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Cards (13)

  • 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 (151000 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