nucleic acid basics
DESCRIPTION
Hybridization. Diagnostic tools. Nucleic acid Basics. PCR. Electrophoresis. DNA-Protein interactions. Chromatin. Gene expression. Six Nucleosides. Cytidine (base: cytosine). 5-methyl Cytidine (base: 5-methy cytosine). Guanosine (base: guanine). Thymidine - PowerPoint PPT PresentationTRANSCRIPT
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Nucleic acid Basics
Hybridization
Electrophoresis
PCRDiagnostic
tools
DNA-Proteininteractions
Chromatin
Gene expression
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Six Nucleosides
Guanosine(base: guanine)
Cytidine(base: cytosine)
Thymidine (base: thymine) thymidine is deoxynucleotide
Uridine(bsae: uracil)
Adenosine(base: adenine)
5-methyl Cytidine(base: 5-methy cytosine)
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Features of Nucleosides
NO
N
OOH
OHNH2
OH
1’ carbon forms aglycosidic linkageto a base (adenineis shown here
2’ carbon is connected to: - H in DNA - OH in RNAIn RNA the OH may functionas a catalyst in some reactions.
3’ oxygen forms a phosphoester bond.
5’ oxygen forms a phosphoester bond.
Cytidine
3’
1’
2’
4’5’
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A Dinucleotide
5’ end
3’ end
O
O
ON
NH
O
O
CH3
P OO
NO
N
N
ON
NH2
Ophosphodiester
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Single Stranded Nucleic Acids
• In cells, RNAs are the most abundant single stranded nucleic acids– secondary structure is largely in the form of
“hairpin loops”.– tertiary structures are important for catalysis.
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The 2’OH as a catalyst
NO
N
OOH
OHNH2
OH
1’ carbon forms aglycosidic linkageto a base (adenineis shown here
2’ carbon is connected to: - H in DNA - OH in RNAIn RNA the OH may functionas a catalyst in some reactions.
3’ oxygen forms a phosphoester bond.
5’ oxygen forms a phosphoester bond.
Cytidine
3’
1’
2’
4’5’
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Single Stranded Nucleic Acids
• Tertiary structures are important for interactions with proteins and can be manipulated to produce designer drugs:– Interference RNAs– Aptamers.
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Rusconi et al, 2002 Nature 419:90-94
RNA inhibitors of clotting factor IXa
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RNA inhibitor of clotting factor IXa
and its antidote
Rusconi et al, 2002 Nature 419:90-94
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Single Stranded Nucleic Acids
• Single stranded DNAs are important in clinical and scientific investigations. Probes and primers are synthetic single stranded DNAs
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Double Stranded Polynucleotides
G:CThree H-bonds
A:TTwo H-bonds
N
N
N
O
N
O
N
O
O
H
H
H
NO
N
O
O
O
N
H
H
NO
N
N
O
N
N
O
HH
O
O
O
NN
O
OCH3
H
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Important Forces
Negative charges on phosphates destabilize
H-bonds stabilize
Base-base stacking interactions stabilize
(bases at the ends lack this stabilizing force)
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Nucleic acid Basics
Hybridization
Electrophoresis
PCRDiagnostic
tools
DNA-Proteininteractions
Chromatin
Gene expression
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DNA “Melting”The DNA strands separate when heated
Strand separation occurs over a narrow temperature range.The midpoint is Tm, the “melting temperature”.
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Factors That Influence TmProperties of the helix
• Base composition:– C:G rich is more stable than A:T rich
• Mismatches:– Sequences with perfect complementarity are
more stable than those with mismatches.
• Length of the helix– Very short helicies are less stable that
moderately long ones.
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Factors That Influence TmProperties of the solution
• Ionic conditions– Solutons with high ionic strength will stabilize.
• Extremes of pH
• Chemicals that disrupt H-bonds– Urea, formamide, formaldehyde
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Factors That Influence TmProperties of cells
• Helix-destabilizing proteins– These proteins play physiologically important
roles in a number of cellular processes.
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Separated Strands Can Rehybridize
- Duplex formation is a bimolecular reaction:thermodynamically favored
- Hair-pin helix formation is a monomolecular reaction:kinetically favored
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Hybridization:Conditions are important
• Concentration is important– Hydridization is a bimolecular reaction. A high
concentration of DNA will favor duplex formation.
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Hybridization:Conditions are important
• Temperature is important– Slow cooling will favor the formation of DNA
duplexes.– Fast cooling will favor the formation of hair-pin
loops, which may prevent duplex formation.– The temperature must be near the Tm if high
stringency is desired (formation of duplexes with perfect complementarity).
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Fluorescence in situ hybridization
FISH
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Biopsy from a patient with breast cancer showing HER-2 amplification
Control probe
HER-2 probe
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HER-2 probe
Control probe