Analytical techniques for DNA

• Many analytical techniques have been useful during the investigation of DNA and RNA

  • Nucleic acids absorb UV light most strongly at 254-260 nm due to the interaction between UV light and the ring systems of the bases
• Nucleic acids can be separated by several gradient centrifugation procedures
• Sedimentation equilibrium centrifugation separates by density gradient
• Sedimentation velocity centrifugation measures the velocity of sedimentation in Svedberg coefficient units
• Heat or other stresses can cause DNA to denature
• Hyperchromic shift during DNA denaturation is used to determine the melting temperature
• Melting temp is a method for estimating the base composition of DNA
• GC content of DNA

  • Higher GC - strong DNA
  • Higher AT - weaker DNA
• The denaturation and renaturation of nucleic acids are the basis for molecular hybridization. This technique have increased  our understanding of gene transcription
• Refinements have helped advance the study of both molecular evolution and organization of DNA in chromosomes.
• Fluorescent in situ hybridization (FISH) uses fluorescent probes to monitor hybridization. 

  • This can be used to identify the chromosomal location of a DNA of interest
  • Ex. the DNA specific to the centromeres of human DNA
• Reassociation kinetics

  • Analyzes the rate of reassociation of complementary single DNA strand
  • It provides info about the size and complexity of genomic DNA from an organism
• Nucleic acid electrophoresis

  • Separates DNA and RNA fragments by size such that smaller fragments migrate through a gel at a faster rate than large fragments
• Electrophoresis

  • Negative charged DNA moves toward electrode (anode), receives negative charges to store (a negative electrode in cells)
  • Kilobase pairs