DNA STRUCTURE AND ANALYSIS

INTRODUCTION

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.

EXPLANATION:

Replication refers to the process of making a copy of the genetic material. It is essential for the
survival and growth of living organisms since it ensures that the genetic information is passed on
accurately to the next generation. During replication, the DNA helix is unwound and a new strand is
synthesized using the existing strand as a template. Once replication is complete, the chromosome is
ready to undergo mitosis and meiosis, two processes that are necessary for cell division and sexual
reproduction.

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.

EXPLANATION:

The genetic material can store vast amounts of information, from the sequences of amino acids
in proteins to regulatory sequences that control gene expression. This information is transmitted from
one generation to the next through the processes of DNA replication and cell division. The chemical
language of the genetic material, consisting of nucleotides and base pairs, allows for the storage of this
diverse information.

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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.

EXPLANATION:
 Gene expression is the process by which the information encoded in a gene is used to produce a
functional protein or RNA molecule. This process involves the transcription of DNA into mRNA and the
translation of mRNA into protein. Gene expression is tightly regulated and varies depending on the
needs of the cell and the organism. The central dogma of molecular biology states that genetic
information flows from DNA to RNA to protein..

4. Variation by mutation. The genetic material is the source of variation brought by mutation.

EXPLANATION:

Mutation refers to any change in the genetic material that can alter the function of a gene or its
regulatory elements. Mutations can occur spontaneously or can be induced by environmental factors
such as radiation and chemicals. Mutations are the source of genetic variation in populations and can
lead to the evolution of new traits over time. Some mutations may be harmful, but others may be
neutral or even beneficial, depending on the context in which they occur. Overall, mutation is a crucial
factor in the evolution of life on Earth.

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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.

EXPLANATION:

Friedrich Miescher was a Swiss physician and biochemist who lived from 1844 to 1895. He is
credited with discovering nucleic acids, which are the basic building blocks of DNA and RNA. In 1869,
Miescher isolated a substance from the nuclei of white blood cells, which he called "nuclein." Later, this
substance was identified as DNA.

Miescher's contribution to genetics was his discovery of nucleic acids, which paved the way for
further research into the role of DNA in inheritance and genetic information transfer.

Miescher's experiment involved isolating the nuclei from white blood cells, which he then
treated with dilute acid to break them down. This released a substance that he called "nuclein," which
was later identified as DNAh

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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.

EXPLANATION:

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.

Phoebus A. Levene was a Russian-American biochemist who lived from 1869 to 1940. He is
known for his research on the structure of nucleic acids, specifically the chemical composition of DNA
and RNA. Levene discovered the basic building blocks of nucleic acids, which he called nucleotides.

Levene's contribution to genetics was his work on the chemical composition of nucleic acids,
which helped to elucidate the structure of DNA and RNA and their roles in genetic information transfer.

Levene's experiment involved isolating and analyzing the components of nucleic acids,
specifically the nucleotides that make up DNA and RNA.

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Evidences Favoring DNA as the genetic material

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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.
EXPLANATION:

Frederick Griffith's Transformation Experiment revolutionized the study of genetics by

demonstrating that genetic material could be transferred between bacteria. Griffith's experiment
involved injecting mice with different strains of the bacteria Streptococcus pneumoniae, and he
observed that a non-virulent strain could be transformed into a virulent strain when it was mixed with
heat-killed virulent bacteria.

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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.

EXPLANATION:

The Oswald-MacLeod-McCarty Experiment revolutionized the study of genetics by providing

definitive proof that DNA is the genetic material. The experiment involved isolating DNA from a virulent
strain of Streptococcus pneumoniae and showing that it was sufficient to transform a non-virulent strain
into a virulent strain.

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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.

EXPLANATION:

The Hershey-Chase Experiment revolutionized the study of genetics by demonstrating that DNA,
not protein, is the genetic material in viruses. The experiment involved infecting bacterial cells with
bacteriophages, and then separating the virus particles from the bacterial cells using a blender. By using
radioactive labeling, Hershey and Chase showed that the DNA of the virus, not the protein coat, was
responsible for infecting the bacterial cells.

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Indirect and Direct Evidence that supports the concept that DNA is the genetic material in Eukaryotes

1. Direct Evidence: Recombinant DNA studies

2. Indirect Evidence
a. 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.

b. 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.

EXPLANATION:

Direct evidence includes experiments that directly demonstrate that DNA is the carrier of
genetic information. One such experiment is the Avery-MacLeod-McCarty experiment, which
demonstrated that DNA is responsible for the transfer of genetic information in bacteria. The
experiment involved isolating different components of bacterial cells and testing their ability to
transform non-virulent bacteria into virulent ones. The researchers showed that DNA was the only
component that had this ability, providing direct evidence that DNA is the genetic material.

Indirect evidence includes experiments that support the idea that DNA is the genetic material by
ruling out alternative possibilities. For example, experiments in which the effects of mutations on
phenotype are observed can indirectly support the idea that DNA is the genetic material. If mutations in
DNA are found to result in changes in phenotype, then this suggests that DNA is responsible for carrying
genetic information.

Other indirect evidence includes the observation that DNA is present in the nucleus of
eukaryotic cells, which is where genetic information is stored. Furthermore, the structure of DNA and its
ability to replicate accurately also support the idea that it is the carrier of genetic information.

Additionally, advances in modern technology have allowed for the direct manipulation of DNA,
providing further evidence that DNA is the genetic material. Techniques such as genetic engineering,
which involves the insertion of foreign DNA into cells to produce new traits or proteins, rely on the fact
that DNA carries genetic information.

In summary, both direct and indirect evidence support the concept that DNA is the genetic
material in eukaryotes. Direct evidence comes from experiments that demonstrate that DNA is
responsible for carrying genetic information, while indirect evidence comes from observations and
ruling out alternative possibilities.

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James Watson and Francis Crick – proposed that the structure of DNA is in the form of a double helix
(1953)

The data on Watson and Crick proposal primarily came from two sources:

1. Base composition analysis of hydrolyzed samples of DNA

2. X-ray diffraction studies of DNA

EXPLANATION:

Past discoveries play a crucial role in advancing our understanding and knowledge of various
scientific breakthroughs. They provide researchers a direct access to the ideas and theories that allow
them to have a comprehensive analysis and interpretation. In this case, two has a significant study
contributes on the creation of Watson and Crick 3D Model of DNA. Which are the study of Base
composition by Erwin Chargaff and the X-ray Diffraction Analysis by Rosalind Franklin.

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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).

EXPLAINATION:

Erwin Chargaff was an Austrian biochemist who lived from 1905 to 2002. He is known for his
work on the chemical composition of DNA, which helped to establish the fundamental principles of DNA
structure and provided evidence for the base-pairing rules of DNA.

Chargaff's base composition analysis involved analyzing the base composition of DNA from
various organisms. Specifically, he measured the relative amounts of the four nucleotide bases (adenine,
thymine, guanine, and cytosine) in DNA samples.

Through his analysis, Chargaff observed that the base composition of DNA varied between
different organisms, but within a given species, the amount of adenine was always roughly equal to the
amount of thymine, and the amount of guanine was always roughly equal to the amount of cytosine.
This became known as Chargaff's rule or the base-pairing rule.

The base-pairing rule was significant because it provided evidence for the double-stranded
nature of DNA and suggested a mechanism for DNA replication. Specifically, the base-pairing rule
suggested that during replication, the two strands of DNA separate and serve as templates for the
synthesis of new complementary strands. The base-pairing rule also helped to establish the chemical
structure of DNA, providing a foundation for the work of Rosalind Franklin, James Watson, and Francis
Crick in determining the 3D structure of DNA

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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.

EXPLAINATION:

In summary, Chargaff's base composition analysis provided important insights into the chemical
composition of DNA and its structure, helping to establish the fundamental principles of DNA and its role
in genetic information transfer.
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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.

EXPLANATION:

Rosalind Franklin was a British biophysicist who played a crucial role in the discovery of the
structure of DNA. One of her key contributions was her use of X-ray diffraction analysis to study the
structure of DNA molecules.

X-ray diffraction is a technique that involves shining X-rays at a crystallized sample of a

substance and analyzing the pattern of scattered X-rays to deduce the arrangement of atoms in the
substance. Franklin used this technique to study the structure of DNA fibers, which were crystallized into
a helical form.

Franklin's X-ray diffraction images of DNA fibers revealed a pattern of X-ray scattering that
suggested a helical structure with regularly spaced cross-sectional units. By analyzing the diffraction
pattern, Franklin was able to determine that the DNA molecule had a regular helical structure with a
repeating pattern.

However, Franklin's images were not immediately recognized as providing evidence for a double
helix structure of DNA. It was only after Franklin's work was shared with James Watson and Francis
Crick, who were working on developing a model for the structure of DNA, that the true significance of
Franklin's work became clear.

Watson and Crick used Franklin's X-ray diffraction images, as well as information from other
scientists, to develop their now-famous double helix model of DNA. Their model was built on the idea of
two complementary strands of nucleotides held together by hydrogen bonds between the base pairs.

In summary, Rosalind Franklin's use of X-ray diffraction analysis was a crucial step in the
discovery of the structure of DNA. Her work provided key insights into the helical structure of the
molecule and served as a foundation for the development of the double helix model of DNA by Watson
and Crick

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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.

EXPLANATION:

The difference between right-handed and left-handed DNA refers to how the molecule twists in
space. Right-handed DNA has a clockwise twist, while left-handed DNA has a counterclockwise twist.

This difference can affect how other molecules interact with the DNA molecule. For example,
enzymes that interact with DNA can have a particular shape that matches with one type of twist, but not
the other. This can affect how efficiently the enzyme can work on the DNA molecule.

Additionally, certain drugs or chemicals may bind specifically to one type of DNA, but not the
other. This can affect how the drug or chemical affects the body or how it interacts with other molecules
in the cell.

Overall, the difference between right-handed and left-handed DNA can have important
implications for how the DNA interacts with other molecules in the cell, and understanding this
difference can have implications for drug development and other areas of research.

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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.

EXPLAINATION:

The two chains are antiparallel:

In the double helix structure of DNA, there are two strands of nucleotides that are twisted
together in a helical shape. The two strands are oriented in opposite directions, meaning that one strand
runs from the 5' end to the 3' end (referred to as the "sense" or "plus" strand), while the other runs
from the 3' end to the 5' end (referred to as the "antisense" or "minus" strand). This orientation is
referred to as antiparallel because the two strands run in opposite directions, like parallel lines that are
pointed in opposite directions. The antiparallel orientation is important because it allows the two
strands to come together and form hydrogen bonds between complementary base pairs.
 The bases of both chains are flat structures lying perpendicular to the axis:

The nucleotide bases, which are adenine, thymine, cytosine, and guanine, are the "letters" of
the genetic code. In the double helix structure, the bases are arranged in pairs, with adenine always
paired with thymine and cytosine always paired with guanine. The bases are flat structures that are
perpendicular to the axis of the helix. They are stacked on top of each other, with a distance of 3.4
angstroms (0.34 nanometers) between each pair. This arrangement allows the bases to form hydrogen
bonds with each other, with adenine always hydrogen-bonding with thymine, and cytosine always
hydrogen-bonding with guanine. This base pairing is crucial to the function of DNA, as it allows the
molecule to replicate and transmit genetic information accurately. The stacking of the bases also helps
to stabilize the double helix structure by minimizing the electrostatic repulsion between the negatively
charged phosphate groups in the nucleotides.

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Other Notes:

The two polynucleotide chains run antiparallel to each other, meaning that one chain runs in the 5’-3’
direction and the other runs in the 3’-5’ direction.

The bases of one polynucleotide chain face inward and bond to complementary bases on the opposing
chain to form hydrogen bonds. Adenine pairs with thymine and guanine pairs with cytosine, following
Chargaff’s base pair rules.

The sugar-phosphate backbone of each polynucleotide chain runs on the outside of the double helix,
forming a helical configuration.

The double helix has a uniform diameter throughout its length. The Watson-Crick model of DNA
structure resolved many earlier discrepancies and provided a structural basis for the mechanisms of
DNA replication, transcription, and translation. Techniques for Analyzing DNA

Gel electrophoresis - is the most common and basic technique used to separate macromolecules like
DNA, RNA, and protein by size and charge.

Southern Blot - is used to detect the presence of specific DNA sequences in a given DNA sample.

Northern Blot - is used to detect the presence of specific RNA sequences in a given RNA sample.

Western Blot - is used to detect the presence of specific protein sequences in a given protein sample.

Polymerase Chain Reaction (PCR) - is the most important and widely used technique in molecular
biology for amplifying (making many copies of) a specific DNA sequence. DNA analysis has revolutionized
the molecular biology field, DNA sequencing, cloning, and genetic engineering. With the emergence of
rapid and high-throughput DNA sequencing technologies, the potential applications of these
technologies are limitless, from personalized medicine to the identification of new therapies, disease
diagnosis, and preventative healthcare.