Science

Kinetics of molecular Hybridization

Hybridization

Hybridization is the process of single stranded nucleic acids binding to another strand with identically complement sequence

Denaturation and Renaturation

  • Heating double-stranded DNA
  • can overcome the hydrogen bonds holding it together
  • and cause the strands to separate resulting in denaturation of the DNA
  • When cooled relatively weak hydrogen bonds between bases can reform and the DNA renatures
  • DNA with a high GC content has more hydrogen bonds
  • GC base pair 3 hydrogen bonds
  • AT base pairs only 2 bonds
  • Higher GC content is reflected in denaturation temperature

Factor affecting Hybridization rate

  • Temperature
  • Concentration of probe
  • Time of incubation
  • Length of target DNA
  • GC content
  • Mismatching

Hybridization temperature

  • For Non denaturing buffer: 50-65 for DNA applications, 55-70 for RNA applications
  • For Denaturing buffer: More common 37-45, Lowest temperature 30 degrees

Hybridization buffer

Classified into two types:

  • denaturing buffers: Example: formamide buffers preferred if probe are known to be less stable at elevated temperatures
  • salt/detergent based buffers: Example: sodium phosphate Buffer Stability may be increased by adding salt decreased by lowering the amount of sodium chloride

Probe concentration

  • The hybridization rate increases with probe concentration
  • Also sensitivity increases with increasing probe concentration.

Time of hybridization

  • Standard buffers 6 and 24 hours.
  • higher concentrations of probe 1 hour
  • Double-stranded probes require longer time

Solution vs. Solid Phase Assays

  • drastically impact the assay
  • PCR requires less than a minute for annealing.
  • Southern blot requires up to 16 hours or longer for hybridization to occur

Length of target DNA sequences

  • The bigger the length higher renaturation rate is
  • Maintain temperature for long duration
  • Less target size increases hybridization efficiency and specificity

Mismatching

  • The Tm of a duplex DNA decreases by 1 °C with every increase of 1% in the mismatched base pairs.

Hybrid stability

  • RNA–RNA hybrids are more stable than RNA–DNA hybrids,
  • Which are, in turn, more stable than DNA–DNA ones.
  • This results in a difference in Tm of approx. 10°C between RNA-RNA and DNA-DNA hybrids.

Factors affecting hybrid stability

The melting temperature of duplex DNA (Tm) is a measure of its thermal stability.

It is dependent on iionic strength

Base composition

Denaturing agents

        Ionic strength

  • Ionic strength affects denaturation and renaturation
  • Provides stable environment for the proper activity of enzymes
  • At high concentrations of salt mismatched hybrids are more stable
  • At high concentrations of salt mismatched hybrids are more stable

   Base composition

  • Base composition of target also affects the hybridization
  • Higher the GC content of the target sequence higher the tm required
  • Higher the AT content of target sequence lower the tm required
  • GC content should be 40 to 60%.

    Denaturing agent

  • It also affects the hybridization
  • Greater the denaturing agent lesser the hybrid stability.
  • Common denaturing agent used in buffers is formamaide.
  • Its concentration depends upon the target length

                                                Stringency  

Generally, hybridization is discussed in terms of stringency and not the T m

  • Stringency is a condition of hybridization
  • It is a relative term that is related to the Tm
  • Reflects the homology between the probe and the target

 

 

 

 

 

Anu

Aneesa Yasmeen is a Content writer who always focuses on exploring better content strategies. She is a passionate blogger and enjoys writing on multiple niches.

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