1. Nucleic Acid Hybridization Nucleic acids Complementary bases Hybridization Complementary strands from any sources Reversible reaction DNA/DNA or DNA/RNA or RNA/RNA

2. Denaturation Denature: helix separation Hydrogen bonds broken / Strands unwind Double strands to Single strands

3. Denaturation

4. Heat ~ 100 C for a short period (completely denatured at 90 C) 97 C + salt Alkaline: pH > 11.3 (0.3 N NaOH)

5. Denaturation Organic solvent: Urea and formamide directly reacting with bases inhibit normal base pairing reversible reaction

6. Denaturation Organic solvent: formaldehyde irreversible denaturation form covalent bond with NH 2 group

7. Renaturation Renaturation / Hybridization / Reassociation Base-pairing reaction of complementary strands Slow cooling at 65 C Fast cooling (100 --> 0 C): stay separated

8. Renaturation Two steps of base-pairing reaction I Nucleation step Quite slow Random reaction of 2 strands collide by incidence Rate-limiting step Short stretches of H bonds

9. Renaturation Two steps of base-pairing reaction II Zippering / Annealing step Fast Extend base-pairing reaction over the whole strands

10. Hybridization AT vs CG regions

11. Hybridization Factors affecting hybridization Rate of reaction Criterion or helix stability

12. Hybridization Rate Factors affecting rate I Concentration of momovalent ion eg. sodium salt (Na+) higher Conc : higher Rate Conc higher than 0.4 M Hybrid destabilize

13. Hybridization Rate Factors affecting rate II Temperature Melting temperature Salt, GC content, Organics Maximum rate = Tm - 25

14. Temperature at which DNA is half folded Melting Temperature Melting curve at 260 nm

15. Melting Temperature Tm (AT-rich) < Tm (GC-rich)

16. Hybridization Rate Factors affecting rate III Fragment Length optimal length at 450 nt Too short: easy mismatch Too long: very slow rate

17. Hybridization Rate Factors affecting rate IV Organic solvent concentration Denaturing agent higher Conc : slightly lower Rate V Solvent Viscosity higher Viscosity : lower Rate

18. Hybridization Rate Factors affecting rate VI GC composition higher GC content : slightly higher Rate VII pH 5-9 no effect > 11 (13) denature

19. Criterion / Thermostability Factors affecting criterion I Temperature Incubation temperature or Ti lower Ti by 1 C : higher mismatch by 1% higher mismatch : lower Tm : lower criterion optimal Ti : Tm - 15

20. Criterion / Thermostability Factors affecting criterion II Concentration of monovalent ion higher salt : higher rate : lower criterion III Fragment length higher length : higher Tm : higher criterion

21. Criterion / Thermostability Factors affecting criterion IV Concentration of organic solvent higher conc : lower Tm : lower criterion V GC composition higher GC content : higher Tm : higher criterion

22. Hybridization Hybrid formation Considered rate and criterion Hybrid specificity Considered hybridization stringency

23. Stringency Conditions for hybridization Effect of degree of mismatch High stringency : best match Low stringency : some mismatch

24. Stringency

25. Factors High stringencyLow stringency Temperature Salt Organic solvent

26. Evaluation of degree of genetic similarity between organisms Evaluation of genome complexity Renaturation analysis

27. DNA with high amounts of satellite DNA Renature much faster When compared to DNA with Mainly single sequences Regardless of genome size Renaturation analysis

28. Multiple-copy sequence of Genome eg. repetitive sequence Easy nucleation step Quick hybridization Renaturation analysis

29. Complex genome High amounts of single sequences Long reaction period Renaturation analysis

30. Eukaryote: 4 DNA groups Foldback DNA Highly repetitive DNA Moderately repetitive DNA Unique / Single copy DNA

31. Hybridization reaction Fundamental tool in molecular study Hybridization partners ssProbe : known sequence and labeled ssTarget: related sequence under study Form ds if complementary (to hybridize)

32. Nucleic acid probe Sequence with known molecular identity Homologous probe: same source Heterologous probe: different source

33. Nucleic acid probe DNA:genomic DNA (by cloning or PCR) complementary DNA RNA: transcription of DNA inserted in plasmid Synthetic oligonucleotide: specific to target sequence sometimes as a set of degenerate probes

34. Nucleic acid probe

35. Probe labeling ds or ss nucleic acid probe to be labeled Working probe: single strands Labeled by incorporating: labeled dNTPs to new DNA strands labeled NTPs to new RNA strands 32 P (or others) to terminal nucleotides

36. Nick Translation Probe labeling

37. Random Primed Labeling Probe labeling

38. Kinase end labeling Probe labeling

39. Fill in labeling by Klenow Probe labeling

40. Riboprobe / RNA probe Probe labeling

41. Types of Label Isotopic label Commonly used: 32 P, 33 P, 35 S or 3 H Non-isotopic label Direct label: Fluorescene dye Indirect label: Digoxygenin Biotin-Strepavidin

42. Choices of Label Sensitivity Resolution Probe stability Safety Ease of Use

43. Radioactive Label TypeHalf Life Maximum Energy of Emission (MeV) 32 P14.3 d1.71 33 P25.5 d0.248 35 S87.4 d0.167 125 I 60 d0.035 3 H12.35 y0.018

44. Radioactive Label Radio-labeled nucleotide Autoradiographic detection Radiation intensity --> signal 32 P: Highly sensitive / Low resolution

45. Non-Radioactive Label Safe / Easy / High resolution / Low sensitivity Direct Label: Fluorescene dye / Fluorophore Indirect Label:Biotin-Strepavidin Digoxigenin Required conjugated marker

46. Non-Radioactive Label Detection Fluorescence Colorimetric assay Alkaline phosphatase + NBT + BCIP Chemiluminescence assay HRP + H 2 O 2 + luminol

47. Fluorophores

48. Indirect Label

50. Nucleic Acid Hybridization Identification of closely related molecules Probe: homogeneous population of identified molecules Target: heterogeneous population of nucleic acid

51. Nucleic Acid Hybridization Liquid / Solution hybridization slow reassociation of single copy in complex genome Solid / Filter hybridization immobilized target to increase reassociation rate Reverse hybridization: unlabeled immobilized probe In situ hybridization: target in tissue

52. Nucleic Acid Hybridization Denaturation of double strands: by heating by alkaline treatment Annealing of complementary strands Formation of Homo or Heteroduplex

53. Nucleic Acid Hybridization

54. Nucleic acid stability Strand length: negligible if exceed 500 bp Base composition: GC / AT content Chemical environment: monovalent cation formamide or urea Factors on energy required for strand separation

55. Melting Temperature Tm as a measure for duplex stability Hybridization at Ti lower than Tm to promote heteroduplex formation

56. Calculation of Tm Hybrids T m (°C) DNA-DNA 81.5 + 16.6 (log 10 [Na + ] a ) + 0.41 (%GC b ) - 500/L c DNA-RNA or 79.8 + 18.5 (log 10 [Na + ] a ) + 0.58 (%GC b ) RNA-RNA + 11.8 (%GC b ) 2 - 820/L c oligo-DNA or For <20 nucleotides: 2 (l n ) oligo-RNA d For 20-35 nucleotides: 22 + 1.46 (l n )

57. Blotting Transfer of Nucleic acid onto solid support Membrane filter: Nylon / Nitrocellulose By capillary force, vacuum or electroblot

58. Blotting

59. Hybridization Southern: electrophoresed DNA Northern: electrophoresed RNA Dot blot: unfractionated target Slot blot: big volume / unfractionated target Colony: bacterial genome Plaque: virus genome

60. Southern/Northern Hybridization

61. Dot Blot Hybridization

62. Slot Blot Hybridization

63. Colony Hybridization

64. DNA Microarray Large-scale gene screening / expression analysis Whole genome study on single pass Hybridization of high-density DNA array Robotic spotting of DNA clones or oligonucleotides

65. Microarray VS Northern

66. Microarray / DNA chip