1. Deoxyribonucleic Acid (DNA)

3. The double helix

4. Nitrogenous Bases and Pentose Sugars

5. Purine and Pyrimidine Structure (1) Pyrimidines are planar (2) Purines are nearly planar (3) Numbering is different

6. Numbering Is Different

8. Bases Have Tautomeric Forms Uracil

10. Nucleosides vs. Nucleotides Glycosidic bond

11. Nucleotides formed by condensation reactions

13. Monophosphates

14. Deoxyribonucleotides

15. Ribonucleotides

16. Only RNA Is Hydrolyzed by Base

17. Nucleoside Diphosphate and Triphosphate

18. Dinucleotides and Polynucleotides Ester bonds

19. Watson-Crick Base Pairs A=T G=C

20. Hoogsteen Base Pairs

21. Other Base Pairs Are Possible Homo PurinesHetero Purines Watson-Crick, Reverse Watson-Crick, Hoogsteen, Reverse Hoogsteen, Wobble, Reverse Wobble

22. Base Pairing Can Result in Alternative DNA Structures Triplex Tetraplex Hairpin Loop Cruciform

23. Periodicity: A pair of strong vertical arcs (C & N atoms) indicate a very regular periodicity of 3.4 Å along the axis of the DNA fiber. Astbury suggested that bases were stacked on top of each other "like a pile of pennies". Helical nature: Cross pattern of electron density indicates DNA helix and angles show how tightly it is wound. Diameter: lateral scattering from electron dense P & O atoms.

24. DNase can only cleave external bond demonstrating periodicity

25. Watson and Crick Model (1953) 2 long polynucleotide chains coiled around a central axis Bases are 3.4 Å (0.34 nm) apart on inside of helix Bases flat & lie perpendicular to the axis Complete turn = 34 Å 10 bases/turn Diameter = 20 Å Alternating major and minor grooves Hydrophobic Hydrophilic Complementarity

26. Base Pairing Results from H-Bonds Only A=T and G  C yield 20 Å Diameter

27. A:C base pair incompatibility

28. Bases Are Flat

29. Chains Are Antiparallel…

30. Base Pairs and Groove Formation

31. Base flipping can occur

32. Helix Is Right-Handed

34. Biologically Significant Form = B-DNA Low Salt = Hydrated, 10.5 bp/turn

35. A- DNA Exists Under High Salt Conditions Side-viewTop-view Base pairs tilted, 23 Å, 11bp/turn

36. Z-DNA Is a Left-Handed Helix Zig-zag conformation, 18 Å, 12 bp/turn, no major groove

38. Propeller Twist Results from Bond Rotation

40. Reassociation Kinetics

41. Denaturation of DNA Strands and the Hyperchromic Shift Denaturation (melting) is the breaking of H, but not covalent, bonds in DNA double helix  duplex unwinds  strands separate Viscosity decreases and bouyant density increases Hyperchromic shift – uv absorption increases with denaturation of duplex Basis for melting curves because G-C pairs have three H bonds but A-T pairs have only two H bonds Duplexes with high G-C content have a higher melting temperature because G-C pairs require a higher temperature for denaturation

43. Molecular Hybridization Reassociation of denatured strands Occurs because of complementary base pairing Can form RNA-DNA Hybrids Can detect sequence homology between species Basis for in situ hybridization, Southern and Northern blotting, and PCR

44. Hybridization

45. Reassociation Kinetics Derive information about the complexity of a genome To study reassociation, genome must first be fragmented (e.g. by shear forces) Next, DNA is heat-denatured Finally, temperature is slowly lowered and rate of strand reassociation (hybridization) is monitored

46. Initially there is a mixture of unique DNA sequence fragments so hybridization occurs slowly. As this pool shrinks, hybridization occurs more quickly C 0 t 1/2 = half-reaction time or the point where one half of the DNA is present as ds fragments and half is present as ss fragments If all pairs of ssDNA hybrids contain unique sequences and all are about the same size, C 0 t 1/2 is directly proportional to the complexity of the DNA Complexity = X represents the length in nucleotide pairs of all unique DNA fragments laid end to end Assuming that the DNA represents the entire genome and all sequences are different from each other, then X = the size of the haploid genome

47. The T m

48. The Hyperchromic Shift (Melting Curve Profile) T m = temperature at which 50% of DNA is denatured Maximum denaturation = 100% single stranded Double stranded 50% double, 50% single stranded

49. High G-C Content Results in a Genome of Greater Bouyant Density

50. Ideal C 0 t Curve 100% ssDNA 100% dsDNA

51. Larger genomes take longer to reassociate because there are more DNA fragments to hybridize Largest genome Smallest genome

52. C 0 t 1/2 Is Directly Proportional to Genome Size

53. Genomes are composed of unique, moderately repetitive and highly repetitive sequences Highly repetitive DNA Moderately repetitive DNA 10 -4 10 -2 10 0 10 2 10 4 Fraction remaining single-stranded (C/C 0 ) Unique DNA sequences 0 100 C 0 t (moles x sec/L)

54. More complex genomes contain more classes of DNA sequences

55. G-C Content Increases T m