1. 710.LC GRADUATE MOLECULAR BIOLOGY 9/15/2010

2. Lecture 4 Competency Test.

4. Name the five components of a PCR reaction.
Template
Buffer
Primers (two of them)
Taq Polymerase
dNTPs

5. The PCR Reaction How does it work?
Heat (94oC) to denature DNA strands
Cool (52oC) to anneal primers to template
Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA
Repeat 35 cycles

6. Denaturing Template DNA
Heat causes DNA
strands to separate 3’ 5’ 3’ 5’ 5’ 3’
Denaturation of DNA at 94oC
In our cells, enzymes (helicases) accomplish the ‘denaturation’ step. 3’ 5’

7. Annealing Primers Primers anneal at 52oC Primers bind to the template
Taq polymerase recognizes 3’ end of primer + template strand 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’
Taq extends at 72oC 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’

8. The exact-length target product is made in the third cycle
Taq polymerase extends…..
Cycle 1
DNA is replicated
Cycle 2
Repeat denaturing, annealing, and extending 35 cycles
Cycle 3
The exact-length target product is made in the third cycle

12. 2) Name two ways to synthesize a gene.
Recombinant PCR
Also: Polymerase cycle assembly
2) Assembly PCR

13. Polymerase cycle assembly

14. Assembly PCR

15. What is Nested PCR?

16. 3) What is the purpose of codon optimizing genes?
To maximize the translation
to the host tRNA population

17. You must know single letter codes
What does
Degree of
Degeneracy
Reflect?

19. eGFP (eucaryotic vs for bacterial expression)
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*
eGFP (eucaryotic vs for bacterial expression)

21. 4) What are the 3 common components of plasmids used in DNA cloning?
Origin [OriC] of replication
Selectable marker [I.e. Kan
Resistance Gene/Amp Resistance
Gene
3) Multiple Cloning Site [MCS]

22. 5) What is the difference between an oligonucleotide and a primer?
Nothing. It is the usage which
differs. A primer is always used
with a polymerase. An oligo
is simply a chain of nucleotides

23. 6) Are oligonucleotides and primers single stranded?
Yes. We use them to anneal to
other single stranded templates.

24. 7) Do oligonucleotides and primers have to be DNA?
No. They can be RNA.
Why do we use RNA sometimes:
Because annealing RNA to DNA
Make very stronger hybrids.

25. 8) Name 4 parameters that affect annealing of two single stranded DNA chains?
Temperature
Salt concentration
DNA concentration
Length of complementarity
Time of re-annealing

26. 9) What does DNA ligase do?
DNA ligase catalyzes the
Phosphodiester bond formation
between two nucleotides.
ATP is used in the reaction to donate
a phosphate.

27. DNA Ligase Covalently Closes Nicks in DNA

28. DNA ligase forms a high energy intermediate that

31. Calf Intestinal Phosphotase?
Aside:
Calf Intestinal Phosphotase?
Cut with EcoR1
GAATTC
CTTAAG
G-OH p-AATTC
CTTAA-p HO-G

32. Calf Intestinal Phosphotase?
Cut with EcoR1
G-OH p-AATTC
CTTAA-p HO-G
G-OH HO-AATTC
CTTAA-OH HO-G

33. Calf Intestinal Phosphotase?
Cut with EcoR1
p-AATTCgatacagagagactcatgacgG-OH
HO-GctatgtctctctgagtactgcCTTAA-p
G-OH HO-AATTC
CTTAA-OH HO-G
Vector won’t religate,
But will take in insert

36. 10) What does a Kinase do?

37. 11) What are Restriction Enzymes?

38. 12) Given one 4-cutter restriction enzyme, how many times might it cut a 1000bp dsDNA molecule?

39. 13) Given one 6-cutter restriction enzyme, how many times might it cut a 1000bp dsDNA molecule?

40. 14) What is the most common type of DNA sequencing?

41. 15) What is NextGen Sequencing?

42. 16) What is a transcriptome?

43. 17) Name two ways to make mutations in plasmid.

44. 18) What is the yeast two-hybrid system?

45. 19) What is the one-hybrid system?

46. 20) Name the protein that binds to the TetO sequence?

47. 21) What is GFP?

48. 22) What is Cherry (protein)?

49. 23) What is Venus (protein) ?

50. 24) What is Cerulean (protein) ?

51. 25) What are molecular beacons?

52. 26) Define I.R.E.S.

53. 27) What does the T2A fragment do?

54. 28) What is Translational Frameshifting?

55. 29) What are CRE and Flp?

56. 30) Cre works with LoxP or FRT? Flp works with LoxP or FRT?

57. 31) What is a Bacterial artificial chromosome?

58. 32) How are Transgenic animals made (in general)? Be brief and specific.

59. 33) What are Zn finger nucleases and what are they good for?

60. 34) What do Double Strand DNA breaks promote?

61. 35) What are ES cells?

62. 36) How were mouse ES cells derived?

63. 37) Why do we use ES cells to make Gene-targeted mice?

64. 38) What is meant by Structure/Function analysis?