10/28/2015

DNA: The String of Life

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
James Watson

Characteristics of the
Genetic Material

Francis Crick

Early Studies on the

Genetic Material
Friedrick Miescher (1868) acid substance from nuclei called nuclein

Replication

Phoebus Levene (1910) tetranucleotide hypothesis (equal amount

of nucleotides)

Storage of
information

Frederick Griffith (1927) In vivo transformation experiment

Oswald Avery, Colin MacLeod, Maclyn McCarty (1944) In vitro
transformation experiment (bacteriophage)

Expression of
information

Alfred Hershey, Martha Chase (1952) Bacteriophage

transformation

Variation by
mutation

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
Central Dogma of Molecular Genetics

In Vivo Transformation Experiment

In Vitro Transformation Experiment

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

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

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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

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

DNA Structure

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

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)

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DNA Structure

Nucleotide Linkage

Precursor molecule in nucleic acid synthesis

Source of energy (ATP)

Base Composition Studies

Chargaff Rule

First studied by Erwin Chargaff (1949-1953)

Agrees with Watson and Crick DNA model

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

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)

Types of DNA
B DNA

A DNA

Z DNA

Bases / 360 turn

Criteria

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

Present

Modified

Absent

Major groove

10 bases every 360 turn

34 A every complete turn
Double helix diameter is 20 A
Semiconservative mode of
replication

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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)
Messenger RNA (mRNA)
Transfer RNA (tRNA)

Small Nuclear RNA (snRNA)

Telomerase RNA
Antisense RNA

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 T m)
Can be measured using spectrophotometer (absorbance at 260 nm)
With increasing temperature, the viscosity of DNA decreases and UV
absorption increase

Differs by sedimentation rate (Svedverg Coefficient)

Molecular Hybridization
Annealing of nucleic acid
(DNA or RNA) strands
sharing nucleotide
sequence similarity
Used to identify
homologous genes in
different species

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

Example: In situ
hybridization or
Fluorescence in situ
hybridization (FISH)

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

Genbank
http://www.ncbi.nlm.nih.gov/genbank/
Under Search look for
nucleotide
Enter accession number,
author or key words in
the 2nd search box

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Take Home Task

Search for the following entries under the nucleotide database of the GenBank:
1. AF525465
2. NM_000207.2
Using the data available in each entry, give the following information
1. Name of the gene
2. Organism where the gene was isolated
3. Taxonomic classification of the organism (include category name, e.g. Kingdom: Animalia)
4. Material used in sequencing the gene
5. Name of the journal paper where the sequence was published (incase of several journals,
give the very first journal that published the sequence)
6. Title of the paper that described the sequence (as answered in #5)
7. Authors of the paper (as answered in #5)
8. How long is the DNA sequence (in base pair)?
9. Give the DNA Sequence
10. Give the amino acid sequence
Note: submit THT typewritten in short bond paper/s