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What is hybridization? Complementary base pairing of two single strands
of nucleic acid double strand product DNA/DNA RNA/RNA DNA/RNA
What holds the two strands together? Hydrogen bonds
between the base pairs
Hydrophobic interactions of stacked bases
van der Waals forces between stacked bases
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
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
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
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
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?
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
Factors affecting the strength of strand pairing
pH [OH- ], ~pH 12
enolic hydroxyl groups on bases ionize• keto-amino H-bonds disrupted
Keto Enol ionizes
G GG
Factors affecting the strength of strand pairing
pH [OH- ], ~pH 12
N3 on thymine and N1 on guanine lose their hydrogens and resulting negative charge is delocalized over the ring
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
Factors affecting the strength of strand pairing
And in our case . . . The presence of the alkaline phosphatase enzyme
causes some steric hindrance.
Combined effects of these factors can be expressed as equations for the Tm – points to be covered
What is Tm? Equation to estimate Tm for DNA oligonucleotides Equation to estimate Tm for polynucleotides
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 occurring. Changing the temperature does not change the Tm!
What is Tm? For complementary oligonucleotides (10 - 23 nt)
Temp at which 50% of complementary molecules exist as single strands
50%
5’ - - - - - - - - - - - - - 3’
3’ - - - - - - - - - - - - - 5’
50%5’ - - - - - - - - - - - - - 3’
3’ -
- - -
- - -
- - -
- - -
5’
What is Tm?
For complementary polynucleotides (>~25nt) Tm is the temp at which 50% of hydrogen bonds
within any one hybrid are broken
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? Difference in strength between G/C vs. A/T bonds? The conditions of the solution?
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
Tm for polynucleotides (cont’d)
How does the equation on the previous slide account for Length? Difference in strength between G/C vs. A/T
bonds? The conditions of the solution?
Membrane hybridization One nucleic acid component is affixed to
membrane; the other is in solution 14;18 translocation: samples affixed; probe(s) in solution
Membrane material binds DNA or RNA nylon charged nylon nitrocellulose
Typical steps in membrane hybridization
blocking or prehybridization hybridization wash or rinse Visualization
Blocking/prehybridization
Why? Remember, membrane binds nucleic acid, so
labeled nucleic acid in hybridization solution can bind everywhere on membrane background
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
Blocking/prehybridization common blocking agents
sodium dodecyl sulfate (SDS) nonfat dry milk bovine serum albumin Ficoll
(carbohydrate polymer)
polyvinylpyrollidone (PVP)
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 Labeled nucleic acid
• If necessary, denatured by – High temperature (95oC) or– Alkaline (high pH) conditions
Hybridization Conditions (cont’d)
Temp set below Tm to optimize rate of hybridization oligonucleotides: 15o below Tm polynucleotides: 15-35o below Tm
Wash/rinse
Why? To remove labeled probe/sample that is
in excess non-specifically bound bound with loose complementarity
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
Wash/rinseHow?
Be aware that wash conditions for oligonucleotide and polynucleotide hybridizations differ because: oligonucleotide hybrids are not in equilibrium
Once separation occurs, if hybridizing strand is not present in excess, hybridization is unlikely to recur. A dilution effect is sufficient.
polynucleotide hybrids are in equilibrium within the strand a temperature, low salt, or denaturant effect is necessary
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
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
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 that will disrupt hybrids