1. DNA HYBRIDISATION Flashcards
Which carbon of the pentose sugar is the nitrogenous base attached to?
C1
Which carbon of the pentose sugar is the phosphate group attached to?
C5
Which carbon of the pentose sugar is the hydroxyl group attached to?
C3
Which bases are purine?
- Adenosine
2. Guanine
Which bases are pyramidine?
- Pyramid = 3
1. Cytosine
2. Thymine
3. Uracil
How many hydrogen bonds between C-G & A-T?
C-G = 3 H A-T = 2 H
** What are phosphodiester bonds & what do they link?
- Phosphodiester bonds link the sugar phosphate
- They form from the oxygen bridge between CO & phosphate (between C1 & C4)
- 3’-5’ phosphodiester linkage between DNA backbone
What is base stacking?
- Base stacking refers to bases being arranged on top of each other because of the hydrophobic interactions which excludes water
Define denaturation
DENATURATION = the conversion of a double stranded DNA molecule into a single stranded molecule. It involves the disruption of H bonds using chemicals or heat
What can induce denaturation?
- Strong alkali
- Urea
- Formamide
** What is hyperchromicity
- Hyperchromicity refers to the increased absorption of UV light at 260nm. A single strand can absorb more UV light than a double stranded molecule
- What is Tm?
- Tm = melting temperature at which 50% of the strands have been separated
- Tm is the temperature for the equilibrium between renaturation & denaturation
What 5 factors can affect Tm?
- GC content
- Length of molecule
- Salt content
- pH
- Mismatches
How does GC content affect Tm?
- More G-C means more bases and therefore more H binds as there are 3H between CG
- More H bonds means the duplex is more stable, and more energy is needed for denaturation
- High GC = High Tm
How does molecule length affect Tm?
- As molecule length increases there will be more bases & more H bonds, so the duplex will be stable requiring more energy
- Tm increases with molecule length but only up to a certain point
- Tm begins to plateau after 300 bp
How can pH affect Tm?
- Strong alkali, urea & formamide can induce denaturation
- OH- ions can disrupt the H bonds in the duplex, so a high pH can destabilize the duplex
- Fewer H bonds = less energy is required therefore Tm is lower
** How does salt concentration affect Tm?
- High [Na+] can stabilize the DNA duplex.
- Na+ is a cation that can interact with the negatively charged backbone of DNA to exclude water which stabilizes the DNA
- Adding high [Na+] to a duplex with mismatches can stabilise it and raise the Tm to that of a perfectly matched duplex
- So high salt can increase the Tm, it reduces the specificity of base pairing
What are mismatches & their effect on Tm?
- Mismatches occur when bases aren’t complementary so they are unable to form H bonds.
- Mismatches have two effects:
1. Distort the structure
2. Reduce the number of H bonds - Fewer H bonds means less energy is needed = lower Tm
- Perfectly matches with no mismatches = high Tm
- Mismatches = lower Tm
What is renaturation?
- Renaturation is the formation of a duplex by the reversal of denaturation vis energy minimisation driven by a change in free net energy
- It is less energetically favorable to maintain a single stranded molecule
What two factors favour renaturation?
- Slow cooling
2. Neutralisation
- What is Hybridisation?
- Both renaturation & hybridisation involve the formation of the duplex
- But hybridisation involves the addition of a third molecule known as a primer which is introduced to the single strands after denaturation
Define stringency
- Stringency refers to the ability to manipulate specificity by manipulating conditions to prevent the hybridisation of mismatched bases
What are the effects of high & low stringency?
HIGH STRINGENCY = perfectly matched duplex with no mismatches. E.g hybridisation occur at a Tm near to the primer-duplex
LOW STRINGENCY = a duplex where mismatches’ are allowed. Although there are mismatches, low stringency conditions are favoured by the kinetics
What is a probe?
- A probe can be either RNA or DNA depending on the technique used (Northern/Southern blotting)
- It can be between 20- 100 bp long
- Probes can be labelled with a fluorescent molecule
Describe the steps of Northern/Southern blotting?
- Extract RNA/DNA
- GEL electrophoresis
- Mass flow by capillary cation carries the nucleic acids which then covalently binds to the membrane
- A voltage is applied across the gel, DNA migrates towards positive electrode - Transfer to a nylon membrane
- Add a labelled probe which can hybridise to the duplex
- Detect hybridisation
