2. Welcome to Molecular Biology Through Discovery Thursday, 1 November 2012 RNA

4. This is a surprising result, as the use of primers to initiate replication is an essential requirement.

5. Polymerase Chain Reaction
Denaturation 95°C
Extension 72°C
Annealing 55°C
Primers
Image courtesy of BioRad

6. DNA Replication
Since the parvovirus is single stranded it is unlikely that it can replicate an identical molecule from a different strand of DNA.
Figure 1 from notes on DNA replication. (image from Teresa Fischer, Indian Rivers State College)

7. DNA Replication
Since the parvovirus is single stranded it is unlikely that it can replicate an identical molecule from a different strand of DNA.

10. What went in?

11. What are the precursors?
DNA Replication
What are the precursors?

12. What are the precursors?
DNA Replication
What are the precursors?
dCTP

13. What went in?
Where to find out?

14. DNA

15. DNA
+ (2) RNAse

16. DNA
+ (2) RNAse
or (3) DNAse

17. DNA
+ (2) RNAse
+ DNA Pol I
or (3) DNAse

18. DNA
+ (2) RNAse
+ DNA Pol I
or (3) DNAse
dGTP dTTP dCTP
dATP

19. What are the precursors?
DNA Replication
What are the precursors?

20. What are the precursors?
DNA Replication
What are the precursors?

21. DNA + (2) RNAse + DNA Pol I + (4) RNAse or (3) DNAse or (5) DNAse
GTP TTP CTP
or (5) DNAse
or (4) nothing
or (5) nothing
ATP

22. DNA + (2) RNAse + DNA Pol I + (4) RNAse or (3) DNAse or (5) DNAse
GTP TTP CTP
or (5) DNAse
or (4) nothing
or (5) nothing
ATP

23. Electrophoresis of DNA through an agarose gel
Magnified view of agarose gel

24. Electrophoresis of DNA through an agarose gel (Gel electrophoresis)
This is like…

25. Electrophoresis of DNA through an agarose gel
(Air graviphoresis)
This is like…
Galileo
Pisa

26. Electrophoresis of DNA through an agarose gel
(Air graviphoresis)
Galileo
Pisa

27. Electrophoresis of DNA through an agarose gel (Gel electrophoresis)
Magnified view of agarose gel

28. Electrophoresis of DNA through an agarose gel (Gel electrophoresis)

29. Electrophoresis of DNA through an agarose gel (Gel electrophoresis)
Standard
???
Calibration
2000
1000
500

30. DNA + (2) RNAse + DNA Pol I + (4) RNAse or (3) DNAse or (5) DNAse
GTP TTP CTP
or (5) DNAse
or (4) nothing
or (5) nothing
ATP

31. Autoradiography
Steve Berg, Winona State University

32. DNA + (2) RNAse + DNA Pol I + (4) RNAse or (3) DNAse or (5) DNAse
GTP TTP CTP
or (5) DNAse
or (4) nothing
or (5) nothing
ATP

33. DNA + (2) RNAse + DNA Pol I + (4) RNAse or (3) DNAse or (5) DNAse
GTP TTP CTP
or (5) DNAse
or (4) nothing
or (5) nothing
ATP

35. Means
DNA isolated from virus
Incubated in vitro w/DNA polymerase + nucleotide (radioactive)
DNA separated by gel electrophoresis to measure size
DNA detected by autoradiography
(Variants) +/- RNAse, +/- DNAse, before and after

36. Result
Radioactive DNA observed near 5200 nt standard
Not observed if +DNAse before or after DNA polymerase
+RNAse doesn't affect result

37. Conclusion
Radioactive band  DNA replication
No primer added  Exogenous primer not needed
No DNA primase added  No primer needed??? or primer from unusual source
(Viral genome is DNA, not RNA)

38. In their experiment, they found out that the virions were disrupted with SDS and proteinase K treatment. The nucleic acid was extracted with phenol, precipitated by ethanol and dissolved in HCL. They incubated the parvovirus’s DNA with a DNA polymerase. Primarily they tested whether ABU virus is a parvovirus by determining the ability of a virion nucleic acid to serve as a primer-template for in vitro polymerization.

39. Don't quote, don't paraphrase
In their experiment, they found out that the virions were disrupted with SDS and proteinase K treatment. The nucleic acid was extracted with phenol, precipitated by ethanol and dissolved in HCL. They incubated the parvovirus’s DNA with a DNA polymerase. Primarily they tested whether ABU virus is a parvovirus by determining the ability of a virion nucleic acid to serve as a primer-template for in vitro polymerization.

40. No experimental basis (or Introduction)
After proving this the new chicken is a part of the parvoviruous family, it was easy to see the parvovirus like characteristic in the DNA of the ABU. There were parvovirus-like particles found in the both the nucleus and the cytoplasm of the effected cells. By knowing some of the characteristics of parvovirus-like particles proves the characteristic of self-replicating fowl parvovirus. From the experiment it was seen, the genome they extracted already has a single stranded DNA molecule that serves as a primer. The particles contained single-stranded DNA of one polarity with a natural primer (that was already present) at the 3'-end of the molecules, it shows it uses its own ends as primers.

41. No experimental basis and irrelevant
After proving this the new chicken is a part of the parvoviruous family, it was easy to see the parvovirus like characteristic in the DNA of the ABU. There were parvovirus-like particles found in the both the nucleus and the cytoplasm of the effected cells. By knowing some of the characteristics of parvovirus-like particles proves the characteristic of self-replicating fowl parvovirus. From the experiment it was seen, the genome they extracted already has a single stranded DNA molecule that serves as a primer. The particles contained single-stranded DNA of one polarity with a natural primer (that was already present) at the 3'-end of the molecules, it shows it uses its own ends as primers.

42. No experimental basis
After proving this the new chicken is a part of the parvoviruous family, it was easy to see the parvovirus like characteristic in the DNA of the ABU. There were parvovirus-like particles found in the both the nucleus and the cytoplasm of the effected cells. By knowing some of the characteristics of parvovirus-like particles proves the characteristic of self-replicating fowl parvovirus. From the experiment it was seen, the genome they extracted already has a single stranded DNA molecule that serves as a primer. The particles contained single-stranded DNA of one polarity with a natural primer (that was already present) at the 3'-end of the molecules, it shows it uses its own ends as primers.

43. No experimental basis
After proving this the new chicken is a part of the parvoviruous family, it was easy to see the parvovirus like characteristic in the DNA of the ABU. There were parvovirus-like particles found in the both the nucleus and the cytoplasm of the effected cells. By knowing some of the characteristics of parvovirus-like particles proves the characteristic of self-replicating fowl parvovirus. From the experiment it was seen, the genome they extracted already has a single stranded DNA molecule that serves as a primer. The particles contained single-stranded DNA of one polarity with a natural primer (that was already present) at the 3'-end of the molecules, it shows it uses its own ends as primers.

44. No experimental basis
After proving this the new chicken is a part of the parvoviruous family, it was easy to see the parvovirus like characteristic in the DNA of the ABU. There were parvovirus-like particles found in the both the nucleus and the cytoplasm of the effected cells. By knowing some of the characteristics of parvovirus-like particles proves the characteristic of self-replicating fowl parvovirus. From the experiment it was seen, the genome they extracted already has a single stranded DNA molecule that serves as a primer. The particles contained single-stranded DNA of one polarity with a natural primer (that was already present) at the 3'-end of the molecules, it shows it uses its own ends as primers.

45. After proving this the new chicken is a part of the parvoviruous family, it was easy to see the parvovirus like characteristic in the DNA of the ABU. There were parvovirus-like particles found in the both the nucleus and the cytoplasm of the effected cells. By knowing some of the characteristics of parvovirus-like particles proves the characteristic of self-replicating fowl parvovirus. From the experiment it was seen, the genome they extracted already has a single stranded DNA molecule that serves as a primer. The particles contained single-stranded DNA of one polarity with a natural primer (that was already present) at the 3'-end of the molecules, it shows it uses its own ends as primers.

46. It appears that the chicken parvovirus described by Kisary et al
It appears that the chicken parvovirus described by Kisary et al. does not require an exogenous primer. This appears to be evidenced by a previous experiment by Bourguignon et al. and is also verified here in that in Figure 2 it is shown that the single stranded DNA did in fact replicate in to a double strand.

47. The single stranded DNA became double stranded with the addition of DNA polymerase, which was shown by the results of electrophoresis bands being equal in length (about 5200 nucleotides).

48. In lanes 4 and 5, the nucleic acid labeled in vitro was resistant to RNase treatment but was completely digested with DNase. This result helps to explain that this particular virus had DNA genetic material and this virus used DNase to replicate inside of the infected cells.

49. Welcome to Molecular Biology Through Discovery Thursday, 1 November 2012 RNA

50. Welcome to Molecular Biology Through Discovery Thursday, 1 November 2012 RNA

51. Belozersky & Spirin (1958)
I could go for a review of reading the table to make sure I actually understood it properly.

52. Belozersky & Spirin (1958)
Belozersky AN, Spirin AS (1958). Nature 182:

53. Welcome to Molecular Biology Through Discovery Thursday, 1 November 2012 RNA

56. 1. Problem Set 8 #7 (frame shifts) 2. Problem Set 8 #8 (triple mutant)
Kaleigh
Jonathan
Michael
Colleen
Abdul Cailin
Sue
Kristen
Bobby
Supriya
Tayab
1. Problem Set 8 #7 (frame shifts)
2. Problem Set 8 #8 (triple mutant)
3. Problem Set 8 #6 (alien world)
Shahroze
Mandi
Shaun
Abdallah
Celeste
Neda
Yordanos Me