DNA hybridisation: DNA Complementarity and its application Flashcards
What do nucleotides make up?
Make up DNA and RNA
Components of a nucleotide?
Nitrogenous base
Pentose sugar
Phosphate group
List the purines
Adenine
Guanine
List the pyrimadines
Cytosine
Thymine
Uracil
What are the 3 bondings in a double helix?
- Sugar phosphates which are linked by phosphodiester bonds
- Base stacking by hydrophobic interactions which excludes water from internal structure
- Van der waal forces are small but contribute to stability
What is double stranded DNA formed from?
From 2 antiparallel strands
In a double stranded DNA, where are the negatively charged phosphate groups located?
Negatively charged phosphates on the outside of the double stranded DNA
When DNA is denatured, what happens to the double stranded molecule?
Conversion of double stranded molecule into a single stranded molecule
What is disrupted due to denaturation?
Disruption of Hydrogen bonds within double helix
What can also induce the disruption of hydrogen bonds?
Induced by strong alkali or urea
How can denaturation be measured?
Can be measured optically by absorbance at 260nm
What increases due to denaturation and what is it called?
Absorption increases and this is called hyperchromicity
What is Tm?
Point at which 50% of all strands separate
What is Tm largely dependent on?
Largely dependent on hydrogen bonds:
- GC content
- Length of DNA molecule
- Salt concentration
- pH
- Mismatches
What does a higher GC content mean?
Means more H bonds therefore higher Tm
Equation for %GC
((G+C)/(G+C+A+T))*100
What happens to Tm value due to a larger duplex and why?
The larger the duplex, the higher the Tm
-There’re more H bonds within the molecule therefore greater stability
When is there no contribution to Tm due to length of DNA molecule?
Beyond 300bp, little or no contribution to Tm
What does salt stabilise?
Salt stabilises DNA duplexes
How does an increase in [Na+] contribute to Tm?
Results in a high Tm
-As it overcomes destabilising effect of mismatched base pairing
What does an alkali pH cause to DNA?
Alkali resorts in destablisation
How does an alkali pH cause denaturation?
The OH- ions disrupt hydrogen bond pairing
The effect of alkali pH on Tm
Fewer H bonds therefore lower Tm
What is mismatch defined as?
Defined as base pair which is unable to form hydrogen bonds
What is renaturation?
Reversal of denaturation
What does the formation of structures in renaturation favour?
Formation of structures favours energy minimisation driven by change in free energy
What is renaturation facilitated by?
- Slow cooling
- Neutralisation
What is hybridization?
Formation of duplex structure of 2 DNA molecules that have been introduced to one another
What is thermodynamically favoured over mismatches?
Perfect matches have a higher Tm therefore favoured more than mismatches
What allows us to manipulate specificity?
Limiting hybridisation between imperfectly matched sequence
What is stable under high stringency and what is it determined by?
Only complementary sequences are stable and is determined by a:
- Temperature near Tm
- Low salt concentration
What does nucleic acid hybridisation technique identify?
Identifies presence of nucleic acid containing specific sequence of bases
What does nucleic acid hybridisation allow?
Allows the absolute or relative quantitation of these sequence in a mixture
What does hybridisation use to form specific duplexes?
Hybridisation uses the ability of nucleic acids to form specific duplexes
What do probes detect?
Probes detect the presence of complementary nucleic acid by hybridisation
What molecules are probes?
ssDNA or RNA molecules
Length of probes
20-1000 bases in length
What are probes labelled with?
Labelled with a fluorescent or luminescent molecules
What does NA blotting technique analyse?
Analyses mRNA or DNA
Why is nucleic acid blotting technique limited?
Limited as they only detect one gene at a time
What is nucleic acid blotting technique superseded by?
Superseded by PCR
What are micro-arrays?
Ordered assembly of thousands of nucleic acid probes
What are micro-arrays fixed to and what do we do to the sample of interest?
Fixed to solid surface then sample of interest is hybridised to the probes
What do micro-arrays detect and what are they used in?
Detect SNP’s and used in genome wide association studies
