Gene Lecture 9 Dna Structure
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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
1
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
2
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!
3
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
4
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
5
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
6
DNA Structure
Precursor molecule in nucleic acid synthesis Source of energy (ATP)
Nucleotide Linkage
7
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
8
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
Present
Modified
Absent
Major groove
9
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
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
10
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)
11
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
12
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
1
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
2
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!
3
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
4
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
5
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
6
DNA Structure
Precursor molecule in nucleic acid synthesis Source of energy (ATP)
Nucleotide Linkage
7
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
8
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
Present
Modified
Absent
Major groove
9
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
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
10
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)
11
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
12
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