Hybridization Conditions

• Degree of homology between the Probe and the Nucleic Acid.

  Stringency
• If the Stringency is high;

  More demanding in terms of complementarity.
• If the Stringency is low;

  Less demanding in terms of complementarity.
• If the Stringency is too high;

  No binding will occur
• If the stringency is too low;

  Nonspecific Binding
• Factors affecting Stringency

  Temperature

  Salt Concentration

  Denaturant Concentration

  Length and Nature of probe
• Stringency is HIGH when there is?

  High Temperature

  Low Salt Concentration

  High Denaturation Concentration
• Stringency is LOW when there is?

  Low Temperature

  High Salt Concentration

  Low Denaturant Concentration
• Formula for Temperature

  Tm=81.5 degrees celcius + 16.6 logM + 0.41 (%GC) - 0.61% (% formamide) - (600/n)
• It is expressed as Cot and is the length of unique (nonrepetitive) nucleotide sequences.

  Sequence Complexity
• Formula for Sequence Complexity


  Cot = [initial Dna] x time for reannealing
• Sequence Complexity: More complex

  = more time to reassociate
• Temperature 25 degrees celcius below Tm for 1-3 Cot 1/2 is optimal for dsDna probes.
• It is the limiting agent during hubridization in the incubation system.

  Probe
• Incumabtion System: Sensitivity Increases with?

  Probe Concentration
• Incubation System: Keep volume of hybridization solution?

  Low
• Incubation System: Recommended hybridization buffer volume ______ of the membrane surface are.

  10 mL/ 100 cm^2
• Incubation within a sealed bag in a water bath or capped glass cylinders in rotary ovens maintains optimal hybridization temperature.