Nucleic acid

Question 1

Summarise viral nucleic acid

Answer 1

• Determines viral properties
• Linear or circular
• Continuous or segmented
• Usually haploid
• May contain alien nucleic acid

Question 2

Give examples of alien nucleic acid

Answer 2

• Polyomaviruses, pestiviruses: Host cell nucleic acid
• Arenaviruses: Ribosomes
• Retroviruses: onc, src genes

Question 3

Nucleic acid purification

Answer 3

• From purified virus suspension
  
  • Proteinase K enyme digestion +
  • Chloroform +
  • Isoamylalcohol →
  • Extraction

or

• Protein lysis/purification by chromatography

Question 4

Describe purification by chromatography

Answer 4

• Gels, filters, beads
• Quicker, simpler, less dangerous

Question 5

Describe early viral nucleic acid investigations

Answer 5

• Morphology studies
• Biochemistry studies

Question 6

Describe later viral nucleic acid investigations

Answer 6

• Manipulation
• Genetic engineering

Question 7

Investigation of RNA infectivity (RNA Polarity)

Answer 7

Question 8

What is required for the transcription of viral RNA?

Answer 8

Viral enzyme

Question 9

Give the examples of nucleic acid EM observation

Answer 9

• Electrophoresis
• Restriction endonuclease analysis

Question 10

EM observation of NA can be…

Answer 10

Linear or circular

Question 11

Electrophoresis

Answer 11

• Size:
  
  • Circovirus = 1.7 kb
  • Pox = 300 kbp
  • Information is proportional to size
• Continuous NA thread
• Segmented genome

Question 12

Restriction endonuclease analysis

Answer 12

• dsDNA
• Endonucleases:
  
  • Bacterial defence enzymes → Recognise certain sequences
  • Sticky end - blunt end
• Enzyme cleavage
• Physical mapping

Question 13

Enzyme cleavage

Answer 13

• More exact size
• Smaller fragments → Handling
• Electrophoresis
• Restriction of fragment length polymorph
  
  • ID + taxonomy
  • Epidemiological investigations

Question 14

Physical mapping

Answer 14

• The localisation of cleavage sites
• Partial digestion with endonucleases

Question 15

Molecular cloning of viral DNA

Answer 15

• Propagation of virus DNA fragments in bacterial plasmids
• Mass DNA production: Quicker + cheaper
• Expression plasmids: Protein production
• Safe bacteria needed

Question 16

After cultivation of viral DNA in a bacterium…

Answer 16

• Plasmid isolation
• Cleavage
• Virus fragment purification

Question 17

Blotting

Answer 17

• Southern blot
• Nylon, hybond filter
• Easier to handle

Question 18

List the methods of NA investigation

Answer 18

• EM observation
• Electrophoresis
• Restriction endonuclease analysis
• Molecular cloning of viral DNA
• Blotting