DNA STRUCTURE AND ANALYSIS

We know that chromosomes contain genes that control phenotypic traits that are transmitted gametes to
future offspring.
Genes must contain some sort of information that, when passed to a new generation, influences the form
and characteristics of each individual, we refer to as the genetic material.
For a molecule to serve as the genetic material, it must exhibit four crucial characteristics:
1. Replication. A genetic material must be able to replicate because it is a fundamental property of all
living organisms. Once the genetic material was replicated, it must be able to undergo mitosis and
meiosis.
2. Storage of information. The molecule to act as a repository of genetic information that may or may
not be expressed by the cell in which it resides. The chemical language of the genetic material must
have the capability of storing such diverse information and transmitting it to progeny cells and
organisms.
3. Expression of information. Gene expression is the process by which the information encoded in a
gene is turned into a function. The initial event in this flow of information is the transcription and
translation, processes serve as the foundation for the central dogma.
4. Variation by mutation. The genetic material is the source of variation brought by mutation.

Proteins vs. Nucleic acid (Which is the real genetic material?) they are the major candidate for the role
Many geneticists favored proteins since they were known to be both diverse and abundant in cells. They
know more about proteins than nucleic acid.
Friedrich Miescher – was the first to study DNA in 1869

- He isolated cell nuclei and derived an acidic substance, now known to contain DNA, that he called
nuclein.
Phoebus A. Levene – concluded that DNA lack the chemical diversity necessary to store extensive
genetic information
He was able to further purify the material. When Levene analyzed the chemical properties of nucleic
acid, he discovered that DNA was abundant in three things: five-carbon sugars, phosphate, and
nitrogen bases (adenine, cytosine, guanine, or thymine). Thus, Levene correctly deduced that the
DNA molecule was made of smaller molecules linked together, and these smaller molecules, which he
named nucleotides. However, he also incorrectly proposed that identical groups of these four
components were repeated over and over, which was the basis of his tetranucleotide hypothesis for
DNA structure. Levene rejected the notion that it could be the genetic material because he thought that
DNA was a simple structure unable to store vast genetic material.
Tetranucleotide hypothesis – DNA is a linear, single-stranded polynucleotide consisting of four
repeating bases (adenine, thymine, guanine, and cytosine) linked to each other by a deoxyribose
phosphate ester backbone.
Levene’s incorrect proposal favored proteins. However, Erwin Chargaff showed that Levene’s proposal was
incorrect when he demonstrated that most organisms do not contain precisely equal proportions of the four
nucleotides.
Evidences Favoring DNA as the genetic material
a. Frederick Griffith’s Transformation Experiment
He performed experiments with several different strains of the bacterium Streptococcus pneumoniae.
The R-strain produced rough colonies on a bacterial plate and did not cause fatal infections. While the
other S-strain was smooth which caused fatal infections when injected into mice. Heat-treated S-strain
cells did not also cause fatal infections. Griffith noticed that upon mixing “heat-treated” S-strain cells
together with some R-type bacteria (neither should kill the mice), the mice died and there were S-strain,
 pathogenic cells recoverable. Thus, some non-living component from the S-type strains contained
genetic information that could be transferred to and transform the living R-type strain cells into S-type
cells. This research provided the foundation for Avery, MacLeod, and McCarty’s work.
b. Oswald-MacLeod-McCarty Experiment
What kind of molecule from within the S-type cells was responsible for the transformation? Researchers
named Avery, MacLeod and McCarty separated the S-type cells into various components, such as
proteins, polysaccharides, lipids, and nucleic acids. Only the nucleic acids from S-type cells were able
to make the R-strains smooth and fatal. Furthermore, when cellular extracts of S-type cells were treated
with DNase (an enzyme that digests DNA), the transformation ability was lost. The researchers
therefore concluded that DNA was the genetic material, which in this case controlled the appearance
(smooth or rough) and pathogenicity of the bacteria.
c. Hershey-Chase Experiment
The researchers studied the transmission of genetic information in a virus called the T2 bacteriophage,
which used Escherichia coli as its host bacterium. The T2 phage itself only contains both protein and
DNA and like all viruses, T2 hijacks the cellular machinery of its host to manufacture more viruses. To
determine which of these two types of molecules contained the genetic blueprint for the virus, Hershey
and Chase grew viral cultures in the presence of radioactive isotopes of either phosphorus ( 32P) or
sulfur (35S). The phage incorporated these isotopes into their DNA and proteins, respectively. The
researchers then infected E. coli with the radiolabeled viruses, and looked to see whether 32P or 35S
entered the bacteria. After ensuring that all viruses had been removed from the surface of the cells, the
researchers observed that infection with 32P labeled viruses (but not the 35S labeled viruses) resulted in
radioactive bacteria.
Hershey and Chase interpreted these results as indicating that the protein of the phage coat remains
outside the host cell and is not involved in directing the production of new phages. On the other hand,
and most important, phage DNA enters the host cell and directs phage reproduction. Hershey and
Chase had demonstrated that the genetic material in phage T2 is DNA, not protein.
These experiments, along with those of Avery and his colleagues, provided convincing evidence that DNA
was the molecule responsible for heredity. This conclusion has since served as the cornerstone of the field
of molecular genetics.
Indirect and Direct Evidence that supports the concept that DNA is the genetic material in
Eukaryotes
Indirect Evidence: Distribution of DNA
The genetic material should be found in the nucleus as part of chromosomes. Both DNA and protein fit
this criterion. However, protein is also abundant in the cytoplasm, whereas DNA is not. Both
mitochondria and chloroplasts are known to perform genetic functions, and DNA is also present in these
organelles. Thus, DNA is found only where primary genetic functions occur. Protein, on the other hand,
is found everywhere in the cell. These observations are consistent with the interpretation favoring DNA
over proteins as the genetic material.
Indirect Evidence: Mutagenesis
Ultraviolet (UV) light is capable of inducing mutations in the genetic material. UV light is most
mutagenic at the wavelength ʎ of 260 nanometers (nm), and both DNA and RNA absorb UV light most
strongly at 260 nm. On the other hand, protein absorbs most strongly at 280 nm, yet no significant
mutagenic effects are observed at that wavelength. This indirect evidence supports the idea that a
nucleic acid, rather than protein, is the genetic material.
Direct Evidence: Recombinant DNA studies
Once they realized that DNA was the genetic material. They put effort in identifying its general structure to
clarify it functions easier.
Deoxyribonucleic Acid (DNA) – a nucleic acid, the genetic material that contains all genetic information
James Watson and Francis Crick – proposed that the structure of DNA is in the form of a double helix
(1953)
The data available to Watson and Crick, crucial to the development of their proposal, came primarily from
two sources:
1. base composition analysis of hydrolyzed samples of DNA
2. X-ray diffraction studies of DNA
Base composition studies by Erwin Chargaff used chromatographic methods to separate the four bases in
DNA samples from various organisms. Chargaff’s data were critical to the success of Watson and Crick as
they devised the double helical model of DNA. On the basis of these data, the following conclusions may
be drawn:
1. The amount of adenine residues is proportional to the amount of thymine residues in DNA. Also, the
amount of guanine residues is proportional to the amount of cytosine residues
2. Based on this proportionality, the sum of the purines (A + G) equals the sum of the pyrimidines (C +
T)
3. The percentage of (G + C) does not necessarily equal the percentage of (A + T).
These conclusions indicate a definite pattern of base composition in DNA molecules. In addition, they
directly refute Levene’s tetranucleotide hypothesis, which stated that all four bases are present in equal
amounts.
X-ray Diffraction Analysis – When fibers of a DNA molecule are subjected to X-ray
bombardment, the X rays scatter in a pattern that depends on the molecule’s
atomic structure. The pattern of diffraction can be captured as spots on
photographic film and analyzed for clues to the overall shape of and regularities
within the molecule.

Rosalind Franklin obtained improved X-ray data from more purified samples of
DNA. She suggested that the structure of DNA was some sort of a helix but she
did not propose a definite model.
The Watson-Crick Model
Watson and Crick published their analysis of DNA structure in 1953. This model has the following major
features:
1. Two long polynucleotide chains are coiled around a central axis, forming a right-handed double
helix. Right handed helix is the most consistent with the data that were available to Watson-Crick
Model. However, Z-DNA, an alternative form of DNA is left handed. How to determine if the DNA is
right handed? Imagine the DNA as a staircase, and to climb that staircase use your right hand for
support.
2. The two chains are antiparallel; that is, their C-5’- to-C-3’ orientations run in opposite directions.
3. The bases of both chains are flat structures lying perpendicular to the axis; they are “stacked” on
one another, 3.4 Å (0.34 nm) apart, on the inside of the double helix.
4. The nitrogenous bases of opposite chains are paired as the result of the formation of hydrogen
bonds; in DNA, only A=T and G ≡C pairs occur.
5. Each complete turn of the helix is 34 Å (3.4 nm) long; thus, each turn of the helix is the length of a
series of 10 base pairs.
 6. A larger major groove alternating with a smaller minor groove
winds along the length of the molecule.
7. The double helix has a diameter of 20 Å (2.0 nm).

Nucleotide – building blocks of all nucleic acid molecules, composed of:

 A pentose sugar (5-carbon sugar)

 A phosphate group
 Nitrogenous base:
o Adenine (A)
o Thymine (T)
o Guanine (G)
o Cytosine (C)

The pentose sugar

DNA contains deoxyribose; RNA contains ribose.
The carbon atoms of a nucleotide’s sugar
molecule are numbered as 1′, 2′, 3′, 4′, and 5′.
Compared with ribose, deoxyribose has a hydrogen
atom rather than a hydroxyl group (OH) at the C-2’
position. The absence of a hydroxyl group at the C-2’
position thus distinguishes DNA from RNA.
In the absence of the C-2’ hydroxyl group, the sugar is
more specifically named 2-deoxyribose.
Base + sugar = nucleoside
Phosphate group + nucleoside = nucleotide
Nucleotide is a monomer, and if these
monomers combined the chain is now called
polynucleotide.
The sugar-phosphate backbone
The phosphate backbone is the portion of the DNA double helix that provides structural support to the
molecule. The phosphate group of one nucleotide bonds covalently with the sugar molecule of the next
nucleotide, and so on, forming a long polymer of nucleotide monomers.
The phosphate group is attached to the 5′ carbon of one nucleotide and the 3′ carbon of the next
nucleotide.
The Base
Purines – nine-member double-ring structure

 Adenine (A)
 Guanine (G)
Pyrimidines- six-member single-ring

 Thymine (T)
 Cytosine (C)

The bases are attached to the C-1’ of the sugar.

If the base is a purine, the N-9 atom is covalently
bonded to the sugar; if the base is a pyrimidine,
the N-1 atom bonds to the sugar.
Adenine can only pair with Thymine and
Guanine can only pair with Cytosine because
these are the only combinations that would
allow for hydrogen bonding to occur.
Watson and Crick discounted the pairing of A with G or of C with T because these would represent purine–
purine and pyrimidine–pyrimidine pairings, respectively. The AC and GT pairings were also discounted,
even though those pairs would each consist of one purine and one pyrimidine.
The base pairs are described as complementary and
attached by hydrogen bonds.
Adenine and Thymine have 2 hydrogen bonds, and Cytosine
and Guanine have 3 hydrogen bonds. This means that the
hydrogen bonding between G and C is stronger in the sense
that it requires more energy to break.
A hydrogen bond is a very weak electrostatic attraction
between a covalently bonded hydrogen atom and an atom
with an unshared electron pair. Even tho two or three
hydrogen bonds are weak, thousands of bonds provide great
stability to the helix.

The antiparallel arrangement of the two chains is a key part

of the double-helix. model. While one chain runs in the 5’-to-
3’ orientation, the other chain goes in the 3’-to-5’ orientation.
The right-handed nature of the helix modeled by Watson and
Crick is best appreciated because it is the standard helix.
Another stabilizing factor is the arrangement of sugars and
bases along the axis. In the Watson–Crick model, the
hydrophobic (“water-fearing”) nitrogenous bases are stacked
almost horizontally on the interior of the axis and are thus
shielded from the watery environment that surrounds the
molecule within the cell. The hydrophilic (“water-loving”)
sugar-phosphate backbones are on the outside of the axis,
where both components may interact with water. These
molecular arrangements provide significant chemical
stabilization to the helix.
A more recent and accurate analysis of the form of DNA that
served as the basis for the Watson–Crick model has revealed
a minor structural difference between the substance and the
model. A precise measurement of the number of base pairs
per turn has demonstrated a value of 10.4, rather than the
10.0 predicted by Watson and Crick. In the classic model,
each base pair is rotated 36° around the helical axis relative
to the adjacent base pair, but the new finding requires a rotation of 34.6°. This results in slightly more than
10 base pairs per 360° turn.
There are two grooves spiraling along outside of the double helix. These grooves are not symmetrical in
size.
Major groove – larger groove
Minor groove – smaller groove