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Sept 22.docx

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Department
Biology
Course
BIOL 3110
Professor
Peter Cheung
Semester
Fall

Description
Sept 22, 2009 Single Stranded DNA Genomes -Some organisms use a single strand of DNA as their form of genetic information storage; only during replication is the genome in a double stranded form -There is an intermediate form of the genome called the replication form (RF) DNA Denaturation -The standard double stranded nature of most genomes can be converted into a single stranded form by heat denaturation Native ds DNA –(100°C `5min) denatured state of ss DNA -The amount of DNA that is denatured can be measured via a spectrometer using a 260nm wavelength light. The absorbency of the light is dependent on the structure of the strands themselves, the more bases that are exposed will result in a grater amount of absorbency. The amount of base stacking that occurs when the double helix is broken is greatly reduced, this increases the absorbency of DNA. -Refer to previous notes for DNA melting Evidence That H-Bonds are Important -Plot melting temperature (Tm) vs. GC content; Tm will increase as the amount of CG content increases (CG pairs have 3 H-bonds vs. AT pairs that only have 2) -Tm in the presence of foramide or urea causes the Tm to decrease because of the competition that occurs between these chemical agents and the H-bonding between the nucleotides. -An increase in pH causes the Tm to decrease because (when pH > 11.3 DNA is fully denatured) the structure of the molecule becomes unstable as the pH rises. The pKa values of DNA comes into play once the pH approaches or exceeds that value since deprotonation occurs and H-bonding becomes unviable. Evidence That Base Stacking is Important -As Na-trifluoroacetate concentration increases the Tm decreases, this is due to the fact that it disrupts the water shell around a hydrophobic substance; this leads to less hydrophobic interactions. -Alcohols and amines decrease the Tm because the solubility of the bases increases which also lowers the hydrophobic interactions Forces That Destabilize the Structure of ds DNA -PO 4roups destabilize the double helix structure due to the negative charge of the phosphates, however, increasing the amount of NaCl will increase
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