1. Hybridization
Diagnostic
tools
Nucleic acid
Basics
PCR
Electrophoresis
DNA-Protein
interactions
Chromatin
Gene expression

2. Six Nucleosides Cytidine (base: cytosine) 5-methyl Cytidine
(base: 5-methy cytosine)
Guanosine
(base: guanine)
Thymidine
(base: thymine)
thymidine is deoxynucleotide
Uridine
(bsae: uracil)
Adenosine
(base: adenine)

3. Features of Nucleosides
1’ carbon forms a
glycosidic linkage
to a base (adenine
is shown here
5’ oxygen forms a
phosphoester bond. 4’ 1’ 5’ 2’ 3’
3’ oxygen forms a
phosphoester bond.
2’ carbon is connected to:
- H in DNA
- OH in RNA
In RNA the OH may function
as a catalyst in some reactions.
Cytidine

4. A Dinucleotide
5’ end
phosphodiester
3’ end

5. 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.

6. The 2’OH as a catalyst Cytidine 1’ carbon forms a glycosidic linkage
to a base (adenine
is shown here
5’ oxygen forms a
phosphoester bond. 4’ 1’ 5’ 2’ 3’
3’ oxygen forms a
phosphoester bond.
2’ carbon is connected to:
- H in DNA
- OH in RNA
In RNA the OH may function
as a catalyst in some reactions.
Cytidine

7. Single Stranded Nucleic Acids
Tertiary structures are important for interactions with proteins and can be manipulated to produce designer drugs:
Interference RNAs
Aptamers.

8. RNA inhibitors of clotting factor IXa
Rusconi et al, 2002 Nature 419:90-94

9. RNA inhibitor of clotting factor IXa and its antidote
Rusconi et al, 2002 Nature 419:90-94

10. Single Stranded Nucleic Acids
Single stranded DNAs are important in clinical and scientific investigations. Probes and primers are synthetic single stranded DNAs

11. Double Stranded Polynucleotides
G:C
Three H-bonds
A:T
Two H-bonds

12. Important Forces H-bonds stabilize Negative charges on
phosphates destabilize
Base-base stacking
interactions stabilize
(bases at the ends lack
this stabilizing force)

13. Hybridization
Diagnostic
tools
Nucleic acid
Basics
PCR
Electrophoresis
DNA-Protein
interactions
Chromatin
Gene expression

14. DNA “Melting” The DNA strands separate when heated
Strand separation occurs over a narrow temperature range.
The midpoint is Tm, the “melting temperature”.

15. Factors That Influence Tm Properties 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.

16. Factors That Influence Tm Properties of the solution
Ionic conditions
Solutons with high ionic strength will stabilize.
Extremes of pH
Chemicals that disrupt H-bonds
Urea, formamide, formaldehyde

17. Factors That Influence Tm Properties of cells
Helix-destabilizing proteins
These proteins play physiologically important roles in a number of cellular processes.

18. Separated Strands Can Rehybridize
- Duplex formation is a bimolecular reaction:
thermodynamically favored
- Hair-pin helix formation is a monomolecular reaction:
kinetically favored

19. Hybridization: Conditions are important
Concentration is important
Hydridization is a bimolecular reaction. A high concentration of DNA will favor duplex formation.

20. 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).

21. Fluorescence in situ hybridization FISH

22. Biopsy from a patient with breast cancer showing HER-2 amplification
Control probe
HER-2 probe

23. Control probe
HER-2 probe