1. 4 DNA : it’s structure, replication, and related assays

3. The components of a nucleotide
Nitrogen containing base, including A, T, G, and C in DNA A, U, G, and C in RNA
Ribose (5 carbon atoms) deoxyribose in DNA ribose in RNA
Connect neucleotides

4. Ribose
1’ has base: A, T, G, C (in DNA)
A, U, G, C (in RNA)
2’ is H in DNA, OH in RNA
5’ has phosphate group to connect with 3’ of another nucleotide
Deoxyribose

5. Why DNA , instead of RNA, carries the genetic information
The 2’ OH in the ribose attacts the phosphodiester bond between nucleotide molecules and results the hydrolysis of RNA.
DNA has no OH on 2’ and is more stable.

7. Nucleotides are joined by linking the phosphate on the 5’ end of the deoxyribose with the 3’ end of next deoxyribose.
Imaging it as the floors of a department store.

8. Makes DNA negatively charged

9. Native DNA is a double helix of complementary antiparallel strands.
A base pair
Native DNA is a double helix of complementary antiparallel strands.
Anti-parallel: one strands goes from 3’ to 5’ and the other goes from 5’ to 3’

10. G C

11. Deoxyribose backbone
Deoxyribose backbone

12. Heating separates DNA into single strands
DNA melts or denatures (meaning separation of double strands) when temperature increases.

13. GC contents affect the DNA melting temperature (Tm)
As GC contents increases, Tm increases.

14. Annealing (hybidizing) of single stranded-DNA
Annealing: the rejoining of separated single strands of DNA to form double helix
Hybride DNA: artificial doubled stranded DNA formed by 2 single stranded DNA from different sources

15. DNA techniques To purify genomic DNA
to separate dna molecules by it’s size
to detect DNA molecules by labeling

16. Obtain genomic DNA 1 Lysis of cell
Use chemical (detergents..) or physical (ultrasonic..) methods 2
Centrifugation
To remove cell debris (large molecules) 3
Extraction
To remove proteins and other hydrophobic molecules 4
Remove RNA
Use ribonuclease to degrade single strand RNA

17. Obtain genomic DNA
3 extraction
4 remove RNA

18. Obtain genomic DNA
The interaction between DNA (negatively charged) and glass or other positively charged material is widely used to separate DNA from solution.

19. DNA purification kit (most convenient )

20. Measure the concentration of your DNA
DNA concentration (µg/ml) = (OD 260) x (dilution factor) x (50 µg DNA/ml)/(1 OD260 unit)
RNA concentration (µg/ml) = (OD 260) x (dilution factor) x (40 µg RNA/ml)/(1 OD260 unit)

21. Electrophoresis knows the size of your DNA?
DNA carries negative charge on their phosphate group, therefore, it moves toward the positive electrode.
Q: can we use free electrophoresis to separate DNA by its size?

22. Size does matter - - - - - - - - - - - -
Small DNA, less charge -
Mobility increases with charge number but decreases with molecule size - - - - - - -
Longer DNA, more charge
Q: can we use free electrophoresis to separate DNA by its size?
A: no, the effects of charge and size cancel each other

23. Gel Electrophoresis knows the size of your DNA

24. Gel Electrophoresis knows the size of your DNA
The DNA fragments are stained with ethidium bromide (EtBr)

25. Detection by radioactivity

27. Duplication of DNA prior to cell division
DNA replication
Duplication of DNA prior to cell division

28. Torsional stress in DNA can be relieved by enzymes
Many prokayrotic genomic DNAs and viral DNAs are circular molecules.
Circular DNAs also are present in mitochondria and chloroplast in eukaryotic cells.
We need to relax the DNA in order to carry out the replication.
DNA Gyrase

29. Enzymes needed to OPEN DNA strands29
DNA helicase
Requires more enzymes than gyrase and helicase

30. DNA helicase and strand binding protein
SSB prevents the annealing of complementary strands

31. Replication overview
III

33. DNA polymerase in action
III
Primer: a short piece of DNA sequence that facilitates the initiation of polymerization
Primer is made by primase.

34. Synthesis goes from 5’ to 3’
Both DNA and RNA synthesis proceed from 5’ to 3’ due to the molecular structure of nucleotide monomer.

35. Replication fork
: the new strand of DNA that is synthesized continuously
III
: fabricate primer
: the new strand of DNA that is synthesized in short pieces and then joined together later

36. Replication fork in action

37. Okazaki fragments: short pieces of DNA that make up the lagging strand
DNA polymerase I: enzyme that makes small stretches of DNA to fill gaps between Okazaki fragments
DNA ligase: an enzyme that joins DNA fragments end to end I

38. Semi-conservative replication

39. Semi-conservative replication: evidence

40. Division of circular bacteria chromosomes

41. Division of bacteria cells

42. Division of higher organism cells
Eukaryotic cells have chromosomes with complicated structure.
The replication of eukaryotic chromosomes initiate at multiple origins spaced along chromosomal DNA.

43. Polymerase Chain Reaction (PCR)
To amplify a specific region (usually known) of DNA sequence from a complex mixture.
PCR depends on the ability of reversible separation (melting) of double –stranded DNA.

44. Polymerase Chain Reaction (PCR)
The copy number of targeted DNA can be calculated by 2x
x is the cycle number.
In order to get accurate amplified sequence, the selection of primer is important.
Taq polymerase: most common polymerase used in PCR
dNTP: deoxynucleotide

45. Why we use hydrophobic surface when combing DNA?

46. Why we use hydrophobic surface when combing DNA?
DNA adsorbs onto this hydrophobic surface by one extremity in a “mushroom” state. This means that the adsorbed DNA has only one attachment point with the surface and retains its fluctuating coil conformation. The reason the DNA binds to the hydrophobic surface is still a matter of discussion. One explanation relies on pH-induced denaturation of the DNA ends, which then expose the hydrophobic part of bases and thus strongly interact with the surface (27)
Allemand J F, Bensimon D, Jullien L, Bensimon A,
Croquette V (1997) Biophys J 73:2064–2070