mid 22 - Basic Molecular Genetic Mechanisms Chapter 4 pp...

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Basic Molecular Genetic Mechanisms Chapter 4, pp 101-106 Chapter 10, pp 405- 408; 424-430
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DNA: deoxyribose nucleic acid C P Sugar Base H DNA Structure
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DNA can denature and renature •Double stranded (ds) DNA can denature (melt) to single stranded (ss) DNA at high temperature, lower salt, or extreme pH conditions •These conditions breakup the H-bonds between complementary bases. •DNA can also renature (anneal) under the reverse conditions
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DNA denaturation • Tm= melting temperature –Temperature at which 50% of the DNA is denatured • The progress of DNA “melting” is usually monitored by following the increase in absorbance of dissolved DNA. The nitrogenous bases of nucleic acid molecule absorb ultraviolet radiation with an absorbance maximum near 260 nm. H 260 nm
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1) DNA molecules with greater proportion of G-C pairs require higher temperatures to denature because of the 3 H-bonds in G-C pairs. Factors that affect DNA denaturation • The % of G-C pairs in a DNA sample can be estimated from its T m .
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2) Ion concentrations also influences the T m because negatively charged phosphate groups in the two strands are shielded by positively charged ions. -When the ion concentration is low, this shielding is decreased, thus increasing the repulsive forces between the negatively charged strands and reducing the T m . 3) Formamide or urea destabilize hydrogen bonds and lower the T m . 4) Extreme pH denature DNA at low temperature. -At low pH, the bases become protonated and thus positively charged, repelling each other. -At high pH, the bases lose protons and become negatively charged, repelling each other. Factors that affect DNA denaturation
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• Denatured DNA form random coils without an organized structure. DNA Renaturation • Conditions that allow DNA strands to renature: -Low temperatures -Increasing ion concentration -Neutralizing the pH • 2 DNA strands not related in sequence: -Will not renature -Will not inhibit complementary DNA strands from finding each other and allowing them to renaturing
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Circular : Many prokaryotes Many viral DNAs Organelles: mitochondria & chloroplasts Some unicellular eukaryotes Linear : Unicellular and multi-cellular eukaryotes • Genomic DNA is associated with protein • Exists in two forms: Chromosome structure
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Circular DNA
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Circular DNA molecules • Each of the two strands in a circular DNA molecule forms a closed structure without free ends. • Circular DNA exists in “relaxed” or “supercoiled” states - “Relaxed” DNA: standard 10 bp/turn - “Supercoiled” DNA: DNA molecule this twisted upon itself • Supercoiling may also occur in linear, eukaryotic DNA.
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Supercoiled DNA • Localized unwinding of a circular DNA molecule (underwound DNA), which occurs during replication, induces torsional stress into the remaining portion of the molecule because the ends of the strands are not free to rotate.
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