DNA complimentary hybridisation Flashcards
What is DNA denaturing?
Conversion of double stranded DNA molecules into single stranded molecules.
How is DNA denatured?
Disruption of Hydrogen bonds within the double helix.
- Occurs when DNA in solution is heated
- Can also be induced by strong alkali or urea
How is the temperature of denaturation measured?
By absorbance at 260nm:
Done by Hyperchromicity: Increased absorption of light at 260nm on denaturation.
Point at which 50% of all strands separate is called the melting temperature or Tm
What does the Tm mostly depend on?
Hydrogen bonds:
- GC content
- Length of DNA molecule
- Salt concentration
- pH (alkali is a denaturant)
- Mismatches (unmatched base pairs)
What is the relationship between Tm and the GC content?
Higher GC content (%) = more hydrogen bonds = higher Tm.
3 H-bonds in GC vs 2 in AT
What is the relationship between Tm and the molecule length?
- Longer the contiguous duplex, the higher Tm
- More Hydrogen bonds within the molecule greater stability
- However little further contribution beyond 300 bp
What is the relationship between Tm and salt cncentration?
Salt stabilises DNA duplexes
High [Na+] = High Tm
increasing the salt concentration stabilises the structure increases the Tm and thus overcomes the destabilising effect of mismatched base pairing
High salt reduces the specificity of base pairing at a given temperature
What is the relationship between Tm and pH?
Chemical denaturants disrupt hydrogen bonds
Alkali, formamide, urea
OH- disrupts H bond pairing
Fewer hydrogen bonds = Lower Tm
High pH (alkalinity) destabilises DNA duplexes
What is the relationship between Tm and mismatches?
-A mismatch is defined as a base pair combination that is unable to form hydrogen bonds
- Reduces Number of Hydrogen bonds, Fewer = lower Tm
- Shorter contiguous stretches of double stranded sequence = lower Tm
- Mismatches also distorts the structure and destabilises adjacent base pairing
How is denaturation reversed?
By renaturisation:
Formation of structure favours energy minimisation driven by change in free energy DG
Facilitated by:
Slow Cooling
Neutralisation
Define hybridisation.
Formation of duplex structure of 2 DNA molecules that have been introduced to one another.
How is a DNA molecule be formed with no mismatches?
Perfect matches have a higher Tm and are thermodynamically favoured over mismatches.
Define stringency.
Manipulating conditions: Limiting hybridisation between imperfectly matched sequences increases specificity.
Under High stringency: only complementary sequences are stable determined by a Temperature near Tm or Low salt concentration
What are nucleic acid hybridisation techniques used for?
- Identifies the presence of NA containing a specific sequence of bases
- Allows the absolute or relative quantitation of these sequences in a mixture
What is a probe?
A ssDNA (or RNA) molecule
Typically 20 – 1000 bases in length
Labelled with a fluorescent or luminescent molecule (less commonly a radioactive isotope)
In some techniques thousands or millions of probes are used simultaneously
