Deoxyribonucleic Acid (DNA):

Deoxyribonucleic acid, also abbreviated as DNA, is the principal

informational macromolecule of the cell, which stores, translates
and transfers the genetic information. In the prokaryotes, the DNA
is found mostly in the nuclear zone. In eukaryotes it is found in the
nucleus, mitochondria and chloroplast. The present understanding
of the storage and utilization of the cell’s genetic information is
based upon the discovery of the structure of DNA by Watson and
Crick in 1953.

Structure of DNA:
DNA is made of two helical chains coiled around the same axis, to
form a right-handed double helix.

2. The two chains in the helix are anti-parallel to each other, i.e., the
5′-end of one polynucleotide chain and the 3′-end of the other
polynucleotide chain is on the same side and close together.
The distance between each turn is 3.6 nm (formerly 3.4 nm).

4. There are 10.5 nucleotides per turn (formerly 10 nucleotides).

5. The spatial relationship between the two strands creates major

and minor grooves between the two strands. In these grooves some
proteins interact.

6. The hydrophilic backbones of alternating deoxyribose and

negatively charged phosphate groups are on the outside of the
double helix.

7. The hydrophobic pyrimidine and purine bases are inside the

double helix, which stabilizes the double helix of the DNA.

8. The double helix is also stabilized by inter-chain hydrogen bond

formed between a purine and pyrimidine base.
9. A particular purine base, pairs by hydrogen bonds, only with a
particular pyrimidine base, i.e., Adenine (A) pairs with Thymine (T)
and Guanine (G) pairs with Cytosine (C) only.

10. Two hydrogen bonds pairs Adenine and Thymine (A = T),

whereas three hydrogen bonds pairs Guanine and Cytosine (G ≡ C).

11. The base pairs A = T and G ≡ C are known as complementary

base pairs.

Due to the presence of complementary base pairing, the two chains

of the DNA double helix are complementary to each other.

Hence the number of A’ bases are equal to the number of T’ bases

(or ‘G’ is equal to ‘C) in a given double stranded DNA.

13. One of the strands in the double helix is known as sense strand,
i.e., which codes for RNA/proteins and the other strand is known as
antisense strand.

The DNA molecules exist in four different structural forms or

organizations under different physiological conditions or in
different cells or at different points in the same DNA.
Functions of DNA:
The base sequence of the DNA constitutes the informational signal
called the genetic material. This nucleotide base sequence enables
the DNA to function, store, express and transfer the genetic
information. Hence it programs and controls all the activities of an
organism directly or indirectly throughout its life cycle.

(a) DNA stores the complete genetic information required to specify

(form) the structure of all the proteins and RNA’s of each organism.

(b) DNA is the source of information for the synthesis of all cellular
body proteins. Some of the proteins are structural proteins and
some are enzymes. These enzymes arrange micro-molecules to form
macromolecules. These macromolecules are arranged to form
supra-molecular complexes or cell organelles which associate to
form cells. These cells group to form tissues which in turn form
different organs of a body, specifically peculiar to that organism
during foetal development, growth and repair. Hence DNA
programs in time and space the orderly biosynthesis of cells and
tissue components.

(c) It determines the activities of an organism throughout its life

cycle, i.e., the period of gestation, birth, maturity, senescence and
death.

(d) It determines the individuality and identity of a given organism.

(e) It duplicates (replicates to form two daughter DNA) itself and

transfers one of the copy to the daughter cell during cell division,
thus maintaining the genetic material from generation to
generation.
 CHEMICAL AND PHYSICAL
PROPERTIES OF DNA

I. ABSORPTION
A. The bases in DNA absorb ultraviolet light at the
wavelength of 260 nm
1. This absorption can be monitored using a
spectrophotometer
2. This is one method used to figure the concentration
of DNA in solution
B. The less ordered the bases the more ultraviolet light is
absorbed
1. Free bases absorb 1.60 units at 260 nm
2. Single stranded DNA absorb 1.37 units at 260 nm
3. Double stranded DNA absorb 1.00 units at 260 nm
II. DENSITY
A. Density can be measured by CsCl-density
ultracentrifugation
1. CsCl, upon ultracentrifugation, will form a density
gradient, with the most dense solution at the bottom
2. Macromolecules, such as DNA, will concentrate in
the area of CsCl that has the same density as
themselves
a. Hence, more dense DNA will migrate downward
and less dense DNA upwards forming bands
B. Density can be used to estimate G+C content
1. GC base pairs are more dense than AT base pairs
2. Therefore, DNA with more GC base pairs will form
bands lower down than an equal number of base
pairs with high AT content
C. Density studies show the existence of satellite DNA
1. If chromosomal DNA is cut into about equal size
pieces and subjected to CsCl-density
ultracentrifugation two bands are formed
a. One band contains most of the DNA from the
genome
b. The second band (the satellite) contains about
5% of the DNA from the genome and has a
highly repetitive sequence
c.
III. DENATURATION
A. Definition
1. DNA is considered denatured when the double
stranded DNA molecule is converted into two single
stranded molecules
2. This can be monitored by noting the increase in
absorption of ultraviolet light
B. Temperature
1. As thermal energy increases, the frequency of
hydrogen bonds breaking between the molecules
increases
a. As temperature increases, the two molecules
will separate into single-stranded molecules
2. The Tm (melting temperature) of a DNA molecule is
the temperature in which half the DNA molecules are
denatures
a. The Tm is used to estimate the G+C content of
a DNA molecule
1. G-C base pairs are held together by three
hydrogen bonds (A-Ts by two) and it
therefore takes more energy (higher
temperatures) to separate molecules with
high GC contents
C. Hydrophobicity of solvent
1. Substances that are hydrophobic tend to decrease
the Tm of DNA molecules
a. Hydrophobic substances will allow the bases in
DNA to dissolve into the solvent
b. Hence, the bases are not constricted to being
stacked upon one another
1. This will make it easier to disrupt the
hydrogen bonding between DNA
molecules
2. Substances that are hydrophilic tend to increase
the Tm of DNA molecules
a. These will keep the bases of DNA stacked upon
one another in the orientation that most favors
hydrogen bonding between DNA strands
D. pH
1. Acids
a. pHs lower then one result in the breakage of
phosphodiester bonds between nucleotides
and breakage of the N-glycosidic bond between
the sugar and purine bases
 b. pH of around 4 results in the selective breakage
of N-glycosidic bonds between the sugar and
purines
1. DNA treated this way is referred to a
apurinic acid, since the purines have
been removed
2. Alkali
a. Base tends to change the polarity of groups
involved in hydrogen bonds
1. Above pH 11.3, all hydrogen bonds are
disrupted and the DNA is totally
denatured
b. DNA is resistant to hydrolysis to about pH 13
1. Unless it is apurinic, then it is hydrolyzed
c. RNA is hydrolyzed into ribonucleotides around
pH 11
E. Ionic strength
1. The phosphates of the DNA sugar-phosphate
backbones are negatively charged
a. Like charges repel each other
b. DNA in distilled water will spontaneously
denature into single stranded DNA
2. Salts that dissociate into ions will neutralize the
charges of the phosphate groups
a. Salts will stabilize the DNA double helix
resulting in a higher Tm
F. G+C content
1. Variation
a. Most higher organisms have a G+C content of
about 0.5 (0.49 - 0.51 for primates)
b. Lower organisms range widely from 0.27 to
0.76 for some bacteria
2. Estimating G+C content
a. G+C content of a DNA molecule can be
estimated from its thermal melting temperature
(Tm)
IV. SOLUBILITY
A. RNA is more soluble in aqueous solutions then DNA
1. Ribose has a 2'-OH group where deoxyribose
contains a 2'-H
a. Hydroxyl groups are polar and dissolve in
water better
b. C-H is a non-polar bond and is therefore
hydrophobic
 B. RNA is less stable then DNA
1. The hydroxyl group on the 2' carbon of ribose is
more reactive the hydrogen found in deoxyribose
V. SIZE
A. Electrophoresis
1. DNA has a negative charge that is proportional to its
size
a. This is due to the negatively charged
phosphates in the sugar-phosphate backbone
b. If DNA is placed in an electrical field it will
migrate towards the positive electrode (the
cathode)
2. If DNA is electrophoresed through a gel, smaller
pieces will migrate faster than larger pieces
a. Larger pieces have trouble squeezing through
the gel matrix and are hence retarded while
smaller pieces migrate easier
b. Type of gels
1. Agarose is used to separate fairly large
DNA molecules
a. 5 million to a few thousands base
pairs
2. Polyacrylamide is used to separate small
pieces of DNA
a. 2 to several hundred base pairs
3. The size of DNA is estimated by comparing its
migration through the gel to DNA molecules of
known size
B. Velocity sedimentation
1. Sedimentation velocity is dependent upon two
variables: density and shape
a. The more dense the DNA the quicker it will
sediment upon centrifugation
b. Globular (more compact) molecules will
sediment faster than linear molecules
C. Electron microscopy
1. The size of DNA molecules can be determined by
electron microscopy
a. The DNA is visualized on a grid of known size
so that the size of the DNA molecule can be
estimated
VI. DNA CONCENTRATION
A. Absorption
1. DNA absorbs ultraviolet light at 260 nm
 2. The more DNA present, the higher the absorption
a. DNA concentrations can be estimated by
comparing its absorption to known
concentrations of DNA
b. DNA most be fairly pure, since many
contaminating substances (e.g., proteins) also
absorb around this wavelength
VII. RENATURATION STUDIES
A. DNA that has been denatured will often come back
together when condition are met
1. This is referred to as renaturation
2. Renaturation occurs because hydrogen bonds of
complimentary base pairs reform
a. Slowly lowering the temperature or adding ions
to solution may lead to renaturation
B. Renaturation rates are dependent on DNA concentration
1. The rate limiting step in renaturation is the collision
of complimentary DNA molecules
a. The more molecules of complimentary DNA
molecules present, the faster they can find
each other and renature
b. DNA molecules in low concentration in solution
will take awhile to find a complimentary
partner, and will therefore renature slower
C. In eukaryotes, three major drops in absorbance occur in
renaturation studies
1. The first drop in absorption is when the highly
repetitive DNA sequence renatures
a. Since these are repeated so often, they are in
the highest concentration
2. The second drop in absorption occurs when the
moderately repetitive DNA renatures
3. Unique DNA sequences are the last to renature
a. These are in the lowest concentration and take
the longest time to find each other
Temperature of Melting (Tm)

The Temperature of Melting (Tm) is defined as the temperature at

which 50% of double stranded DNA is changed to single-
standard DNA. The higher the melting temperature the greater the
guanine-cytosine (GC) content of the DNA.

The temperature at which the DNA strands are half denatured,

meaning half double-stranded, half single-stranded, is called
the melting temperature (Tm). The amount of strand separation,
or melting, is measured by the absorbance of the DNA solution at
260nm.