LAB6.NEW - RECOMBINANT DNA SESSION 06: HYBRIDIZATION OF THE...

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RECOMBINANT DNA SESSION 06: HYBRIDIZATION OF THE BLOT BACKGROUND Melting of DNA sequences The principal forces holding the DNA double helix together are hydrogen bonds between bases in opposite strands and hydrophobic bases stacking interactions between bases within the same and opposite strands. Phosphate groups tend to destabilize dsDNA. The melting temperature of DNA depends on its base composition. G-C base pairs in double-stranded DNA can form 3 hydrogen bonds as opposed to 2 hydrogen bonds for A-T base pairs. There is a linear increasing relationship between the percent G-C content and the melting temperature of DNA. The negative charges of phosphate groups on phosphate-sugar backbone cause electrostatic repulsion and tend to blow dsDNA apart. A high cation concentration stabilizes double-stranded DNA by shielding the phosphate groups from each other and raises the melting temperature.The length of the sequence being melted influences the melting temperature. Short sequences, because there are fewer forces to hold them together, are less stable. Annealing If, after DNA is melted, the temperature is rapidly dropped below the melting point, single strands of DNA will be "frozen" in base pairing with other sequences of only partial homology. If instead, the temperature is held optimally about 25 degrees below the melting temperature, there will be "creep" or annealing. Partially frozen structures will be competed out by invasion of better base pairing strands by "zipping up" from
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LAB6.NEW - RECOMBINANT DNA SESSION 06: HYBRIDIZATION OF THE...

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