Fundamental Genetics

Lecture 9

DNA Structure
and Analysis
John Donnie A. Ramos, Ph.D.
Dept. of Biological Sciences
College of Science
University of Santo Tomas

DNA: The String of Life

James Watson Francis Crick

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 Characteristics of the
Genetic Material
‰ Replication
‰ Storage of
information
‰ Expression of
information
‰ Variation by
mutation

Central Dogma of Molecular Genetics

Early Studies on the

Genetic Material
‰ Friedrick Miescher (1868) – acid substance from nuclei called nuclein
‰ Phoebus Levene (1910) – tetranucleotide hypothesis (equal amount
of nucleotides)
‰ Frederick Griffith (1927) – In vivo transformation experiment
‰ Oswald Avery, Colin MacLeod, Maclyn McCarty (1944) – In vitro
transformation experiment (bacteriophage)
‰ Alfred Hershey, Martha Chase (1952) – Bacteriophage
transformation
‰ William Astbury (1938) – X-ray diffraction analysis of DNA
‰ Rosalind Franklin (1950) – improved X-ray diffraction analysis of
DNA
‰ James Watson and Francis Crick (1953) – DNA double helix
structure

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In Vivo Transformation Experiment

“Transformation
might be due to the
polysaccharide
capsule or some
compound required
for capsule
synthesis”

In Vitro Transformation Experiment

DNA is responsible for the transformation of
avirulent strain to a virulent type!

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 Hershey-Chase Experiment
DNA (and not protein) is the genetic
material in phage T2.

Evidences Favoring DNA as the

Genetic Material
‰ DNA is found only where genetic function is known to occur
but protein is ubiquitous.
‰ DNA content of cells is directly correlated with the number of
sets of chromosomes present but not for proteins

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 Evidences Favoring DNA as the
Genetic Material
‰ DNA absorbs UV at the
same wavelength where
mutation occurs (action
spectrum) but proteins
absorbs at different
wavelength
‰ Recombinant DNA
Technology (transgenic
organisms) – direct
evidence

RNA: Genetic Material in

Some Viruses

‰ First identified in 1956 in

tobacco mosaic virus (TMV)
‰ Uses RNA replicase to duplicate
genetic material
‰ Retroviruses – undergo reverse
transcription (RNA to cDNA)
using reverse transcriptase

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 DNA Structure
‰ Proposed by Watson
and Crick in 1953
based on:
‰ Base composition
analysis of
hydrolyzed samples
of DNA
‰ X-ray diffraction
studies of DNA
‰ Sequence of
nucleotides codes for
the genetic
information (4n where
n refers to the no. of
nucleotides)

DNA Structure

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 DNA Structure

‰ Precursor molecule in nucleic acid synthesis

‰ Source of energy (ATP)

Nucleotide Linkage

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Base Composition Studies
‰ First studied by Erwin Chargaff (1949-1953)
‰ Agrees with Watson and Crick DNA model

Chargaff Rule

‰ Amount of A is proportional to T
while C is proportional to G
‰ Sum of purines (A+G) equal to
sum of pyrimidines (C + T)
‰ Percentage of G + C does not
necessarily equal to percentage of
A+T

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 The Watson-Crick DNA Model
‰ Right-handed double helix
‰ Antiparallel chains
‰ Nitrogenous bases as flat
structures inside the helix
‰ Bases are 3.4 A apart
‰ Base complementarity (A-T
and G-C)
‰ 10 bases every 360° turn
‰ 34 A every complete turn
‰ Double helix diameter is 20 A
‰ Semiconservative mode of
replication

Types of DNA
Criteria B DNA A DNA Z DNA
Bases / 360° turn 10 bp 11 bp 12 bp
Length / 360° turn 34 A 37.4 A 40.8
Diameter of helix 20 A 23 A 18 A
Direction of turn Right-handed Right-handed Left-handed
Major groove Present Modified Absent

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 RNA Structure
‰ Ribose sugar
‰ Same nitrogenous bases as DNA except that T replaced by U
‰ Single stranded (but can form double strands)
‰ Forms:
‰ Ribosomal RNA (rRNA) ‰ Small Nuclear RNA (snRNA)
‰ Messenger RNA (mRNA) ‰ Telomerase RNA
‰ Transfer RNA (tRNA) ‰ Antisense RNA

‰ Differs by sedimentation rate (Svedverg Coefficient)

Nucleic Acid Unique Characteristics

‰ Hydrogen bonds breaks at high temperature (denaturation or
unwinding)
‰ Hydrogen bonds reform at lower temperature (annealing)
‰ Melting Temperature (Tm)= temperature at which 50 % of H bonds are
broken (DNA with higher GC content has higher Tm)
‰ Can be measured using spectrophotometer (absorbance at 260 nm)
‰ With increasing temperature, the viscosity of DNA decreases and UV
absorption increase

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 Molecular Hybridization

‰ Annealing of nucleic acid

(DNA or RNA) strands
sharing nucleotide
sequence similarity
‰ Used to identify
homologous genes in
different species
‰ Example: In situ
hybridization or
Fluorescence in situ
hybridization (FISH)

Reassociation Kinetics
‰ Measures the rate of annealing between
complementary strands
‰ Measures half reaction time (point when
½ of the reaction are double stranded)
‰ Half Reaction is lower in smaller genomes
‰ Used to measure repetitive DNA
sequences (characteristic of eukaryotes)

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 Electrophoresis
‰ Agarose gel
electrophoresis
‰ Polyacrylaminde gel
electrophoresis
‰ Separates nucleic acids
by size under an
electrical field
‰ DNA is negatively
charged (travels to +
charge)
‰ Southern Blot –
detection of DNA
‰ Northern Blot –
detection of RNA

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