Chapter

10
DNA Structure
and Analysis
Lecture Presentation byDr.
Cindy Malone,California State
University Northridge

© 2017 Pearson Education, Ltd.

Section 10.1: Criteria for Genetic
Material
To serve as genetic material,
molecule must be able to:
Replicate
Store information
Express information
Allow variation by mutation

The Genetic Material Must Exhibit these 4

Characteristics

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Section 10.1: The Central Dogma

Central dogma of molecular genetics

DNA → RNA → protein
DNA makes RNA (transcription), which
makes proteins (translation) (Figure 10-1)

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© 2017 Pearson Education, Ltd. Figure 10-1
 10.2 Until 1944, Observations Favored
Protein as the Genetic Material

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Section 10.2: Protein as Genetic
Material
In the 1940s, geneticists favored proteins
as genetic material
Proteins and nucleic acids were major
candidates for genetic material
Proteins were diverse and abundant in cells

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Section 10.2: Tetranucleotide
Hypothesis
Tetranucleotide hypothesis
DNA contains equal amounts of four
nucleotides
Postulated identical groups and repeats of
four components was basis for DNA
structure
Lack of chemical diversity in DNA
suggested it could not store extensive
genetic information
Proteins favored as genetic material

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 Nucleic
acids

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Section 10.3: Avery, MacLeod, and
McCarty
Avery, MacLeod, and McCarty
1944 publication on chemical nature of
transforming principle in bacteria
First direct experimental proof that DNA is
biomolecule responsible for heredity

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Section 10.3: Griffith’s Transformation
Experiment (HORIZONTAL gene transfer)
Griffith (1927)
Provided foundation for Avery, MacLeod,
and McCarty’s research
Showed avirulent strains of Diplococcus
pneumoniae could be transformed to
virulence (Figure 10-2)
Speculated transforming principle could
be part of polysaccharide capsule or
compound required for capsule synthesis

https://www.jove.com/science-education/10573/
bacterial-transformation
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 Smooth=vir
ulent

Rough=
non-
virulent

© 2017 Pearson Education, Ltd. Figure 10-2

 Ways of transformation used
routinely today

Figure from:
thermofischer
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Section 10.3: Transforming Principle is
DNA
Avery, MacLeod, and McCarty (1940’s)

DNase (deoxyribonuclease) utilized to

destroy transforming activity

Demonstrated transforming principle was

DNA, not protein (Figure 10-3)

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© 2017 Pearson Education, Ltd. Figure 10-3
Section 10.3: Hershey and Chase

Hershey and Chase (1952)

Used Escherichia coli and bacteriophage T2
Demonstrated that DNA, not protein, is the
genetic material
Used radioisotopes 32P and 35S
Demonstrated DNA enters bacterial cell
during infection an directs viral
reproduction
(Figures 10-4 and 10-5)

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Hershey and Chase experiment

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© 2017 Pearson Education, Ltd. Figure 10-4
© 2017 Pearson Education, Ltd. Figure 10-5
 Protoplasts

E. coli is treated with lysozyme

Outer wall is removed without destroying
bacterium – naked cell

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Transfection :is the process of introducing naked
or purified nucleic acids into eukaryotic cells.

Infection : by only viral nucleic acid

Proves conclusively that viral DNA alone
contains all necessary information for
production of mature viruses

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 10.4 Indirect and Direct Evidence
Supports the Concept That DNA Is
the Genetic Material in Eukaryotes

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Section 10.4: Indirect Evidence of DNA
as Genetic Material
Indirect evidence: Distribution of DNA
Close correlation between gametes and
diploids in amount of DNA and number of
chromosome sets
No such correlation between gametes and
diploids for proteins

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Section 10.4: Indirect Evidence:
Mutagenesis
Indirect evidence: Mutagenesis
UV light: Most mutagenic at
wavelength 260 nm (action spectrum)
DNA absorbs UV at 260 nm
Protein absorbs UV at 280 nm
A wavelength at which no significant mutagenic
effects are observed
Molecule serving as genetic material
expected to absorb at mutagenic
wavelength (Figure 10-6)

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© 2017 Pearson Education, Ltd. Figure 10-6
Section 10.4: Direct Evidence:
Recombinant DNA Studies
Recombinant DNA technology
Segments of eukaryotic DNA corresponding
to specific genes isolated and spliced into
the bacterial DNA
Complex inserted into bacterial cell and
monitored

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Section 10.4: Direct Evidence

Eukaryotic DNA now functional in

bacterial cell
Eukaryotic gene product in bacteria
containing eukaryotic gene provides direct
evidence: DNA is present and functional in
bacterial cell
Example: Insulin and interferon production
by bacteria

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Section 10.4: Direct Evidence:
Transgenic Animals
Transgenic animal example
Human DNA microinjected into fertilized
mouse egg
DNA encoded human β-globin gene
Now present and expressed in mouse and
transmitted to progeny

Where do you think the transgene is inserted in

the genome?

images.finea
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rtamerica.co
m
 10.5 RNA Serves as the Genetic
Material in Some Viruses

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Section 10.5: RNA as Genetic Material
in Some Viruses
Some viruses have RNA core, not DNA
TMV: Tobacco mosaic virus (1956)
Demonstrated RNA serves as genetic
material for these viruses

RNA replicase
Enzyme isolated from E. coli (1965, Pace
and Spiegelman)
Replication of the viral RNA is dependent
on RNA replicase

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Section 10.5: Retroviruses and
Reverse Transcriptase
Retroviruses
Replicate unusually
RNA serves as template for DNA synthesis
Complementary synthesis of DNA by RNA-
dependent DNA polymerase reverse
transcriptase
Reverse transcriptase
RNA-dependent DNA polymerase enzyme

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 COV-2 belongs to Coronavirus family. It is
+ strand RNA virus.
A Great lecture
https://biology.mit.edu/undergraduate/current-students/subject-offerings/covid-19-
sars-cov-2-and-the-pandemic/?fbclid=IwAR2Xswvm1a1s1i1xfBQJ-
TfNxqhUwuVR74jJXjGN-v1H248M9ouU7L9t-RQ

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 10.6 Knowledge of Nucleic Acid
Chemistry Is Essential to the
Understanding of DNA Structure

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Section 10.6: Nucleotides

Nucleotides
DNA is nucleic acid
Nucleotides: Building blocks of nucleic acid
Nucleotides are building blocks of DNA

Nucleotides consist of:

Nitrogenous base (two kinds)
Pentose sugar
Phosphate group

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Section 10.6: Nitrogenous Bases

Two kinds of nitrogenous bases

Purines (nine-member ring)
Adenine (A)
Guanine (G)
Pyrimidines (six-member ring)
Cytosine (C)
Thymine (T)
Uracil (U)
Figure 10-7

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© 2017 Pearson Education, Ltd. Figure 10-7
© 2017 Pearson Education, Ltd. Figure 10-7a
 Learn the C numbers and why they are
important!

© 2017 Pearson Education, Ltd. Figure 10-7b

Section 10.6: The Bases of DNA and
RNA
DNA
Bases are A, C, T, G

RNA
Bases are A, C, U, G

Only DNA contains T

Only RNA contains U

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Section 10.6: Ribose and Deoxyribose

RNA contains ribose sugar

DNA contains deoxyribose
“Deoxy” (without an oxygen) (Figure 10-
7b)

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 Nucleoside Nucleotide

Contains Nucleoside
nitrogenous base with
and pentose sugar phosphate
Molecule is group
composed of added
purine or
pyrimidine base
and ribose or
deoxyribose sugar

© 2017 Pearson Education, Ltd. Figure 10-8

Section 10.6: Mono-, Di-, and
Triphosphates
Nucleoside monophosphates (NMP)
A nucleotide

Nucleoside diphosphates (NDP)

Nucleotide with addition of two phosphate
groups

Nucleoside triphosphates (NTP)

Nucleotide with addition of three phosphate
groups

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Section 10.6: Triphosphates

Triphosphates
Serve as precursor molecule during nucleic
acid synthesis

ATP and GTP: Adenosine triphosphate

and guanine triphosphate
Large amount of energy involved in
adding/removing terminal phosphate group

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© 2017 Pearson Education, Ltd. Figure 10-9
Section 10.6: Phosphodiester Bonds

Phosphodiester bonds
Nucleotides are linked by phosphodiester
bonds between phosphate group at C-5′
position and OH group on C-3′ position
(Figure 10-10)

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© 2017 Pearson Education, Ltd. Figure 10-10a
 10.7 The Structure of DNA Holds the
Key to Understanding Its Function

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Section 10.7: Structure of DNA

Watson and Crick, 1953

Proposed structure of DNA as a double
helix
Chargaff (1949–1953)
Proposed base composition
Amount of A is proportional to T
Amount of C is proportional to G
Percentage of C + G does not equal
percentage of A + T (Table 10.3)

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© 2017 Pearson Education, Ltd. Table 10.3
Section 10.7: X-Ray Diffraction

Base composition analysis (Chargaff)

and X-ray diffraction provided crucial
data to Watson and Crick
X-ray diffraction
Studies by Rosalind Franklin (1950–1953)
showed DNA had a 3.4-angstrom
periodicity, characteristic of helical
structure (Figure 10-11)

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© 2017 Pearson Education, Ltd. Figure 10-11
Section 10.7: Structure of DNA

Watson and Crick model (1953)

Proposed DNA as:
Double helix
Two anti-parallel strands connected by base
pairing
Stacked nitrogenous bases (Figure 10-12)

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© 2017 Pearson Education, Ltd. Figure 10-12
© 2017 Pearson Education, Ltd. Figure 10-12a
© 2017 Pearson Education, Ltd. Figure 10-12b
Section 10.7: Base Pairing – Hydrogen
Bonds
Chemical affinity produces hydrogen
bonds in pair of bases
A-T and G-C base pairing provides
complementarity of two strands and
chemical stability to the helix
A-T: Double bond
G-C: Triple bond (Figure 10-14)

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© 2017 Pearson Education, Ltd. Figure 10-14
Section 10.7: Semiconservative Model
of Replication
Watson and Crick: Semiconservative
model
Storage of genetic information in sequence
of bases
Mutations or genetic changes that could
result in alteration of bases

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 10.8 Alternative Forms of DNA Exist

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Section 10.8: Alternative Forms of
DNA
Watson-Crick DNA model of B-DNA
Seen under aqueous, low-salt conditions
Alternative forms of DNA
Different conformations of DNA observed
under different conditions of isolation

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Section 10.8: Alternative Forms of
DNA
Under different conditions of isolation,
different conformations of DNA are seen
A-DNA
B-DNA
C-DNA
D-DNA
E-DNA
P-DNA
Z-DNA

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Section 10.8: Alternative Forms of
DNA
A-DNA
Slightly more compact than B-DNA
(Figure 10-15)
Prevalent under high-salt or dehydration
conditions
C-DNA, D-DNA, E-DNA, and P-DNA
Right-handed forms of DNA
Less compact than B-DNA

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Section 10.7: Alternative Forms of
DNA
D-DNA and E-DNA
Lack guanine

Z-DNA
Left-handed double helix

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© 2017 Pearson Education, Ltd. Figure 10-15
 10.9 The Structure of RNA Is Chemically
Similar to DNA, but Single Stranded

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Section 10.9: Structure of RNA

RNA
Sugar ribose replaces deoxyribose of DNA
Nitrogenous base uracil replaces thymine
of DNA
Most are single-stranded (ss)
Exception: Animal viruses have double-stranded
helices

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Section 10.9: Three Classes of RNAs

Three classes of cellular RNAs (function

during gene expression)
Messenger RNA (mRNA)
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
(Table 10.4)

Originate as complementary copies of one

of two DNA strands during transcription

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© 2017 Pearson Education, Ltd. Table 10.4
Section 10.9: Three Major Classes of
RNAs
rRNAs: Ribosomal RNAs
Structural components of ribosomes for
protein synthesis
mRNAs: Messenger RNAs
Template for protein synthesis
Carry genetic information from gene to
ribosome
tRNAs: Transfer RNAs
Carry amino acids for protein synthesis

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Section 10.9: Unique RNAs in
Eukaryotes
Unique RNAs
Telomerase RNA and RNA primers
Involved in DNA replication at chromosome
ends
snRNA: Small nuclear RNA
Process mRNAs
Antisense RNA, microRNA, siRNA
Involved in gene regulation

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 10.10 Many Analytical Techniques
Have Been Useful during the
Investigation of DNA and RNA

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Section 10.1: Analytical Techniques

Analytical techniques useful during DNA

and RNA investigation
Absorption of UV light
Denaturation and renaturation of nucleic
acids
Molecular hybridization
FISH: Fluorescent in situ hybridization
Reassociation kinetics
Electrophoresis of nucleic acids

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Section 10.10: Absorption of UV Light

Nucleic acids
Absorb UV light strongly at 254–260 nm
due to interaction between UV light and
ring systems of the bases (Figure 10-16)
UV light used in localization, isolation,
and characterization
Use of UV critical to isolation of nucleic
acids following separation

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Section 10.10: Denaturation and
Renaturation of Nucleic Acids
DNA can denature due to heat or
stress
Hyperchromic shift
Increase in UV absorption of heated DNA in
solution
Denaturation used to determine melting
temperature (Tm) (Figure 10-16)
Graph of absorption vs temperature gives
melting profile of DNA

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© 2017 Pearson Education, Ltd. Figure 10-16
Section 10.10: Molecular Hybridization

Molecular hybridization
Denaturation and renaturation of nucleic
acids are the basis for molecular
hybridization (Figure 10-17)
Example: Single strands of nucleic acids
combine duplex structures, yet are not
from same source
If DNA is isolated from two distinct sources,
double- stranded hybrids will form

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© 2017 Pearson Education, Ltd. Figure 10-17
Section 10.10: FISH

Fluorescent in situ hybridization

(FISH)
Uses fluorescent probes to monitor
hybridization
Mitotic cells fixed to slides and subjected to
hybridization
ssDNA is added and hybridization is
monitored
Probes are nucleic acids that will hybridize
ONLY with specific chromosomal areas
(Figure 10-18)
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© 2017 Pearson Education, Ltd.
Figure 10-18
Section 10.10: Reassociation Kinetics

Reassociation kinetics
Analyzes rate of reassociation of
complementary single DNA strands
DNA fragmented into small
defragmented/dissociated pieces
Reassociation of ssDNA provides
information on size and genomic
complexity of organism

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Section 10.10: Electrophoresis

Nucleic acid electrophoresis

Separates DNA and RNA fragments by size
Smaller fragments migrate through gel at
faster rate than large fragments (Figure
10-19)
Agarose gel
Porous matrix restricts migration of larger
molecules more than it restricts smaller ones

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© 2017 Pearson Education, Ltd. Figure 10-19