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• Denaturation:

  • Process of strand separation
  • Involves disruption of hydrogen bonds between base pairs
  • Agents of DNA denaturation: Heat, Alkali, Melting proteins (e.g. RNA polymerase, helicase)
• Melting Curve and T m:

  • Melting curve is a plot of extent of denaturation vs. temperature
  • Melting temperature (T m) is the midpoint of the melting curve
  • T m is the temperature at which half of the DNA is denatured
• Factors Influencing T m:

  • Size and base composition of the DNA
  • High GC content leads to high T m
  • Shorter DNA results in low T m
  • Conditions employed for denaturation: ionic strength, pH, urea, methanol, etc.
• Monitoring Denaturation:

  • Monitor A 260 with increasing temperature
  • A 260 is expected to increase with increasing temperature
  • As DNA gets denatured, nitrogenous bases become more exposed and strongly absorb UV light
  • Hyperchromic shift
• DNA Breathing:

  • Transient denaturation of a small fraction of base pairs in the genome
  • Occurs more frequently between bases with fewer H-bonds
  • Necessary for gene regulation
  • Requires no molecules for the event to occur
• DNA Renaturation:

  • Ability of the two separated complementary strands to reform into the ordered state (double helix)
  • Two steps: nucleation and zippering
  • Upon renaturation, A 260 decreases
  • Hypochromic shift
• DNA Renaturation Kinetics:

  • The rate of heat-denatured DNA sequences in solution to renature depends on DNA concentration, reassociation temperature, cation concentration, and viscosity
• C o t Analysis:

  • C o - initial ssDNA concentration (in moles nucleotide per liter)
  • t - time for renaturation to be completed (in secs)
  • From analysis of a C o t plot, one can determine genome size and genome complexity (relative proportions of single-copy and repetitive sequences)
• Classes of Eukaryotic DNA based on sequence complexity:

  • Unique sequence: 1 to 3 copies per genome, single copy, low copy, non-repetitive DNA (e.g. most coding genes, regulatory sequences)
  • Moderately repetitive DNA: 10 to 10,000 copies per genome, generally dispersed repeats, occasionally clustered (i.e. gene families) (e.g. rRNA and tRNA genes, histone genes)
  • Highly repetitive DNA: 100,000 to 1 M copies per genome, generally found as tandem repeats (e.g. centromeric DNA, telomeres DNA, satellite DNA, some transposons)
• Applications of Denaturation and Renaturation:

  1. Studying Genome Relatedness:

  • Denatured DNA of two different organisms are allowed to renature
  • Percent homology is determined to reflect the similarity in the sequence of DNA molecules in the genomes of the species being compared

  2. RNA Looping:

  • Used to detect introns within the cloned gene
  • mRNA + corresponding gene hybridize & view under EM
  • Intron loop
• Probing:

  • Probe: ssDNA or RNA fragment (15 – 1000 nt) of known sequence, complementary to the target DNA/RNA, labeled radioactively or non-radioactively
  • Used for detection of specific nucleotide sequences in the DNA/RNA sample
• Southern Blotting + Hybridization:

  • Devised by Edward Southern in 1975
  • DNA fragments separated on a gel, blotted to a charged surface/membrane (e.g. nitrocellulose membrane)
  • Hybridized with a DNA probe
  • Location of DNA + probe on the filter detected by autoradiography
  • Applications: RFLP Mapping, Forensic Investigation, Genetic Screening
• Other Blotting Techniques:

  • Northern Blotting: RNA blotted on membrane with DNA probe
  • Western Blotting: proteins blotted, labeled antibody specific for the protein
  • Eastern Blotting: Used to detect glycoproteins and lipoproteins