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GEN > 1. DNA HYBRIDISATION > Flashcards

1. DNA HYBRIDISATION Flashcards

1
Q

Which carbon of the pentose sugar is the nitrogenous base attached to?

A

C1

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2
Q

Which carbon of the pentose sugar is the phosphate group attached to?

A

C5

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3
Q

Which carbon of the pentose sugar is the hydroxyl group attached to?

A

C3

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4
Q

Which bases are purine?

A
  1. Adenosine

2. Guanine

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5
Q

Which bases are pyramidine?

A
  • Pyramid = 3
    1. Cytosine
    2. Thymine
    3. Uracil
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6
Q

How many hydrogen bonds between C-G & A-T?

A 
C-G = 3 H
A-T = 2 H
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7
Q

** What are phosphodiester bonds & what do they link?

A
  • 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
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8
Q

What is base stacking?

A
  • Base stacking refers to bases being arranged on top of each other because of the hydrophobic interactions which excludes water
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9
Q

Define denaturation

A

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

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10
Q

What can induce denaturation?

A
  1. Strong alkali
  2. Urea
  3. Formamide
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11
Q

** What is hyperchromicity

A
  • Hyperchromicity refers to the increased absorption of UV light at 260nm. A single strand can absorb more UV light than a double stranded molecule
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12
Q
  • What is Tm?
A
  • Tm = melting temperature at which 50% of the strands have been separated
  • Tm is the temperature for the equilibrium between renaturation & denaturation
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13
Q

What 5 factors can affect Tm?

A
  1. GC content
  2. Length of molecule
  3. Salt content
  4. pH
  5. Mismatches
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14
Q

How does GC content affect Tm?

A
  • 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
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15
Q

How does molecule length affect Tm?

A
  • 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
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16
Q

How can pH affect Tm?

A
  • 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
17
Q

** How does salt concentration affect Tm?

A
  • 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
18
Q

What are mismatches & their effect on Tm?

A
  • 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
19
Q

What is renaturation?

A
  • 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
20
Q

What two factors favour renaturation?

A
  1. Slow cooling

2. Neutralisation

21
Q
  • What is Hybridisation?
A
  • 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
22
Q

Define stringency

A
  • Stringency refers to the ability to manipulate specificity by manipulating conditions to prevent the hybridisation of mismatched bases
23
Q

What are the effects of high & low stringency?

A

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

24
Q

What is a probe?

A
  • 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
25
Q

Describe the steps of Northern/Southern blotting?

A
  1. Extract RNA/DNA
  2. 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
  3. Transfer to a nylon membrane
  4. Add a labelled probe which can hybridise to the duplex
  5. Detect hybridisation

GEN (18 decks)

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