Post on 31-Dec-2015
description
Nucleic acid Basics
Hybridization
Electrophoresis
PCRDiagnostic
tools
DNA-Proteininteractions
Chromatin
Gene expression
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)
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’
A Dinucleotide
5’ end
3’ end
O
O
ON
NH
O
O
CH3
P OO
NO
N
N
ON
NH2
Ophosphodiester
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.
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’
Single Stranded Nucleic Acids
• Tertiary structures are important for interactions with proteins and can be manipulated to produce designer drugs:– Interference RNAs– Aptamers.
Rusconi et al, 2002 Nature 419:90-94
RNA inhibitors of clotting factor IXa
RNA inhibitor of clotting factor IXa
and its antidote
Rusconi et al, 2002 Nature 419:90-94
Single Stranded Nucleic Acids
• Single stranded DNAs are important in clinical and scientific investigations. Probes and primers are synthetic single stranded DNAs
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
Important Forces
Negative charges on phosphates destabilize
H-bonds stabilize
Base-base stacking interactions stabilize
(bases at the ends lack this stabilizing force)
Nucleic acid Basics
Hybridization
Electrophoresis
PCRDiagnostic
tools
DNA-Proteininteractions
Chromatin
Gene expression
DNA “Melting”The DNA strands separate when heated
Strand separation occurs over a narrow temperature range.The midpoint is Tm, the “melting temperature”.
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.
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
Factors That Influence TmProperties of cells
• Helix-destabilizing proteins– These proteins play physiologically important
roles in a number of cellular processes.
Separated Strands Can Rehybridize
- Duplex formation is a bimolecular reaction:thermodynamically favored
- Hair-pin helix formation is a monomolecular reaction:kinetically favored
Hybridization:Conditions are important
• Concentration is important– Hydridization is a bimolecular reaction. A high
concentration of DNA will favor duplex formation.
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).
Fluorescence in situ hybridization
FISH
Biopsy from a patient with breast cancer showing HER-2 amplification
Control probe
HER-2 probe
HER-2 probe
Control probe