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Dna in basic
1. DNA: The Secret Of Our Life
(An Introduction Lecture)
lecture 1
2. 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
4. We enter the 20th
century with an
understanding of the DNA building
block.
5. 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
19. 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
20.
21. Most Common Secondary Structure (3D structure)
• B-DNA
• Alpha Helix
• Right Handed Turn
• 10 bases per 360º turn
23. 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. 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. 25
Nucleotides
• Subunits of DNA
and RNA
– Nucleosides
linked to
phosphate group
via ester bond
– “dNTP’s”: DNA
– “rNTP’s”: RNA
26. 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.
29. 29
DNA Structure
The 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)
31. 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.
33. 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)
34. • Southern blotting of DNA fragments
APPLICATION 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 Restriction
fragments I II III
I Normal
-globin
allele
II Sickle-cell
allele
III Heterozygote
Preparation of restriction fragments. Gel electrophoresis. Blotting.
Gel
Sponge
Alkaline
solution
Nitrocellulose
paper (blot)
Heavy
weight
Paper
towels
1 2 3
Figure 20.10
35. 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).
Radioactively
labeled probe
for -globin
gene is added
to solution in
a plastic bag
Probe hydrogen-
bonds to fragments
containing normal
or mutant -globin
Fragment from
sickle-cell
-globin allele
Fragment from
normal -globin
allele
Paper blot
Film over
paper blot
Hybridization with radioactive probe. Autoradiography.
I II III
I II III
1 2
36. • DNA microarray assay of gene expression levels
APPLICATION
TECHNIQUE
Tissue sample
mRNA molecules
Labeled cDNA molecules
(single strands)
DNA
microarray
Size of an actual
DNA microarray
with all the genes
of yeast (6,400
spots)
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
