Transcript CHMI 2227E Biochemistry I

CHMI 2227E
Biochemistry I
Nucleic acids:
structure
- physico-chemical properties
-
CHMI 2227 - E.R. Gauthier, Ph.D.
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Nucleic acids
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Hershey and Chase
The Waring blender experiment…
32P
in nucleic acids only
35S
in proteins only (Met/Cys)
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Nature of nucleic acids
1. nitrogenated bases
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Nature of nucleic acids
2. sugars
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Nature of nucleic acids
3. nucleosides Glycosidic
bond
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H
1
H
1’
Deoxyadenosine
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1’
Cytidine
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Nature of nucleic acids
3. nucleosides
Purines
Base
DNA
RNA
Adenine
Deoxyadenosine
Adenosine
Guanine
Deoxyguanosine
Guanosine
Pyrimidines
Base
DNA
Cytosine
Thymine
Uracil
Deoxycytidine
RNA
Cytidine
Deoxythymidine
-
Uridine
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Nature of nucleic acids
3. nucleosides
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Nature of nucleic acids
4. nucleotides
Gamma
g
Beta
b
Alpha
a
Deoxythymidine 5’triphosphate
Guanosine 5’monophosphateCHMI 2227 - E.R. Gauthier, Ph.D.
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Nature of nucleic acids
4. nucleotides
Number of
phosphate
groups
Deoxynucleotide/Nucleotide
1
Deoxyadenosine-5’-monophosphate
2
Deoxyguanosine-5’-diphosphate
3
Adenosine-5’-triphosphate
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Nature of nucleic acids
5. polynucleotides
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Nature of nucleic acids
5. phosphodiester bond
g
b
a
CHMI 2227 - E.R. Gauthier, Ph.D.
http://www.mbi.ufl.edu/facilities/msg/bch4024m/lecture45.pdf
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Nature of nucleic acids
6. Chargaff’s rules
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Structure of nucleic acids
1. DNA
bases
Sugar/phosphate backbone
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Structure of nucleic acids
1. DNA
CHMI 2227 - E.R. Gauthier,
1 Å Ph.D.
(Ångstrom)
15 m
= 0.1 nm = 1 x 10-10
Structure of nucleic acids
1. DNA
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Structure of nucleic acids
2. DNA/RNA hybrids and RNA duplexes
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Structure of nucleic acids
2. DNA/RNA hybrids and RNA duplexes
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Structure of nucleic acids

Java applets:
 http://www.moleculesinmotion.com/
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DNA absorbance
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DNA denaturation
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Hyperchromic shift
DNA denaturation
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DNA#1
DNA#2
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DNA denaturation
G+C-rich
A+T-rich
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Hybridization
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Agarose gel electrophoresis
DNA
-
Power
+
• Polymerized agarose is porous,
allowing for the movement of DNA
Scanning Electron Micrograph
of Agarose Gel (1×1 µm)

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DNA molecules
of known length
Agarose gel electrophoresis
Staining with
ethidium bromide
Standard curve: Log length vs
distance migrated
Ethidium bromide fluoresces red only when
it intercalates between baseCHMI
pairs.
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Southern blot and Northern blot
Annealing
32P-labeled
nucleic
acid probe is
complementary to one
of the DNA molecules
on the gel
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Southern blot
Genomic DNA
Different allele?
cut with restriction enzymeCHMI 2227 - E.R. Gauthier, Ph.D.
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FISH
Fluorescent in-situ hybridization
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Microarrays aka DNA chips
RNA labeling
CHMI
2227 - E.R.
1000’s
ofGauthier,
genesPh.D.
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DNA denaturation
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DNA denaturation
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The «C value» paradox
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The «C value» paradox
The fruit fly
Drosophila
melanogaster
180 Mb
The mountain
grasshopper
Podisma pedestris
18,000 Mb
kbp
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The «C value» paradox
Highly repeated
Moderately repeated
Single copy
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Human genome
Human genome
3000 Mb
Genes and other related
sequences
Extra-genic DNA
2100 Mb
900 Mb
Coding DNA
Non-coding DNA
Repeated DNA
90 Mb
810 Mb
420 Mb
Single/low copy
number DNA
1680 Mb
Pseudogenes
Gene
fragments
Introns, etc
Tandem
repeats
Satellite DNA
Interspersed
repeats
Microsatellite
DNA
Minisatellite
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Gauthier, Ph.D.
DNA
LTR elements
LINE elements
SINE elements
Transposons
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Human genome
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