapid cooling does not reverse
denaturation, but if the cooled solution is again heated and then
cooled slowly, renaturation takes place.
 (ii) Denaturation by Chemical Agents:

• Denaturation of DNA double helix can also be brought about by

certain chemical agents such as urea and formamide.

• These chemical reagents enhance the aqueous solubility of the

purine and pyrimidine groups.

• The Tm value is lowered by the addition of urea. In 8M urea,

Tm is decreased by nearly 20°C. DNA can be completely
denatured by 95% formamide at room temperature only.
 (iii) Effect of pH on Denaturation:
• Denaturation also occurs at acidic and alkaline solutions in which ionic
changes of the purine and pyrimidine bases can occur.

• In acidic solutions at pH values 2-3 the amino groups bind with protons and
the DNA double helix is disrupted.

• Similarly, in alkaline solutions at pH 12, the enolic hydroxyl groups ionize,

thus preventing the keto-amino hydrogen bonding.
 Hyperchromicity

• Hyperchromicity is the increase of absorbance (optical density) of a

material.
• The most famous example is the hyperchromicity of DNA that occurs when
the DNA duplex is denatured.
• UV absorption is increased when the two single DNA strands are being
separated, either by heat or by addition of denaturant or by increasing the
pH level.
• The opposite, a decrease of absorbance is called hypochromicity.
 Hyperchromicity in DNA denaturation

• Heat denaturation of DNA, also called melting, causes the double helix
structure to unwind to form single stranded DNA.
• When DNA in solution is heated above its melting temperature (usually
more than 80 °C), the double-stranded DNA unwinds to form single-
stranded DNA.
• The bases become unstacked and can thus absorb more light. In their
native state, the bases of DNA absorb light in the 260-nm wavelength
region.
• When the bases become unstacked, the wavelength of maximum
absorbance does not change, but the amount absorbed increases by 37%.
• A double stranded DNA strand dissociating to two single strands produces
a sharp cooperative transition.
• Hyperchromicity can be used to track the condition of DNA as temperature
changes.
• The transition/melting temperature (Tm) is the temperature where the
absorbance of UV light is 50% between the maximum and minimum, i.e.
where 50% of the DNA is denatured.
• A ten fold increase of monovalent cation concentration increases the
temperature by 16.6 oC.
• The hyperchromic effect is the striking increase in absorbance of DNA
upon denaturation.
• The two strands of DNA are bound together mainly by the stacking
interactions, hydrogen bonds and hydrophobic effect between the
complementary bases.
• The hydrogen bond limits the resonance of the aromatic ring so the
absorbance of the sample is limited as well.
• When the DNA double helix is treated with denatured agents, the
interaction force holding the double helical structure is disrupted.
• The double helix then separates into two single strands which are in the
random coiled conformation.
• At this time, the base-base interaction will be reduced, increasing the UV
absorbance of DNA solution because many bases are in free form and do
not form hydrogen bonds with complementary bases.
• As a result, the absorbance for single-stranded DNA will be 37% higher
than that for double stranded DNA at the same concentration.
Nucleic acid melting curve showing hyperchromicity as a function of temperature
 Hypochromic Effect

• Hypochromicity describes a material’s decreasing ability to absorb light.

• Hyperchromicity is the material’s increasing ability to absorb light.
• The Hypochromic Effect describes the decrease in the absorbance of
ultraviolet light in a double stranded DNA compared to its single stranded
counterpart.
• Compared to a single stranded DNA, a double stranded DNA consists of
stacked bases that contribute to the stability and the hypochromicity of the
DNA.
• When a double stranded DNA is denatured, the stacked bases break apart
and thus becomes less stable.
• It also absorbs more ultraviolet light since the bases no longer forms
hydrogens bonds and therefore are free to absorb light.
• Ways to denature DNA include high temperature, addition of denaturant,
and increasing the pH level.
Difference in absorbance of ultraviolet light between single stranded and double
stranded DNA
 Importance of Hypochromic Effect

• The measurement of absorption of light is important in monitoring the

melting and annealing of DNA.
• At the melting temperature (Tm), the DNA is half denatured and half
double stranded. By lowering the temperature below the Tm, the denatured
DNA strands would anneal back into a double stranded DNA. When
temperature is above the Tm, the DNA is denatured.
• Because melting occurs almost instantly at a certain temperature,
monitoring the absorbance of the DNA at various temperature would
indicate the melting temperature.
• By being able to find the temperature at which DNA
melted and annealed, scientists are able to separate
DNA strands and anneal them with other DNA strands.
• This is important in creating hybrid DNAs, which
consists of two DNA strands from different sources.
Since DNA strands can only anneal if they are similar,
the creation of hybrid DNAs can indicate similarities
between genomes of different organisms.
Nucleic acid melting curve showing hyperchromicity as a function of temperature
 Increase in absorption of ultraviolet light (= Hyperchromic effect)

• As a result of resonance, all the bases in nucleic acids absorb ultraviolet light.
And all nucleic acids are characterized by a maximum absorption of UV light
at wavelengths near 260 nm.

• When the DNA is denatured, there occurs a marked increase in optical

absorbancy of UV light by pyrimidine and purine bases, an effect called
hyperchromicity or hyperchromism which is due to unstacking of the base
pairs.
• This change reflects a decrease in hydrogen-
bonding. Hyperchromicity is observed not
only with DNA but with other nucleic acids
and with many synthetic polynucleotides
which also possess a hydrogen-bonded
structure.
The absorbance spectra of a DNA solution at 260 nm and pH 7.0
 Decrease in absorption of ultraviolet light (=
Hypochromic effect)
• It describes the decrease in the absorbance of ultraviolet light in a double
stranded DNA compared to its single stranded counterpart.

• Compared to a single stranded DNA, a double stranded DNA consists of

stacked bases that contribute to the stability and the hypochromicity of the
DNA.

• When a double stranded DNA is denatured, the stacked bases break apart and
thus becomes less stable. It also absorbs more ultraviolet light since the bases
no longer forms hydrogens bonds and therefore are free to absorb light.

• Ways to denature DNA include high temperature, addition of denaturant, and

increasing the pH level.
 Renaturation of DNA
• It is the formation of base pair repairs and complementary strands of DNA
come back together.

• Renaturation occurs if double stranded DNA is heated above Tm, then the
temperature is slowly decreased under appropriate conditions.
• When preparations of double-stranded DNA are denatured and allowed to
renature, the rate of renaturation can give valuable information about the
complexity of the DNA if there are repetitive sequences in the DNA, it shows
less complexity in comparison to its total length, but the complexity is equal
to its total length if all sequences are unique.
• The 1kb DNA fragments are denatured by heating above its Tm and then
renatured at a temperature 10°C below the Tm and monitored either by
decrease in absorbance at 260 nm (hypochromic effect), or by passing samples
at intervals through a column of hydroxylapatite, which retains only double
stranded DNAs, and estimating how much of the sample is retained.
• The degree of renaturation after a given time depends on C0, the
concentration of double stranded DNA prior to denaturation, and t, the
duration of the renaturation in seconds.

• The concentration is measured in nucleotides per unit volume. In order

to compare the rates of renaturation of different samples of DNA it is
usual to measure C0 and the time taken for renaturation to proceed half
way to completion, t1/2, and to multiply these values together to give a
C0t1/2 value. The larger the C0t1/2, the greater the complexity of the
DNA; hence λ DNA has a far lower C0t1/2 than does human DNA.
• if the extent of renaturation is plotted against log C0t (known as
Cot curve), it is observed that part of the DNA is renatured quite
rapidly while the rest is very slow to renature.

• This indicates that some sequences have a higher concentration

than others i.e., part of the genome consists of repetitive
sequences.
• These repetitive sequences can be separated from the single-copy
unique DNA by passing the renaturating sample through a
hydroxylapatite column early in the renaturation process, at a
time which gives a low value of C0t.

• At this stage only the rapidly renaturating sequences will be

double stranded, and will, therefore, bind to the column.
• The renaturation rate of DNA is an excellent indicator of the sequence
complexity and the size of the DNA.

• For example, bacteriophage T4 DNA contains about 2x105 nucleotide

pairs, whereas Escherichia coli DNA possesses 4.64x106. E. coli DNA
is considerably more complex in that it encodes more information. Or
we may say that for any given amount of DNA (in grams), the
sequences represented in an E. coli sample are more heterogeneous,
that is, more dissimilar from one another, than those in an equal weight
of phage T4 DNA.

• Therefore, it will take the E. coli DNA strands longer to find their
complementary partners and reanneal. This situation can be analysed
quantitatively through C0t Curves.
• Since a sequence of single-stranded DNA needs to find its
complementary strand to reform a double helix, common
sequences renature more rapidly than rare sequences.

• Indeed, the rate at which a sequence will reassociate is

proportional to the number of copies of that sequence in the DNA
sample. A sample with a highly repetitive sequence will renature
rapidly, while complex sequences will renature slowly.
• However, instead of simply measuring the percentage of double-stranded
DNA versus time, the amount of renaturation is measured relative to a C0t
value.

• The C0t value is the product of C0 (the initial concentration of DNA), t (time
in seconds), and a constant that depends on the concentration of cations in the
buffer.

• Repetitive DNA will renature at low C0t values, while complex and unique
DNA sequences will renature at high C0t values. The fast renaturation of the
repetitive DNA is because of the availability of numerous complementary
sequences.
C0t Curve of DNA
 C0t analysis

• C0t analysis, a technique based on the principles of DNA

reassociation kinetics, is a biochemical technique that measures
how much repetitive DNA is in a DNA sample such as a genome.

• It is used to study genome structure and organization and has

also been used to simplify the sequencing of genomes that
contain large amounts of repetitive sequence.
• Since a sequence of single-stranded DNA needs to find its complementary
strand to reform a double helix, common sequences renature more rapidly
than rare sequences.

• Indeed, the rate at which a sequence will reassociate is proportional to the

number of copies of that sequence in the DNA sample.

• A sample with a highly-repetitive sequence will renature rapidly, while

complex sequences will renature slowly.
• However, instead of simply measuring the percentage of double-stranded
DNA versus time, the amount of renaturation is measured relative to a C0t
value.
• The C0t value is the product of C0 (the initial concentration of DNA), t (time
in seconds), and a constant that depends on the concentration of cations in the
buffer.
• Repetitive DNA will renature at low C0t values, while complex and unique
DNA sequences will renature at high C0t values.
• The fast renaturation of the repetitive DNA is because of the availability of
numerous complementary sequences.
DNA ISOLATION
 INTRODUCTION
• DNA isolation is a process of purification of DNA from sample using a
combination of physical and chemical methods.
• The first isolation of DNA was done in 1869 by Friedrich Miescher.
• Methods used to isolate DNA are dependent on the source, age, and size
of the sample.
• In general, they aim to separate DNA present in the nucleus of the cell
from other cellular components.
• Isolation of DNA is needed for genetic analysis, which is used for
scientific, medical, or forensic purposes.
• Scientists use DNA in a number of applications, such as introduction of
DNA into cells and animals or plants, or for diagnostic purposes, in
medicine the latter application is the most common.
• On the other hand, forensic science needs to recover DNA for
identification of individuals (for example rapists, petty thieves, accident, or
war victims), paternity determination, and plant or animal identification.
• Presence of proteins, lipids, polysaccharides and some other organic or
inorganic compounds in the DNA preparation can interfere with DNA
analysis methods.
• They can also reduce the quality of DNA leading to its shorter storage life.
 SOURCES
• Sources for DNA isolation are very diverse.
• Basically it can be isolated from any living or dead organism.
• Common sources for DNA isolation include whole blood , hair, sperm ,
bones, nails, tissues, blood stains, saliva , buccal (cheek) swabs, epithelial
cells, urine, paper cards used for sample collection, bacteria, animal
tissues, or plants.
• Stored samples can come from archived tissue samples, frozen blood or
tissue, exhumed bones or tissues, and ancient human, animal, or plant
samples.
 PROCEDURE

• Isolation of DNA basically consists of four major steps.

i. Preparation of a cell extract.

ii. Purification of DNA from cell extract.
iii. Concentration of DNA samples.
iv. Measurement of purity of DNA concentration.
 1. Preparation of acell extract:

• To extract DNA from a tissue/cells of interest, the cells have to be separated

and the cell membranes have to be disrupted.
• The "Extraction buffer" helps in carrying out these processes.
• Chemicals such as EDTA (Ethylene Diamine Tetra Acetate) which removes
Mg2+ ions that are essential for preserving the overall structure of the cell
membrane, and SDS (Sodium Dodecyl Sulfate) which aids in disrupting the
cell membranes by removing the lipids of the cell membranes are included in
the extraction buffer.
• Having lysed the cells, the final step in the preparation of a cell extract is
removal of insoluble cell debris.
• Cell debris and partially digested organelles etc. can be pelleted by
centrifugation leaving the cell extract as a reasonably clear supernatant.
 2. Purification of DNA from cell extract.

• In addition to DNA the cell extract will contain significant quantities of

detergents, proteins, salts and reagents used during cell lysis step and
RNA.
• A variety of procedures can be used to remove these contaminants,
leaving the DNA in a pure form.
• The most commonly used procedures are:
i. Ethanol precipitation.
ii. Phenol–chloroform extraction.
iii. Minicolumn purification.
 1. Ethanol precipitation

• Ethanol precipitation usually by ice-cold ethanol or isopropanol.

• Since DNA is insoluble in these alcohols, it will aggregate together,
giving a pellet upon centrifugation.
• Precipitation of DNA is improved by increasing of ionic strength,
usually by adding sodium acetate.
 2. Phenol–chloroformextraction.

• Phenol–chloroform extraction in which phenol denatures proteins in the

sample.
• After centrifugation of the sample, denaturated proteins stay in the organic
phase while aqueous phase containing nucleic acid is mixed with the
chloroform that removes phenol residues from solution.

.
3. Minicolumn purification.
• Minicolumn purification that relies on the fact that the nucleic acids may bind
(adsorption) to the solid phase (silica or other) depending on the pH and the salt
concentration of the buffer.
 3. Concentration of DNA samples

• The most frequently used method of concentration is ethanol precipitation.

• In the presence of salt and at a temperature of -20°C or less, absolute
ethanol will efficiently precipitate polymeric nucleic acids.
• With a concentrated solution of DNA one can use a glass rod to pull out
the adhering DNA strands while for dilute solutions precipitated DNA can
be collected by centrifugation and redissolving in an appropriate volume
of water.
 4. Measurement of purity of DNA
concentration.

• DNA concentrations can beaccurately measured by UV absorbance spectrometry.

• The amount of UV radiation absorbed by a solution of DNA is directly proportional to
the amount of DNA sample.
• Usually absorbance is measured at 260 nm, at this wave length an absorbance of 1.0
corresponds to 50 µg of double-stranded DNA per ml.
• With a pure sample of DNA the ratio of the absorbancies at 260 nm and 280 nm
(A260/A280) is 1.8.
• Ratios of less than 1.8 indicate that the preparation is contaminated, either with protein
or with phenol.
 RNA STRUCTURE AND FUNCTIONS
• RNA, abbreviation of ribonucleic acid, complex compound of high molecular weight that
functions in cellular protein synthesis and replaces DNA (deoxyribonucleic acid) as a carrier
of genetic codes in some viruses.
• RNA consists of ribose nucleotides (nitrogenous bases appended to a ribose sugar) attached
by phosphodiester bonds, forming strands of varying lengths. The nitrogenous bases in
RNA are adenine, guanine, cytosine, and uracil, which replaces thymine in DNA.
• The ribose sugar of RNA is a cyclical structure consisting of five carbons and one oxygen.
The presence of a chemically reactive hydroxyl (−OH) group attached to the second carbon
group in the ribose sugar molecule makes RNA prone to hydrolysis.
• This chemical lability of RNA, compared with DNA, which does not have a reactive −OH
group in the same position on the sugar moiety (deoxyribose), is thought to be one reason
why DNA evolved to be the preferred carrier of genetic information in most organisms. The
structure of the RNA molecule was described by R.W. Holley in 1965.
• RNA typically is a single-stranded biopolymer.
• The presence of self-complementary sequences in the RNA
strand leads to intrachain base-pairing and folding of the
ribonucleotide chain into complex structural forms consisting
of bulges and helices.
• The 3D structure of RNA is critical to its stability and
function, allowing the ribose sugar and the nitrogenous bases
to be modified in numerous different ways by cellular enzymes
that attach chemical groups (e.g., methyl groups) to the chain.
• Such modifications enable the formation of chemical bonds
between distant regions in the RNA strand, leading to complex
contortions in the RNA chain.
• This further stabilizes the RNA structure.
• Molecules with weak structural modifications and stabilization
may be readily destroyed.
• As an example, in an initiator transfer RNA (tRNA) molecule
that lacks a methyl group (tRNAiMet), modification at
position 58 of the tRNA chain renders the molecule unstable
and hence nonfunctional; the nonfunctional chain is destroyed
by cellular tRNA quality control mechanisms.
• RNAs can also form complexes with molecules known as
ribonucleoproteins (RNPs).
• The RNA portion of at least one cellular RNP has been shown
to act as a biological catalyst, a function previously ascribed
only to proteins.
 RNA (Ribonucleic acid )

RNA is a polymer of
ribonucleotides linked
together by 3’-5’
phosphodiester linkage
RNA V/S DNA

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 Differences between RNA and DNA
S.No. RNA DNA

1) Single stranded mainly except Double stranded (Except for

when self complementary certain viral DNA s which
sequences are there it forms a are single stranded)
double stranded structure (Hair
pin structure)
2) Ribose is the main sugar The sugar moiety is deoxy
ribose
3) Pyrimidine components differ. Thymine is always there but
Thymine is never uracil is never found
found(Except tRNA)
4) Being single stranded structure- It does follow Chargaff's rule.
It does not follow Chargaff’s rule The total purine content in a
double stranded DNA is always
equal to pyrimidine content.

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S.No. RNA
5) RNA can be easily destroyed
by alkalies to cyclic diesters of
mono nucleotides.
6) RNA is a relatively a labile
molecule, undergoes easy and
spontaneous degradation
7) Mainly cytoplasmic, but also
present in nucleus (primary
transcript and small nuclear
RNA)
8) The base content varies from
100- 5000. The size is
variable.
DNA
DNA resists alkali action due to the
absence of OH group at 2’ position
DNA is a stable molecule. The
spontaneous degradation is very 2
slow. The genetic information can be
stored for years together without any
change.
Mainly found in nucleus, extra
nuclear DNA is found in
mitochondria, and plasmids etc
Millions of base pairs are there
depending upon the organism
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S.No. RNA
9) There are various types of RNA –
mRNA, r RNA, t RNA, Sn RNA, Si
RNA, mi RNA and hn RNA. These
RNAs perform different and
specific functions.
10) No variable physiological forms
of RNA are found. The different
types of RNA do not change their
forms
11) RNA is synthesized from DNA, it
can not form DNA(except by the
action of reverse transcriptase). It
can not duplicate (except in certain
viruses where it is a genomic
material )
12) Many copies of RNA are present
per cell
DNA
DNA is always of one type and
performs the function of storage
and transfer of genetic
information.
There are variable forms of
DNA (A to E and Z)
DNA can form DNA by
replication, it can also form
RNA by transcription.
Single copy of DNA is present
per cell.
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 Types of RNA
In all prokaryotic and eukaryotic organisms, three main
classes of RNA molecules exist-
1) Messenger RNA(m RNA)
2) Transfer RNA (t RNA)
3)Ribosomal RNA (r RNA) The other
are –
o small nuclear RNA (SnRNA),
o micro RNA(mi RNA) and
o small interfering RNA(Si RNA) and
o heterogeneous nuclear RNA (hnRNA).

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 Messenger RNA (m-RNA)

 Comprises only 5% of the RNA in the cell

 Most heterogeneous in size and base sequence
All members of the class function as messengers carrying the
information in a gene to the protein synthesizing machinery

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 Structural Characteristics of m-RNA

The 5’ terminal end is capped by 7- methyl guanosine

triphosphate cap.
The cap is involved in the recognition of
mRNA by the translating machinery
It stabilizes m RNA by protecting it from 5’exonuclease

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 Structural Characteristics of m-RNA(contd.)

The 3’end of most m-RNAs have a polymer of Adenylate

residues( 20-250)
 The tail prevents the attack by 3’ exonucleases
Histones and interferons do not contain poly A tails
On both 5’ and 3’ end there are non coding sequences which are
not translated (NCS)
The intervening region between non coding sequences present
between 5’ and 3’ end is called coding region. This region encodes
for the synthesis of a protein.

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 Structural Characteristics of m-RNA

5’ cap and 3’ tail impart stability to m RNAby protecting from specific exo
nucleases.
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 Structural Characteristics of m-RNA(Contd.)

The m- RNA molecules are formed with the help of DNA

template during the process of transcription.
The sequence of nucleotides in m RNA is complementary
to the sequence of nucleotides on template DNA.
The sequence carried on m -RNA is read in the form of codons.
 A codon is made up of 3 nucleotides
The m-RNA is formed after processing of
heterogeneous nuclear RNA

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 Heterogeneous nuclear RNA (hnRNA)

In mammalian nuclei , hnRNA is the

immediate product of gene transcription
The nuclear product is heterogeneous in size (Variable) and is
very large.
Molecular weight may be more than 107, while the molecular
weight of m RNA is less than 2x 106
75 % of hnRNA is degraded in the nucleus, only 25% is
processed to mature
m RNA

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Heterogeneous nuclear RNA (hnRNA)

• Mature m –RNA is formed from primary transcript by capping, tailing,

splicing and base modification.
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 Transfer RNA (t- RNA)

Transfer RNA are the smallest of three major species of RNA molecules
 They have 74-95 nucleotide residues
They are synthesized by the nuclear processing of a precursor molecule
They transfer the amino acids from cytoplasm to the protein synthesizing
machinery, hence the name t RNA.
 They are easily soluble , hence called “Soluble RNAors
RNA
They are also called Adapter molecules, since they act as adapters for the
translation of the sequence of nucleotides of the m RNA in to specific amino
acids
There are at least 20 species of t RNA one corresponding to each of the 20
amino acids required for protein synthesis.

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 Structural characteristics of t- RNA

1)Primary structure- The nucleotide sequence of all the t RNA

molecules allows extensive intrastand complimentarity that
generates a secondary structure.
2)Secondary structure- Each single t- RNA shows extensive
internal base pairing and acquires a clover leaf like structure. The
structure is stabilized by hydrogen bonding between the bases
and is a consistent feature.

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 Structural characteristics of t-RNA

Secondary structure (Clover leaf structure)

All t-RNA contain 5 main arms or loops which are as follows-
a) Acceptor arm
b) Anticodon arm
c) D HU arm
d) TΨ C arm
e) Extra arm

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 Secondary structure of t- RNA

a) Acceptor arm
 The acceptor arm is at 3’ end
 It has 7 base pairs
 The end sequence is unpaired
Cytosine, Cytosine-Adenine at the 3’ end
 The 3’ OHgroup terminal of Adenine bindswith carboxyl group of
amino acids
 The t RNA bound with amino acid is called Amino acyl t
RNA
 CCA attachment is done post transcriptionally

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 Secondary structure of t-RNA

The carboxyl group of amino acid is attached to 3’OH group of Adenine

nucleotide of the acceptor arm. The anticodon arm base pairs with the codon
present on the m- RNA
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 Secondary structure of t-RNA(contd.)

b) Anticodon arm
 Lies at the opposite end of acceptor arm
 5 base pairs long
Recognizes the triplet codon present in the m RNA
Base sequence of anticodon arm is complementary to the base
sequence of m RNA codon.
Due to complimentarity it can bind specifically with m RNA by
hydrogen bonds.

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 Secondary structure of t-RNA(contd.)

c) DHU arm
 It has 3-4 base pairs
Serves as the recognition site for the enzyme (amino acyl t RNA
synthetase) that adds the amino acid to the acceptor arm.
d) TΨC arm
 This arm is opposite to DHU arm
Since it contains pseudo uridine that is why it is so named
 It is involved in the binding of t RNA to the ribosomes

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 Secondary structure of t-RNA(contd.)

• e) Extra arm or Variable arm

 About 75 % of t RNA molecules possess a short extra arm
 If about 3-5 base pairs are present the t-RNA is said to be belonging to
class 1. Majority t -RNA belong to class 1.
 The t –RNA belonging to class 2 have long extra arm, 13-21 base pairs in
length.

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 Tertiary structure of t- RNA

The L shaped tertiary structure is

formed by further folding of the clover
leaf due to hydrogen bonds between T
and D arms.
The base paired double helical stems get
arranged in to two double helical columns,
continuous and perpendicular to one
another.

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 Ribosomal RNA (rRNA)
The mammalian ribosome contains two major nucleoprotein subunits—
a larger one with a molecular weight of 2.8 x 106 (60S) and a smaller
subunit with a molecular weight of 1.4 x 106 (40S).
The 60S subunit contains a 5S ribosomal RNA (rRNA), a 5.8S rRNA,
and a 28S rRNA; there are also probably more than 50 specific
polypeptides.
The 40S subunit is smaller and contains a single 18S rRNA and
approximately 30 distinct polypeptide chains.
All of the ribosomal RNA molecules except the 5S rRNA are processed
from a single 45S precursor RNA molecule in the nucleolus .
 5S rRNA is independently transcribed.

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Ribosomal RNA (rRNA)

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 Ribosomal RNA (rRNA)

The functions of the ribosomal RNA molecules in the

ribosomal particle are not fully understood, but they are necessary
for ribosomal assembly and seem to play key roles in the binding
of mRNA to ribosomes and its translation
Recent studies suggest that an rRNA component performs the
peptidyl transferase activity and thus is an enzyme (a ribozyme).

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 Small RNA

Most of these molecules are complexed with proteins to form

ribonucleoproteins and are distributed in the nucleus, in the
cytoplasm, or in both.
They range in size from 20 to 300 nucleotides and are present
in 100,000– 1,000,000 copies per cell.

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 Small Nuclear RNAs (snRNAs)

snRNAs, a subset of the small RNAs, are significantly

involved in mRNA processing and gene regulation
Of the several snRNAs, U1, U2, U4, U5, and U6 are involved
in intron removal and the processing of hnRNA into mRNA
The U7 snRNA is involved in production of the correct 3'
ends of histone mRNA—which lacks a poly(A) tail.

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 Small Nuclear RNAs (snRNAs).

Sn RNA s are involved in the process of splicing (intron removal) of primary

transcript to form mature m RNA. The Sn RNA s form complexes with
proteins to form Ribonucleoprotein particles called snRNPs

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 Micro RNAs, miRNAs, and Small Interfering RNAs,
siRNAs

These two classes of RNAs represent a subset of small RNAs;

both play important roles in gene regulation.
miRNAs and siRNAs cause inhibition of gene expression by
decreasing specific protein production albeit apparently via distinct
mechanisms

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 Micro RNAs (miRNAs)

miRNAs are typically 21–25 nucleotides in length and are

generated by nucleolytic processing of the products of distinct
genes/transcription units
The small processed mature miRNAs typically hybridize, via the
formation of imperfect RNA-RNA duplexes within the 3'-
untranslated regions of specific target mRNAs, leading via
unknown mechanisms to translation arrest.

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 Micro RNAs (miRNAs)

microRNAs, short non-coding RNAs present in all living organisms, have

been shown to regulate the expression of at least half of all human genes.
These single-stranded RNAs exert their regulatory action by binding
messenger RNAs and preventing their translation into
proteins.
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Small Interfering RNAs (siRNAs)
siRNAs are derived by the specific nucleolytic cleavage of larger,
double-stranded RNAs to again form small 21– 25 nucleotide-long
products.
These short siRNAs usually form perfect RNA-RNA hybrids
with their distinct targets potentially anywhere within the length of
the mRNA where the complementary sequence exists.
Formation of such RNA-RNA duplexes between siRNA and
mRNA results in reduced specific protein production because the
siRNA-mRNA complexes are degraded by dedicated nucleolytic
machinery;
some or all of this mRNA degradation occurs in specific organelles
termed P bodies.

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 Small Interfering RNAs (siRNAs)

Small interfering RNA (siRNA) are 20-25 nucleotide-long double-stranded

RNA molecules that have a variety of roles in the cell. They are involved in
the RNA interference (RNAi) pathway, where it interferes with the expression
of a specific gene by hybridizing to its corresponding RNA sequence in the
target mRNA. This then activates the degrading mRNA. Once the target
mRNA is degraded, the mRNA Cannot be translated into protein.

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 Significance of mi RNAs and si RNAs

Both miRNAs and siRNAs represent exciting new potential

targets for therapeutic drug development in humans.

In addition, siRNAs are frequently used to decrease or "knock-

down" specific protein levels in experimental procedures in the
laboratory, an extremely useful and powerful alternative to gene-
knockout technology.

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Thank you