Basics of hybridization

1. Basics of hybridization

2. What is hybridization? • Complementary base pairing of two single strands of nucleic acid  double strand product • DNA/DNA • RNA/RNA • DNA/RNA

3. What holds the two strands together? • Hydrogen bonds between the base pairs

4. What holds the two strands together? • Hydrophobic interactions of stacked bases • van der Waals forces between stacked bases

5. Factors affecting the strength of strand pairing • Number of GC pairs vs. AT pairs • Mismatch • Length of hybridizing strands • [Salt] of hybridization solution • Temperature • Concentrations of denaturants

6. Factors affecting the strength of strand pairing • Number of GC pairs vs. number of AT pairs • The more H-bonds between strands, the more strongly they are held together • 3 H-bonds between G and C • 2 H-bonds between A and T • So…the more GC pairs, the more H-bonds between strands

7. Factors affecting the strength of strand pairing • % Mismatch • the greater the lack of complementarity, • the fewer hydrogen bonds • the lower the strength of the hybrid

8. Factors affecting the strength of strand pairing • Length of hybridizing strands • the longer the strands, • the more hydrogen bonds and • the more hydrophobic interactions, so • the greater the strength of the hybrid

9. Factors affecting the strength of strand pairing • [salt] of solution •  [salt]  strength of the hybrid • negative charges of the phosphate moieties of the sugar-phosphate backbones repel each other • + ions from salts in solution act as counterions to reduce repulsion • Monovalent cations (Na+) • Divalent cations (1 mM Mg++ = 100 mM Na+) • Why does [Mg++] affect specificity of PCR priming?

11. Factors affecting the strength of strand pairing • Temperature • heat increases the kinetic energy of each of the two strands • sufficient heat makes kinetic energy > H-bond energy • strands separate

12. Factors affecting the strength of strand pairing • pH • [OH-], ~pH 12 • enolic hydroxyl groups on bases ionize • keto-amino H-bonds disrupted • Concentration of denaturants • formamide, urea

13. Combined effects of these factors can be expressed as equations for the Tm • What is Tm? • Equation to estimate Tm for DNA oligonucleotides • Equation to estimate Tm for polynucleotides

14. What is Tm? Tm = temperature of melting or separation of strands Tm is a function of the DNA fragment or RNA strand under consideration and the solution in which the hybridization is occuring. Changing the temperature does not change the Tm!

15. What is Tm? For complementary oligonucleotides (10 - 23 nt) Temp at which 50% of complementary molecules exist as single strands 50% 50% 5’ - - - - - - - - - - - - - 3’ 3’ - - - - - - - - - - - - - 5’ 5’ - - - - - - - - - - - - - 3’ 3’ - - - - - - - - - - - - - 5’

16. What is Tm? • For complementary polynucleotides (>~25nt) • Tm is the temp at which 50% of hydrogen bonds within any one hybrid are broken

17. Combined effects of factors contributing to strength of a hybrid can be expressed as equations for Tm • for DNA oligonucleotides in 1.0M Na+ Tm (oC) = 4 (G+C) + 2 (A+T) • Note: how does this equation account for • length? • % GC? • The conditions of the solution

18. Combined effects can be expressed as equations for Tm • for DNA polynucleotides and oligos as short as 14 nt Tm = 81.4 + 16.6 log [(M+)/1+0.7(M+)] + 0.41 (%G+C) - 600/L - %mismatch - 0.65 (% formamide) M+ = monovalent cation concentration L = length of probe sequence

19. Tm for polynucleotides (cont’d) • How does the equation on the previous slide account for • length? • % GC? • The conditions of the solution

20. Membrane hybridization • One nucleic acid component is affixed to membrane; the other is in solution • probe(s) affixed; sample in solution • HLA-DQalpha • samples affixed; probe(s) in solution • 14;18 translocation • Membrane material binds DNA or RNA • nylon • charged nylon • nitrocellulose

21. Steps in membrane hybridization • blocking or prehybridization • hybridization • wash or rinse • visualization

22. Blocking/prehybridization • Why? • Remember, membrane binds nucleic acid, so • labeled nucleic acid in hybridization solution can bind everywhere on membrane   background

23. Blocking/prehybridization • How? • Membrane with affixed nucleic acid is bathed in blocking solution at hybridization temperature • Components of blocking solution bind non-specifically to membrane to prevent labeled nucleic acid from binding except to complementary strands

24. Blocking/prehybridization • common blocking agents • sodium dodecyl sulfate (SDS) • nonfat dry milk • bovine serum albumin • Ficoll (carbohydrate polymer) • polyvinylpyrollidone (PVP)

25. Hybridization • What? • Labeled nucleic acid in solution is allowed to anneal to affixed complementary strands • Conditions • Must be determined empirically • Hybridization solution includes • [Salt] determined from Tm formulas • Membrane blocking agents • Denatured labeled nucleic acid; denatured by • High temperature (95oC) or • Alkaline (high pH) conditions

26. Hybridization • Conditions (cont’d) • Temp set below Tm to optimize rate of hybridization • oligonucleotides: 15o below Tm • polynucleotides: 15-35o below Tm

27. Wash/rinse • Why? • To remove labeled probe/sample that is • in excess • non-specifically bound • bound with loose complementarity

28. Wash/rinseHow? • Bathe membrane in solution lacking labeled probe/sample • Use stringency conditions that minimize non-specific hybridization • stringency = likelihood that two strands will separate • Be aware that wash conditions for oligonucleotide and polynucleotide hybridizations differ because: • oligonucleotide hybrids are not in equilibrium • polynucleotide hybrids are in equilibrium

29. Choosing wash conditions • To wash polynucleotide hybridizations (equilibrium) • raise stringency conditions to make it harder for imperfect hybrids to remain annealed • perform washes just below the Tm •  stringency  likelihood that two strands will separate • Lower the salt concentration • Raise the temperature • Include denaturants

30. Choosing wash conditions (cont’d) • To wash oligonucleotide hybridizations • Use stringency similar to or lower than hybridization condtions • Same or lower temperature • Same or higher salt concentrations • Short time periods In the HLA-DQ alpha assay, is the hybridization oligo or poly? How do the hybridization and wash conditions compare?

31. Visualization • requires a visible signal • radioactive • non-radioactive, enzyme linked • non-radioactive, non-enzymatic • e.g., use of fluorescent label • for enzyme-linked signal generation • additional block and rinse steps required • avoid conditions which will disrupt hybrids

32. How were conditions for HLA-DQ determined? • Use buffer recipes, probe sequences, and Tm equations to estimate Tm for one or more oligonucleotide probes for the • hybridization solution • wash solution • citrate buffer • Compare calculated Tms to temps used for each step. • How does the Tm change if there is a single nucleotide allelic difference?

34. How were conditions for HLA-DQ determined? (cont’d) • What do your answers tell you about the design of the protocol? • How do you think the intensity of color at the control probe is made to be less than the intensities of positive hybridization at shorter probes?