Module 4

• Nucleic acid analyses


  Techniques used to characterize DNA and RNA

  
• Nucleic acid analyses


  • Electrophoresis
  • Hybridization Assays
  • Amplification techniques
  • DNA Sequencing
  • Polymorphism-based Analysis
• Nucleic acid degradation


  Denaturation: separation of dsDNA to ssDNA, breaking of H bonds between base pairs

  Degradation: breaks between individual nucleotides, or in the phosphate backbone of DNA molecule (dsDNA or ssDNA)

  
• Causes of DNA degradation

  • Incorrect handling and storage
  • Lack of temperature control during extraction
  • Presence of nucleases

  
• Steps to prevent DNA degradation


  • Correct handling and storage of materials
  • Perform extractions at 4°C, on ice or in the cold
  • Inhibit nuclease activity (low temperature, chemical inhibitors, protein precipitation)
  • Store purified DNA correctly

  
• DNA storage temperatures


  • Short-term (weeks): 4°C in Tris-EDTA

  Medium-term (months): -80°C in Tris-EDTA

  Long-term (years): -80°C as a precipitate under ethanol

  Long-term (decades): -164°C or dried
• In 1978, Werner Arber was awarded the Nobel Prize for Physiology or Medicine together with Americans Daniel Nathans and Hamilton Smith for proving that molecular scissors in bacteria, so-called restriction enzymes, recognize foreign DNA molecules and cut them at certain points.
• Nucleic acid


  • Has unique biochemical properties

  Base pairing forms the basis for DNA replication, RNA transcription and translation into protein

  Enzymes (polymerases, transcriptase, nucleases, ligases) are used as tools for molecular biology and molecular diagnostics

  dNTPs are the monomeric substrates for the polymerization reaction
• Molecular basis of pathogen identification and subtyping

  Molecular methods seek to detect and visualize DNA/RNA unique to the target pathogen

  The ultimate discriminatory test would be to sequence the entire genome of every organism

  The detection of nucleotide differences among shared and uniquely present genomic regions
• Homology


  Conserved characters that are relatively unchanged among taxa or certain group of organisms

  Variable characters that are modified
• 16S rRNA gene


  Primary gene target for molecular detection of bacterial species as it contains a mixture of conserved, variable and hypervariable regions
• Primers


  Target the highly conserved regions of the 16S and 23S rRNA genes
• Pathogen subtyping


  Further differentiation below the species (type) or subspecies level (subtype) based on a particular characteristic that is widely accepted per species (e.g. epidemiological lineages, serotypes, drug resistance, toxigenic activity)
• Knowledge of a pathogen's biology will be crucial in the development and selection of a molecular diagnostic assay. The selection of particular genetic targets for molecular diagnosis is dependent on the diagnostic objective.
• Pathogen detection assay


  Used for qualitative detection and/or pathogen load determination, targeting short regions specific to the desired taxonomic class of the infectious agent
• Gene targets for molecular detection of Flavivirus and Dengue Virus

  • Commonly used targets in infectious disease diagnostics (green box)

  Target sequences for generic detection of all Dengue Serotypes (yellow, blue)

  Target sequences for serotype-specific detection of DENV-1, DENV-2, DENV-3, DENV-4 (purple, green, red, grey)
• Pathogen variant trait detection assay

  Used to detect mutations that confer a particular trait, such as drug resistance or increased virulence, by directly targeting the related gene that codes for the particular trait
• Target genomic regions for drug resistance genotyping of HIV-1

  • The numbers represent the codon position where the mutations are associated with HIV drug resistance
• Molecular diagnostics has a tremendous impact on the diagnosis of infectious disease and genetic disorders. The real power of the technology lies in its ability to cut across traditional disciplines in laboratory medicine. Consequently, an understanding the principles underlying the various nucleic acid amplification technology is important for all involved in the practice of laboratory medicine.