chmi 2227 - e.r. gauthier, ph.d. 1 chmi 2227e biochemistry i nucleic acids: - structure -...
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CHMI 2227 - E.R. Gauthier, Ph.D. 1
CHMI 2227EBiochemistry I
Nucleic acids:- structure- physico-chemical properties
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Nucleic acids
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Hershey and ChaseThe Waring blender experiment…32P in nucleic acids only 35S in proteins only (Met/Cys)
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Nature of nucleic acids1. nitrogenated bases
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Nature of nucleic acids2. sugars
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Nature of nucleic acids3. nucleosides
H
Deoxyadenosine
Glycosidicbond
1’
91
1’
Cytidine
H
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Purines
Base DNA RNA
Adenine Deoxyadenosine Adenosine
Guanine Deoxyguanosine Guanosine
Nature of nucleic acids3. nucleosides
Pyrimidines
Base DNA RNA
CytosineDeoxycytidine Cytidine
Thymine Deoxythymidine -
Uracil - Uridine
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Nature of nucleic acids3. nucleosides
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Nature of nucleic acids4. nucleotides
Guanosine 5’monophosphateDeoxythymidine 5’triphosphate
Alpha
Beta
Gamma
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Number of phosphate
groupsDeoxynucleotide/Nucleotide
1 Deoxyadenosine-5’-monophosphate
2 Deoxyguanosine-5’-diphosphate
3 Adenosine-5’-triphosphate
Nature of nucleic acids4. nucleotides
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Nature of nucleic acids5. polynucleotides
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Nature of nucleic acids5. phosphodiester bond
http://www.mbi.ufl.edu/facilities/msg/bch4024m/lecture45.pdf
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Nature of nucleic acids6. Chargaff’s rules
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Structure of nucleic acids1. DNA
bases
Sugar/phosphate backbone
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Structure of nucleic acids1. DNA
1 Å (Ångstrom) = 0.1 nm = 1 x 10-10 m
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Structure of nucleic acids1. DNA
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Structure of nucleic acids2. DNA/RNA hybrids and RNA duplexes
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Structure of nucleic acids2. 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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DNA denaturation
Hyp
erc
hro
mic
sh
ift
DNA#1
DNA#2
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DNA denaturation
A+T-rich
G+C-rich
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Hybridization
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Agarose gel electrophoresis
+-
Power
Scanning Electron Micrograph
of Agarose Gel (1×1 µm)
• Polymerized agarose is porous,
allowing for the movement of DNA
DNA
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Agarose gel electrophoresisDNA moleculesof known length
Standard curve: Log length vs distance migrated
Staining with ethidium bromide
Ethidium bromide fluoresces red only whenit intercalates between base pairs.
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Southern blot and Northern blot
32P-labeled nucleic acid probe is complementary to one of the DNA molecules on the gel
Annealing
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Southern blot
Genomic DNAcut with restriction enzyme
Different allele?
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FISHFluorescent in-situ hybridization
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Microarrays aka DNA chips
RNA labeling
1000’s of genes
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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» paradoxThe mountain grasshopper
Podisma pedestris
The fruit fly Drosophila
melanogaster
18,000 Mb 180 Mb
kbp
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The «C value» paradox
Single copy
Moderately repeated
Highly repeated
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Human genomeHuman genome
3000 Mb
Genes and other related sequences
900 Mb
Extra-genic DNA
2100 Mb
Coding DNA
90 Mb
Non-coding DNA
810 Mb
Repeated DNA
420 Mb
Single/low copy number DNA
1680 Mb
Pseudogenes Gene fragments
Introns, etc Tandem repeats
Interspersed repeats
Satellite DNA
Minisatellite DNA
Microsatellite
DNALTR elements SINE elements
LINE elements Transposons
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Human genome
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