6 - General characteristics and purification of viral nucleic acid. Methods of nucleic acid investigation I. Flashcards
General characteristics and purification of viral nucleic acid. Methods of nucleic acid investigation I. (size, morphological characteristics, restriction enzyme analysis, bacterial cloning, Southern blot)
1
Q
Characteristics of viral nucleic acid
A
- Virion: core
- Carries genetic information, and determines the viral properties
- Relatively small
- Easy to handle → early genetic investigation
- dsDNA, ssDNA, dsRNA, ssRNA
- Linear or circular
- Continuousorsegmented
- Usually haploid
- May contain alien NA
- Polymaviruses, pestiviruses: host cell nucleic acid
- Arenaviruses: ribosomes
- Retroviruses: onc, src genes
- NA deficiency: incomplete or defective particles
2
Q
Nucleic acid purification
A
- From purified virus suspension ➔ NA purification
- 2 methods:
- Proteinase K enzyme digestion ➔ pure NA
-
Protein lysis + chromatography ➔ pure NA
- Faster, simpler, less dangerous
3
Q
Investigation of viral nucleic acid
A
- Morphology + biochemical structure studied initially
- Later manipulated with genetic engineering
4
Q
Give the methods of nucleic acid investigation
Riktig??
A
- Digestion whit nucleases
- Electrophoresis
- Restriction endonuclease analysis
- Molecular cloning of viral DNA
- Blotting - southern blot
5
Q
Digestion with nucleases
A
- DNA or RNA virus
- 5’===========→ AAA 3’
- “+” sense
- E.g. Picorna virus – mRNA
- 5’←=========== 3’ =
- “-“sense
- E.g. Orthomyxo virus
- For transcrpition viral enzyme is needed
- If purified RNA injected into cell → is there virus production?
- Shows up as linear or circular under electron microscopes
6
Q
Electrophoresis
A
- Agarose gel electrophoresis or polyacrylamide gel electrophoresis
- Size: varies greatly but more size ➔ more information (information is proportional with size)
- Continous NA thread (eg. picornaviridae)
- Segmented genome (eg. retroviridae)
7
Q
Restriction endonuclease analysis
A
- dsDNA
- Endonucleases:
- Enzymes that defend bacteria
- Recognize key sequences, have sticky and blunt ends
- Enzyme cleavage:
- To get a more exact size
- Smaller fragments → easier to handle
- Sorted by Agarose-gel electrophoresis –
- Gives identification + taxonomy
- Physical mapping:
- Locates cleavage sites
8
Q
Molecular cloning of viral DNA
A
- Propagation of virus DNA fragments in bacterial plasmids (they merge!)
- Allows plasmid isolation, cleavage and virus fragment purification
- Mass DNA production: quicker and cheaper
- Safe bacteria needed
9
Q
Blotting: southern blot
A
- Nylon, hybond fliter
- Easier to handle (eg. for NA hybridization)
