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DNA: The Secret Of Our Life(An Introduction Lecture)
lecture 1
Lecture Objectives
• At the of this Lecture you should be able to learn:
• 1. The history of DNA discovery with their importance for the new concepts and application techniques
• 2. The chemical and physical properties of DNA and how these properties were exploited for DNA technologies
Early historical perspective
We enter the 20th century with an understanding of the DNA building
block.
Some Experimental Data leading to DNA as biological source of DNA
• Griffith’s • Avery et al.
• Hershey and Chase
• Chargaff
• Wilkins and Franklin
• Watson and Crick
Griffith’s Experiment: 1928
Conclusion: A Transformation “factor” exists
Support for nucleic acid transfer
Hershey and Chase Experiment, 1952: Confirms DNA as genetic material
Conclusion: DNA identified as source of genetic information
Franklin and Wilkins 1947
1920-1958 1916-2004
Chargaff’s Rule 1948
1905-2002 1905-2002 1905-2002
1905-2002
Watson and Crick, 1953 inferred the DNA structure
Nobel Prize: 1962
1928-1916-2004 1916-2004
The building blocks of DNA are nucleotides.
RNA’s Sugar DNA’s Sugar
Nitrogenous Bases
DNA nucleotides
Polarity and Anti-Parallel
Back to Franklin and Wilkins Data: Pairing of specific classes of bases can account for diameter of DNA
Just right!
6 sided ring
6 sided ring + 5 sided ring
Most Common Secondary Structure (3D structure)
• B-DNA
• Alpha Helix
• Right Handed Turn
• 10 bases per 360º turn
A function of Major and Minor Grooves
23
Nucleosides• Nucleosides: nitrogenous base linked to specific sugar
– RNA: adenosine, guanosine, cytidine, uridine– DNA: deoxyadenosine, deoxyguanosine, deoxycytidine,
(deoxy)thymidine
138.192.68.68/.../Nucleosides.gif
DNA nucleoside RNA nucleoside
24
Nucleotides
The nucleotide structure consists of– the nitrogenous base attached to the 1’ carbon
of deoxyribose– the phosphate group attached to the 5’ carbon
of deoxyribose– a free hydroxyl group (-OH) at the 3’ carbon of
deoxyribose
25
Nucleotides
• Subunits of DNA and RNA– Nucleosides
linked to phosphate group via ester bond
– “dNTP’s”: DNA– “rNTP’s”: RNA
26
DNA Structure
Nucleotides are connected to each other to form a long chain
phosphodiester bond: bond between adjacent nucleotides– formed between the phosphate group of one
nucleotide and the 3’ –OH of the next nucleotide
The chain of nucleotides has a 5’ to 3’ orientation.
27
28
DNA structure determinationChargaff's Rules
– Erwin Chargaff determined that • amount of adenine = amount of thymine
• amount of cytosine = amount of guanine
29
DNA StructureThe double helix consists of:
– 2 sugar-phosphate backbones– nitrogenous bases toward the interior of the
molecule– bases form hydrogen bonds with complementary
bases on the opposite sugar-phosphate backbone• Adenine pairs with Thymine (2 H bonds)• Cytosine pairs with Guanine (3 H Bonds)
30
31
DNA Structure
The two strands of nucleotides are antiparallel to each other– one is oriented 5’ to 3’, the other 3’ to 5’
The two strands wrap around each other to create the helical shape of the molecule.
32
33
Chemical Properties of DNA
• Factors that affect DNA structure– Temperature: denaturation (can be reversible)– pH: high pH can denature DNA– Salt concentration: lowering salt concentration
can denature DNA– Molecular Hybridization (DNA:DNA) and
(DNA:RNA)– UV absorption (230-260nm)
• Southern blotting of DNA fragmentsAPPLICATION Researchers can detect specific nucleotide sequences within a DNA sample with this method. In
particular, Southern blotting is useful for comparing the restriction fragments produced from different samples of genomic DNA.
TECHNIQUE In this example, we compare genomic DNA samples from three individuals: a homozygote for the normal -globin allele (I), a homozygote for the mutant sickle-cell allele (II), and a heterozygote (III).
DNA + restriction enzyme Restrictionfragments I II III
I Normal-globinallele
II Sickle-cellallele
III Heterozygote
Preparation of restriction fragments. Gel electrophoresis. Blotting.
Gel
Sponge
Alkalinesolution
Nitrocellulosepaper (blot)
Heavyweight
Papertowels
1 2 3
Figure 20.10
RESULTS Because the band patterns for the three samples are clearly different, this method can be used to identify heterozygous carriers of the sickle-cell allele (III), as well as those with the disease, who have two mutant alleles (II), and unaffected individuals, who have two normal alleles (I). The band patterns for samples I and II resemble those observed for the purified normal and mutant alleles, respectively, seen in Figure 20.9b. The band pattern for the sample from the heterozygote (III) is a combination of the patterns for the two homozygotes (I and II).
Radioactivelylabeled probefor -globingene is addedto solution ina plastic bag
Probe hydrogen-bonds to fragmentscontaining normalor mutant -globin
Fragment fromsickle-cell-globin allele
Fragment fromnormal -globinallele
Paper blot
Film overpaper blot
Hybridization with radioactive probe. Autoradiography.
I II IIII II III
1 2
• DNA microarray assay of gene expression levelsAPPLICATION
TECHNIQUE
Tissue sample
mRNA molecules
Labeled cDNA molecules(single strands)
DNAmicroarray
Size of an actualDNA microarraywith all the genesof yeast (6,400spots)
Isolate mRNA.1
With this method, researchers can test thousands of genes simultaneously to determine which ones are expressed in a particular tissue, under different environmental conditions in various disease states, or at different developmental stages. They can also look for coordinated gene expression.
Make cDNA by reverse transcription, using fluores-cently labeled nucleotides.2
Apply the cDNA mixture to a microarray, a microscope slide on which copies of single-stranded DNA fragments from the organism‘s genes are fixed, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray.
3
Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample.
4
RESULT The intensity of fluorescence at each spot is a measure of the expression of the gene represented by that spot in the tissue sample. Commonly, two different samples are tested together by labeling the cDNAs prepared from each sample with a differently colored fluorescence label. The resulting color at a spot reveals the relative levels of expression of a particular gene in the two samples, which may be from different tissues or the same tissue under different conditions.
Figure 20.14
Central Dogma
Information Transfer