Hybridization is the process of single-stranded nucleic acids binding to another strand with an 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 is 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 a probe 1 hour
- Double-stranded probes require a 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 the renaturation rate is
- Maintain temperature for a 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 ionic strength
Base composition
Denaturing agents
Ionic strength
- Ionic strength affects denaturation and renaturation
- Provides a 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
- The base composition of the target also affects the hybridization
- The 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.
- A common denaturing agent used in buffers is formamide.
- 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
