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Analysis and Characterization of Nucleic Acids and Proteins

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PROBES Single-stranded DNA or RNA molecules used to detect

complementary sequences. carry radioactive or chemical markers to facilitate their

detection. can be anywhere from fifteen to thousands of

nucleotides long. Hybridization reactions using DNA/RNA probes are so

sensitive and selective that they can detect complementary sequences present at a concentration as low as one molecule per cell.

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Type

Origins

Characteristic of starting

material

Labeling

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DNA LABELING Incorporation of labeled nucleotides:

Random primed labeling with klenow. Nick translation with DNA polymerase I and DNAse I.

End labeleing: 3’end labeling with terminal transferase. 5’end labeling with polynucleotide kinase.

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Random primed labeling with klenow

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Nick translation with DNA polymerase I and DNAse I

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3’fill-end labeling by klenow

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5’end labeling with polynucleotide kinase

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Run-off transcription from cloned DNA inserts in specialized plasmid vectors

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labeling

1. Isotopic labeling: often 32P, 33P, 35S or 3H

2. None isotopic labeling:A. Direct nonisotopic labeling:

Often involve incorporation of modified nucleotides containing a fluorophore.

B. Indirect nonisotopic labeling. The biotin-streptavidine system Digoxigenin system.

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General Hybridization Times/ Temperatures

ON=overnight

Optimal Hybridization Times

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Hybridization Conditions Three steps of hybridization reaction

Prehybridization to block non-specific binding Hybridization under appropriate conditions Post-hybridization to remove unbound probe

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Hybridization Stringency Stringency is the combination of conditions in which

the target is exposed to the probe. Conditions of high stringency are more demanding of

probe/target complementarity. If conditions of stringency are set too high, the probe will not

bind to its target. Low stringency conditions are more forgiving.

If conditions are set too low, the probe will bind unrelated targets, complicating interpretation of the final results.

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Hybridization Stringency Closely related genes are not identical in

sequence, but are similar Conserved sequence relationship is indicator of

functional importance Use lower temperature hybridization to identify

DNAs with limited sequence homology: reduced stringency

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Several factors affect stringency

Temperature of hybridization Calculate Tm of the probe sequence

The hybridization temperature of oligonucleotide probes is about 5C below the melting temperature

Salt concentration of the hybridization buffer The concentration of denaturant such as formamide

in the buffer. The nature of the probe sequence can also impinge

on the level of stringency. A probe with a higher percentage of G and C bases will

bind under more stringent conditions than one with greater numbers of A and T bases.

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Several factors affect stringency Amount of genomic DNA Proportion of the genome that is complementary

to the probe Size of the probe (short probe = low signal) Labeling efficiency of the probe Amount of DNA transferred to membrane

High Stringency for well matched hybrids High temp (65o-68oC) or 42oC in presence of 50%

formamide Washing with low salt (0.1X SSC), high temp (25oC)

Low Stringency Low temp, low formamide Washing with high salt

Formamide concentration increases stringency. Low salt increases stringency. Heat increases stringency.

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Detection Methods Isotopic labels (3H, 32P, 35S, 125I)

Photographic exposure (X-ray film) Quantification (scintillation

counting, densitometry)

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Detection Methods Non-isotopic labels (enzymes,

lumiphores) Enzymatic reactions (peroxidase,

alkaline phosphatase) Luminescence (Adamantyl

Phosphate derivatives, “Lumi-Phos”)

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Radioactive orchemiluminescent detection(autoradiography film)

Chromogenic detection(nitrocellulose membrane)

Southern Blot Results

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Southern Blot Applications

Genetics, oncology (translocations, gene rearrangements)

Typing/classification of organisms Cloning/verification of cloned DNA Forensic, parentage testing (RFLP, VNTR)

Line probe assay (LIPA): Based on reverse hybridization Available commercially Homework:

List at least 3 specific applications for LIPA

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