7 - Methods of nucleic acid investigation II.

Question 1

Theory

Answer 1

• dsDNA heating → the double helix splits
• Cooling down: the complementary threads rejoin
• Probe: labeled oligonucleotide (DNA or mRNA)
  
  • Complementary to the viral genome
  • Labeling: isotope [32P] or enzyme
• Sample + probe
• Heating – cooling
• Washing (removal of unbound probe)
• Autoradiography or substrate
• → classification of viruses, relatedness, diagnostics

Question 2

List the different methods

Answer 2

1. ​Nucleic acid hybridization
2. DNA microarry technique
3. Polymerase chain reaction
4. Sequencing

Question 3

Nucleic acid hybridization

Answer 3

• Theory:
  
  • dsDNA heating → the double helix splits
  • cooling down: the complementary threads rejoin
• Probe: labeled oligonucleotide (DNA or mRNA) with a labelled isotope [32P] or enzyme
  
  • One strand of DNA and the probe – bind on cooling then probe washed out
  • Autoradiography or substrate used to aid virus diagnostics and classification
    
    • But – only targeted at 1 nucleic acid with known viruses

Question 4

DNA microarry technique

Answer 4

• DNA samples bound to glass slides or membrane filters → “DNA chip”
• Hybridized with fluorescent probes after heating/cooling
• Laser scanning
• Computer analyzed
  
  • Identification, “typing” of viruses
  • Comparison of virus strains, genetic relationships

Question 5

Polymerase chain reaction

Answer 5

• Amplification of specific DNA fragments
• Requirments:
  
  • Template of nucleic acid
  • Primers - single stranded DNA (2)
  • Free deoxy-nucleotides
  • Thermo-resistant polymerase (Taq)
• Heating and cooling in cycles (n)
• → 2ⁿ copies of the fragment

Question 6

Sequencing

Answer 6

• Determination of the nucleotide sequence
• The complete information about the nucleic acid
• Sanger’s method: polymerization of DNA sequence
  
  • Needs:
    
    • Template
    • Primer
    • Deoxy-nucleotides (1, not a pair)
    • Labeled dideoxy-nucleotides
    • Polymerase enzyme
  • → polymerization of the complementary thread
• Dideoxy-nucleotide: chain termination
• Polyacrylamide gel-electrophoresis

Question 7

(Real time PCR)

Answer 7

• Fluorescent labeling
• Laser detection of amplification products, computer analysis
• Quantification

Question 8

Investigation of viruses with RNA genome

Answer 8

• Oligonucleotide fingerprint technique:
  
  • Investigation of RNA viruses
  • Creates many short fragments
  • Digestion with ribonuclease A, or T1 ribonuclease
  • Two dimensional PAGE → relation, epidemiology investigations
• Reverse transcriptase conversion:
  
  • ​Investigation of RNA viruses
  • RNA dependant DNA polymerase
  • → transcription to dsDNA
  • Allows us to use DNA investigation methods