Chapter 6: Analysis and Characterization of Nucleic Acids and Proteins

1. Chapter 6: Analysis and Characterization of Nucleic Acids and Proteins Donna C. Sullivan, PhD Division of Infectious Diseases University of Mississippi Medical Center

2. Objectives • Describe how restriction enzyme sites are mapped on DNA. • Construct a restriction enzyme map of a DNA plasmid or fragment. • Diagram the Southern blot procedure. • Define hybridization, stringency, and melting temperature. • Calculate the melting temperature of a given sequence of dsDNA. • Describe comparative genomic hybridization (CGH).

3. Restriction Enzymes • Type I • Methylation/cleavage (3 subunits) • >1000 bp from binding site • e.g., Eco AI GAGNNNNNNNGTCA • Type II • Cleavage at specific recognition sites • Type III • Methylation/cleavage (2 subunits) • 24–26 bp from binding site • e.g., Hinf III CGAAT

4. Restriction Endonucleases: Type II

5. There are hundreds of restriction enzymes

6. Cohesive Ends (3´ Overhang) Cohesive Ends (5´ Overhang) Blunt Ends (No Overhang) BamH1 GGATCC CCTAGG KpnI GGTACC CCATGG HaeIII GGCC CCGG Restriction Enzymes

7. GGATCC CCTAGG BamHI (5’ Overhang) CTCGTG GAGCAG BssSI (5’ Overhang) NNCAGTGNN NNGTCACNN TspRI (3’ Overhang) AGATCT TCTAGA BglII (5’ Overhang) Enzymes Recognizing Non palindromic Sequences Enzymes Generating Compatible Cohesive Ends CCCGGG GGGCCC XmaI (5’ Overhang) GATC CTAG DpnI (Requires methylation) GGCC CCGG HaeIII (Inhibited by methylation) CCCGGG GGGCCC SmaI (Blunt Ends) Methylation-sensitive Enzymes Isoschizomers Restriction Enzymes

8. Sticky ends must match (be complementary) for optimal re-ligation. Blunt ends can be re-ligated with less efficiency than sticky ends. Sticky ends can be converted to blunt ends with nuclease or polymerase. Blunt ends can be converted to sticky ends by ligating to synthetic adaptors. Ligation of Restriction Enzyme Digested DNA

9. Cloning into Plasmid Vectors

10. Restriction Enzyme Mapping • Digest DNA with a restriction enzyme. • Resolve the fragments by gel electrophoresis. • The number of bands indicates the number of restriction sites. • The size of the bands indicates the distance between restriction sites.

11. XhoI BamH1 XhoI BamH1 4.3 kb 3.7 kb 2.3 kb 1.9 kb 1.4 kb 1.3 kb 0.7 kb 4.0 kb BamH1 2.8 kb 1.7 kb 2.8 kb 1.1 kb 1.2 kb 1.2 kb 1.7 kb 1.1 kb XhoI 1.2 kb XhoI Restriction Enzyme Mapping

12. Southern Blot • Developed by Edwin Southern. • The Southern blot procedure allows analysis of any specific gene or region without having to clone it from a complex background.

13. Denaturation of DNA: Breaking the Hydrogen Bonds

14. Denaturation and Annealing (Re-forming the Hydrogen Bonds)

15. Denaturation/Annealing: An Equilibrium Reaction

16. HYBRIDIZATION: Denaturation and Annealing of DNA

17. Basic Techniques for Analysisof Nucleic Acids • Enzymatic modification (polymerase, kinase, phosphatase, ligase) • Endonuclease digestion (DNAse, RNase, restriction enzymes) • Electrophoresis (agarose and polyacrylamide gel electrophoresis)

18. Molecular Search Tools: Blots • Southern blots • DNA immobilized on solid support • Northern blots • RNA immobilized on solid support • Western blots • Proteins immobilized on solid support

19. Southern Blot Hybridization • Transfer DNA from a gel matrix to a filter (nitrocellulose, nylon) • Fix DNA to filter (Heat under a vacuum, UV cross-link • Hybridize with single stranded radiolabeled probe

20. Southern Blot • Extract DNA from cells, etc • Cut with RE • Run on gel (usually agarose) • Denature DNA with alkali • Transfer to nylon (usually capillary action) • Autoradiograph

21. Blotting a Gel • Separate restriction enzyme-digested DNA by gel electrophoresis • Soak gel in strongly alkali solution (0.5 N NaOH) to melt double stranded DNA into single stranded form • Neutralize pH in a high salt concentration (3 M NaCl) to prevent re-hybridization

22. Blot to Solid Support • Originally used nitrocellulose paper, now use chemically modified nylon paper • Binds ssDNA strongly • Transferred out of gel by passive diffusion during fluid flow to dry paper toweling • Block excess binding sites with foreign DNA (salmon sperm DNA)

23. DNA Binding Media • Electrostatic and hydrophobic: • Nitrocellulose • Nylon • Reinforced nitrocellulose • Electrostatic • Nylon, nytran • Positively charged nylon

24. Transfer of DNA to Membrane

25. Dry paper Nitrocellulose membrane Gel Soaked paper Reservoir Capillary Transfer

26. Whatman paper - Nitrocellulose filter + Buffer Buffer Gel Glass plates Electrophoretic Transfer

27. Gel Recirculating buffer Nitrocellulose filter Vacuum Porous plate Vacuum Transfer

28. Southern Blot • Block with excess DNA (unrelated) • Hybridize with labeled DNA probe • Wash unbound probe (controls stringency)

29. Theprobe determines what region is seen. • DNA, RNA, or protein • Covalently attached signal molecule • radioactive (32P, 33P, 35S) • nonradioactive (digoxygenin, biotin, fluorescent) • Specific (complementary) to target gene

30. The Probe Determines What Region Is Seen • DNA, RNA, or protein • Covalently attached signal molecule • radioactive (32P, 33P, 35S) • nonradioactive (digoxygenin, biotin, fluorescent) • Specific (complementary) to target gene

31. Complementary Sequences • Complementary sequences are not identical. • Complementary strands are antiparallel. P5′ - GTAGCTCGCTGAT - 3′OH OH3′ - CATCGAGCGACTA -5′P

32. Southern Blot Hybridization: Overview

33. Types Of Nucleic Acid Probes • dsDNA probes • Must be denatured prior to use (boiling, 10 min) • Two competing reactions: hybridization to target, reassociation of probe to itself • ssDNA probes • RNA probe • Rarely used due to RNAses, small quantities • PCR generated probes • ss or ds, usually use asymmetric PCR

34. Detection Methods • Isotopic labels (3H, 32P, 35S, 125I) • Photographic exposure (X-ray film) • Quantification (scintillation counting, densitometry) • Non-isotopic labels (enzymes, lumiphores) • Enzymatic reactions (peroxidase, alkaline phosphatase) • Luminescence (Adamantyl Phosphate derivatives, “Lumi-Phos”)

35. Radioactive Labels • 32P: t1/2 = 14.3 days • High energy beta emitter • With good probe (106 cpm/ml), overnight signal • 33P: t1/2 = 25.4 days • Lower energy • 3-7 days for signal • 35S: t1/2 = 87.4 days • More diffuse signal • 3H: t1/2 = 12.4 years • Very weak • Got grand kids?

36. Radiolabeling Probes • Nick translation • DNase to create single strand gaps • DNA pol to repair gaps in presence of  32P ATP • Random primer • Denature probe to single stranded form • Add random 6 mers, 32P ATP, and DNA pol • 5’ End label • Remove 5’ Phosphate with Alkaline phosphatase • Transfer32P from  32P ATP with T4 polynucleotide kinase

37. Melting Temperature (Tm) • The temperature at which 50% of a nucleic acid is hybridized to its complementary strand. DS DS = SS SS Tm Increasing temperature

38. Melting Temperature and Hybridization • Your hybridization results are directly related to the number of degrees below the melting temperature (Tm) of DNA at which the experiment is performed. • For a aqueous solution of DNA (no salt) the formula for Tm is: • Tm = 69.3oC + 0.41(% G + C)oC

39. Tm in Solution is a Function of: • Length of DNA • GC content (%GC) • Salt concentration (M) • Formamide concentration Tm = 81.5°C + 16.6 logM + 0.41 (%G + C) - 0.61 (%formamide) - 600/n (DNA:DNA)

40. Denaturation: Melting Temperatures

41. G + C Content (as a %) • GC content has a direct effect on Tm. • The following examples, demonstrate the point. • Tm = 69.3oC + 0.41(45)oC = 87.5oC (for wheat germ) • Tm = 69.3oC + 0.41(40)oC = 85.7oC • Tm = 69.3oC + 0.41(60)oC = 93.9oC

42. Tm • For short (14–20 bp) oligomers: • Tm = 4° (GC) + 2° (AT)

43. Melting Temperature (Tm) andG + C Content

44. Formula Which That Takes The Salt Concentration Into Account • Hybridizations though are always performed with salt. • Under salt-containing hybridization conditions, the effective Tm is what controls the degeree of homology between the probe and the filter bound DNA is required for successful hybridization. • The formula for the Effective Tm (Eff Tm). • Eff Tm = 81.5 + 16.6(log M [Na+]) + 0.41(%G+C) - 0.72(% formamide)

45. Optimal Hybridization Times Optimal Hybridization Temperatures General Hybridization Times/ Temperatures ON=overnight

46. Hybridization Conditions • Three steps of hybridization reaction • Prehybridization to block non-specific binding • Hybridization under appropriate conditions • Post-hybridization to remove unbound probe • High Stringency for well matched hybrids • High temp (65o-68oC) or 42oC in presence of 50% formamide • Washing with low salt (0.1X SSC), high temp (25oC) • Low Stringency • Low temp, low formamide • Washing with high salt

47. Stringency • Stringency describes the conditions under which hybridization takes place. • Formamide concentration increases stringency. • Low salt increases stringency. • Heat increases stringency.

48. Hybridization Stringency • Closely related genes are not identical in sequence, but are similar • Conserved sequence relationship is indicator of functional importance • Use lower temperature hybridization to identify DNAs with limited sequence homology: reduced stringency

50. Stringency • Stringency describes the conditions under which hybridization takes place. • Formamide concentration increases stringency. • Low salt increases stringency. • Heat increases stringency.