DNA hybridisation and DNA complementarity Flashcards
What is DNA comprised of?
- 4 nucleotides
- Phosphate
- Hydroxyl group
- Pentose sugar
- Phosphate group
What is the nitrogenous base?
- A ring structure composed of carbon and nitrogen.
- Can be double or single ringed.
What is the pentose sugar?
-5 carbons that form a cyclical structure with oxygen bridge between C1 and C4 carbons.
Which carbon is the nitrogenous base attached to?
-Carbon 1
Which carbon is phosphate group attached to?
-Carbon 5 via ester bond
Which carbon is hydroxyl group attached to?
-Carbon 3
How many hydroxyl groups in RNA?
2
What is hydroxyl groups important for?
-Key to polymerisation of nucleotides in DNA or RNA
What are pyrimidines?
-Cytosine and Thymine
What are purines?
-Thymine and Adenine
On what basis does hydrogen bonds form?
-On the basis of Watson Crick pairing
How are sugar phosphates linked?
-linked by phosphodiester bonds between 3 prime and 5 prime.
How does DNA gain its stability?
- Sugar phosphates
- Base stacking
- VDW
- Hydrogen bonds
What is the double stranded helix formed from?
-Two antiparallel strands
How are the DNA strands arranged?
- Bases stacked
- Negative phosphates on outside
What is denaturation?
- When double stranded DNA becomes single stranded
- Due to disruption of Hydrogen bonds
Under what circumstances does denaturation occur?
- Occurs when DNA in solution is heated
- Can also be induced by strong alkali or urea
What is hyperchromicity?
-Increased absorption of light at 260nm On denaturation
What is Tm?
-Point at which 50% of all strands separate is called the melting temperature or Tm.
How can we measure denaturation?
-Denaturation can be measured optically by absorbance at 260nm
How does hypochromicity?
- As temperature increases the duplex melts and the optical density increases
- Single stranded DNA absorbs UV light to a greater extent than double stranded DNA, this property is termed hyperchromicity
- The denaturation of a DNA duplex depends upon the stability of the structure determined by its sequence of bases
What does Tm depend on?
- GC content
- Length of DNA molecule
- Salt concentration
- pH (alkali is a denaturant)
- Mismatches (unmatched base pairs)
What is the result of higher GC content?
- Hydrogen bonds
- Higher Tm
What is formula to find percent of GC?
%GC = ((𝐺+𝐶))/((𝐺+𝐶+𝐴+𝑇)) x 100
-Where G is the number of guanine nucleotides, C is the number of cytosine nucleotides etc
How does length of molecule affect Tm?
- 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 Na+ and Tm?
-Increasing the salt concentration stabilises the structure increases the Tm and thus overcomes the destabilising effect of mismatched base pairing
Which chemical denaturants disrupt hydrogen bonds?
-Alkali, formamide, urea
How does NaOH disrupt hydrogen bonds?
- OH- disrupts H bond pairing
- Fewer hydrogen bonds = Lower Tm
- High pH (alkalinity) destabilises DNA duplexes
What is a mismatch?
-A mismatch is defined as a base pair combination that is unable to form hydrogen bonds
What are the effects of mismatches?
- 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
What is the reversal of denaturation?
-Formation of structure favours energy minimisation driven by change in free energy DG
What is renaturation facilitated by?
- Slow Cooling
- Neutralisation
Define hybridisation
-Formation of duplex structure of two DNA molecules that have been introduced to one another
At what temperature do we get perfect hybrid?
-85 degrees
What is the relationship between perfect matches and Tm?
- Perfect matches have a higher Tm
- Thermodynamically favoured over Mismatches
What are the effects of manipulating conditions?
-Limiting hybridisation between imperfectly matched sequences allows us to manipulate specificity
What is the effect of high stringency?
-Only a perfectly matched duplex can form
What is the effect of low stridency?
-Hybridisation occurs with mismatches allowed.
What are the conditions of high stridency?
- Temp near Tm
- Low Na concentration
