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DNA Structure

and Analysis

Fundamental Genetics Lecture 9

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

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

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

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

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

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

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)

Messenger RNA (mRNA)

Transfer RNA (tRNA)

Differs by sedimentation rate (Svedverg Coefficient)

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 Tm)

Can be measured using spectrophotometer (absorbance at 260 nm)

With increasing temperature, the viscosity of DNA decreases and UV

absorption increase

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)

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