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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