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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

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Nucleic acid Basics

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Nucleic acid basics

Hybridization

Diagnostic

tools

Nucleic acid

Basics

PCR

Electrophoresis

DNA-Protein

interactions

Chromatin

Gene expression


Six nucleosides

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)


Features of nucleosides

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


A dinucleotide

A Dinucleotide

5’ end

phosphodiester

3’ end


Single stranded nucleic acids

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

The 2’OH as a catalyst

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


Single stranded nucleic acids1

Single Stranded Nucleic Acids

  • Tertiary structures are important for interactions with proteins and can be manipulated to produce designer drugs:

    • Interference RNAs

    • Aptamers.


Rna inhibitors of clotting factor ixa

RNA inhibitors of clotting factor IXa

Rusconi et al, 2002 Nature 419:90-94


Rna inhibitor of clotting factor ixa and its antidote

RNA inhibitor of clotting factor IXa and its antidote

Rusconi et al, 2002 Nature 419:90-94


Single stranded nucleic acids2

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

Double Stranded Polynucleotides

G:C

Three H-bonds

A:T

Two H-bonds


Important forces

Important Forces

H-bonds stabilize

Negative charges on

phosphates destabilize

Base-base stacking

interactions stabilize

(bases at the ends lack

this stabilizing force)


Nucleic acid basics

Hybridization

Diagnostic

tools

Nucleic acid

Basics

PCR

Electrophoresis

DNA-Protein

interactions

Chromatin

Gene expression


Dna melting the dna strands separate when heated

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 tm properties of the helix

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 tm properties of the solution

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 tm properties of cells

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

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

Hybridization:Conditions are important

  • Concentration is important

    • Hydridization is a bimolecular reaction. A high concentration of DNA will favor duplex formation.


Hybridization conditions are important1

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

Fluorescence in situ hybridizationFISH


Biopsy from a patient with breast cancer showing her 2 amplification

Biopsy from a patient with breast cancer showing HER-2 amplification

Control probe

HER-2 probe


Nucleic acid basics

Control probe

HER-2 probe


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