DNA Complimentary Hybridisation & Its Application Flashcards
Describe the pentose sugar structure in the DNA/RNA nucleotide.
• 5 carbons that form a cyclical structure with oxygen bridge
• Carbons are numbered 1-5
• Nitrogenous base joined to carbon 1
• Phosphate group joined to carbon 5
• Hydroxyl group carbon 3
What DNA Nucleotides are pyrimidine?
Cytosine + Thymine
What DNA Nucleotides are purines?
Adenine + Guanine
How are sugar phosphates linked in DNA?
Phosphodiester Bonds
Describe how Base stacking occurs in a Nucleotide Chain of DNA
Hydrophobic interactions lead to the arrangement of bases set above each other internalised to the structure.
What is the function of Van der Walls forces?
Contributes to the stability.
What are the two main features of Double Stranded DNA?
- Negatively charged Phosphates on the outside
- Forms two Antiparallel strands
What is DNA denaturation?
The process of separating the DNA double helix into to 2 separate strands due to the disruption of hydrogen bonds between bases.
How can DNA be dentured?
- Occurs when DNA in solution is heated
- Can also be induced by strong alkali or urea
How can Denaturation be measured optically?
- by measuring the absorbence at 260nm
- Increased absorption of light at 260nm on denaturation.
What is the Tm?
The temperature at which 50% of all DNA strands separate.
What does the Tm depend on?
- GC content
- Length of DNA molecule
- Salt concentration
- pH (alkali is denaturant)
- Mismatches (unmatched base pairs)
How does GC content effect Tm?
Higher the GC content = more hydrogen bonds = higher Tm
How does the length of DNA affect Tm?
The longer the continuous duplex = the higher the Tm
- More hydrogen bonds within the molecule means more stability
- However less contribution to change in Tm beyond 300bp
How does salt concentration [Na+] effect Tm?
- Salt stabilises DNA duplexes
- High [Na+] = High Tm - This overcomes the destabilising effect of mismatched base pairing.
How does salt concentration affect specificity of base pairing?
High salt reduces the specificity of base pairing at a given temperature and makes the duplex stable.
How does pH affect the Tm?
- High pH (alkalinity) destabilises DNA duplexes
- OH disrupts H bond pairing
- Fewer hydrogen bonds = Lower Tm
What is a mismatch?
A base pair combination that is unable to form hydrogen bonds.
How do Mismatches effect Tm?
- Reduces Number of Hydrogen bonds
- Fewer hydrogen bonds = lower Tm
- Mismatches also distorts the structure and destabilises adjacent base pairing
What is Renaturation facilitated by?
- Slow Cooling
- Neutralisation
What energy does Renaturation favour?
Favours energy minimisation driven by change in Free energy DG
What is Hybridisation?
Formation of duplex structure of two different DNA molecules that have been introduced to one another.
How is complimentary base paring and Tm the basis of specificity?
- Perfect matches have a higher Tm
- Perfect matches are thermodynamically favoured over mismatches
How can we form a complimentary duplex with no mis-matches?
We can use the property of perfect matches have a higher Tm by hybridising at the higher Tm of both duplex’s.
What is Stringency?
Manipulating conditions: Limiting hybridisation between imperfectly matched sequences allows us to manipulate specificity
High Stringency = the better the match.
Low Stringency = mismatching
What occurs under High Stringency?
- only complementary sequences are stable
- Temperature near Tm
- Low salt concentration
What is the function of Nucleic acid hybridisation techniques?
Identifies the presence of NA containing a specific sequence of bases allowing for the absolute or relative quantitation of these sequences in a mixture
How does hybridisation work?
It Uses the complementarity and hybridisation of labelled nucleic acids (by attaching probes)
What is a Probe?
- A short stranded DNA/RNA molecule
- Labelled with a fluorescent or luminescent molecule
What are two examples of High Throughput techniques?
- Microarrays
- Next generation sequencing
What occurs during the process of Microarrays?
- An ordered assembly of thousands nucleic acid probes are fixed to a solid surface.
- Then sample of interest is hybridised
What occurs during the process of Next Generation Sequencing?
The Parallel sequencing of millions of molecules captured in a surface by hybridisation
