basics of hybridization

Basics of hybridization

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

What holds the two strands together? 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


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


% Mismatch the greater the lack of complementarity,

the fewer hydrogen bonds the lower the strength of the hybrid


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?


Temperature heat increases the kinetic energy of each of the two

strands sufficient heat makes kinetic energy > H-bond energy strands separate


pH [OH- ], ~pH 12

enolic hydroxyl groups on bases ionize keto-amino H-bonds disrupted

Concentration of denaturants formamide, urea

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

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!

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? % GC? 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? % GC? The conditions of the solution

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

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 Denatured labeled nucleic acid; 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

Be aware that wash conditions for oligonucleotide and polynucleotide hybridizations differ because: oligonucleotide hybrids are not in equilibrium polynucleotide hybrids are in equilibrium

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

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?

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

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?

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?

basics of hybridization

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