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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