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Genomics > DNA hybridisation and DNA complementarity > Flashcards

DNA hybridisation and DNA complementarity Flashcards

1
Q

What is DNA comprised of?

A
  • 4 nucleotides
  • Phosphate
  • Hydroxyl group
  • Pentose sugar
  • Phosphate group
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2
Q

What is the nitrogenous base?

A
  • A ring structure composed of carbon and nitrogen.

- Can be double or single ringed.

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3
Q

What is the pentose sugar?

A

-5 carbons that form a cyclical structure with oxygen bridge between C1 and C4 carbons.

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4
Q

Which carbon is the nitrogenous base attached to?

A

-Carbon 1

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5
Q

Which carbon is phosphate group attached to?

A

-Carbon 5 via ester bond

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6
Q

Which carbon is hydroxyl group attached to?

A

-Carbon 3

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7
Q

How many hydroxyl groups in RNA?

A

2

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8
Q

What is hydroxyl groups important for?

A

-Key to polymerisation of nucleotides in DNA or RNA

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9
Q

What are pyrimidines?

A

-Cytosine and Thymine

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10
Q

What are purines?

A

-Thymine and Adenine

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11
Q

On what basis does hydrogen bonds form?

A

-On the basis of Watson Crick pairing

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12
Q

How are sugar phosphates linked?

A

-linked by phosphodiester bonds between 3 prime and 5 prime.

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13
Q

How does DNA gain its stability?

A
  • Sugar phosphates
  • Base stacking
  • VDW
  • Hydrogen bonds
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14
Q

What is the double stranded helix formed from?

A

-Two antiparallel strands

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15
Q

How are the DNA strands arranged?

A
  • Bases stacked

- Negative phosphates on outside

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16
Q

What is denaturation?

A
  • When double stranded DNA becomes single stranded

- Due to disruption of Hydrogen bonds

17
Q

Under what circumstances does denaturation occur?

A
  • Occurs when DNA in solution is heated

- Can also be induced by strong alkali or urea

18
Q

What is hyperchromicity?

A

-Increased absorption of light at 260nm On denaturation

19
Q

What is Tm?

A

-Point at which 50% of all strands separate is called the melting temperature or Tm.

20
Q

How can we measure denaturation?

A

-Denaturation can be measured optically by absorbance at 260nm

21
Q

How does hypochromicity?

A
  • 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
22
Q

What does Tm depend on?

A
  • GC content
  • Length of DNA molecule
  • Salt concentration
  • pH (alkali is a denaturant)
  • Mismatches (unmatched base pairs)
23
Q

What is the result of higher GC content?

A
  • Hydrogen bonds

- Higher Tm

24
Q

What is formula to find percent of GC?

A

%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

25
Q

How does length of molecule affect Tm?

A
  • Longer the contiguous duplex, the higher Tm
  • More Hydrogen bonds within the molecule greater stability
  • However little further contribution beyond 300 bp
26
Q

What is the relationship between Na+ and Tm?

A

-Increasing the salt concentration stabilises the structure increases the Tm and thus overcomes the destabilising effect of mismatched base pairing

27
Q

Which chemical denaturants disrupt hydrogen bonds?

A

-Alkali, formamide, urea

28
Q

How does NaOH disrupt hydrogen bonds?

A
  • OH- disrupts H bond pairing
  • Fewer hydrogen bonds = Lower Tm
  • High pH (alkalinity) destabilises DNA duplexes
29
Q

What is a mismatch?

A

-A mismatch is defined as a base pair combination that is unable to form hydrogen bonds

30
Q

What are the effects of mismatches?

A
  • 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
31
Q

What is the reversal of denaturation?

A

-Formation of structure favours energy minimisation driven by change in free energy DG

32
Q

What is renaturation facilitated by?

A
  • Slow Cooling

- Neutralisation

33
Q

Define hybridisation

A

-Formation of duplex structure of two DNA molecules that have been introduced to one another

34
Q

At what temperature do we get perfect hybrid?

A

-85 degrees

35
Q

What is the relationship between perfect matches and Tm?

A
  • Perfect matches have a higher Tm

- Thermodynamically favoured over Mismatches

36
Q

What are the effects of manipulating conditions?

A

-Limiting hybridisation between imperfectly matched sequences allows us to manipulate specificity

37
Q

What is the effect of high stringency?

A

-Only a perfectly matched duplex can form

38
Q

What is the effect of low stridency?

A

-Hybridisation occurs with mismatches allowed.

39
Q

What are the conditions of high stridency?

A
  • Temp near Tm

- Low Na concentration

Genomics (4 decks)

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