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Genomics > DNA Complimentary Hybridisation & Its Application > Flashcards

DNA Complimentary Hybridisation & Its Application Flashcards

1
Q

Describe the pentose sugar structure in the DNA/RNA nucleotide.

A

• 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

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2
Q

What DNA Nucleotides are pyrimidine?

A

Cytosine + Thymine

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3
Q

What DNA Nucleotides are purines?

A

Adenine + Guanine

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4
Q

How are sugar phosphates linked in DNA?

A

Phosphodiester Bonds

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5
Q

Describe how Base stacking occurs in a Nucleotide Chain of DNA

A

Hydrophobic interactions lead to the arrangement of bases set above each other internalised to the structure.

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6
Q

What is the function of Van der Walls forces?

A

Contributes to the stability.

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7
Q

What are the two main features of Double Stranded DNA?

A
  • Negatively charged Phosphates on the outside
  • Forms two Antiparallel strands
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8
Q

What is DNA denaturation?

A

The process of separating the DNA double helix into to 2 separate strands due to the disruption of hydrogen bonds between bases.

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9
Q

How can DNA be dentured?

A
  • Occurs when DNA in solution is heated
  • Can also be induced by strong alkali or urea
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10
Q

How can Denaturation be measured optically?

A
  • by measuring the absorbence at 260nm
  • Increased absorption of light at 260nm on denaturation.
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11
Q

What is the Tm?

A

The temperature at which 50% of all DNA strands separate.

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12
Q

What does the Tm depend on?

A
  • GC content
  • Length of DNA molecule
  • Salt concentration
  • pH (alkali is denaturant)
    - Mismatches (unmatched base pairs)
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13
Q

How does GC content effect Tm?

A

Higher the GC content = more hydrogen bonds = higher Tm

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14
Q

How does the length of DNA affect Tm?

A

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
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15
Q

How does salt concentration [Na+] effect Tm?

A
  • Salt stabilises DNA duplexes
    - High [Na+] = High Tm
  • This overcomes the destabilising effect of mismatched base pairing.
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16
Q

How does salt concentration affect specificity of base pairing?

A

High salt reduces the specificity of base pairing at a given temperature and makes the duplex stable.

17
Q

How does pH affect the Tm?

A
  • High pH (alkalinity) destabilises DNA duplexes
  • OH disrupts H bond pairing
  • Fewer hydrogen bonds = Lower Tm
18
Q

What is a mismatch?

A

A base pair combination that is unable to form hydrogen bonds.

19
Q

How do Mismatches effect Tm?

A
  • Reduces Number of Hydrogen bonds
  • Fewer hydrogen bonds = lower Tm
  • Mismatches also distorts the structure and destabilises adjacent base pairing
20
Q

What is Renaturation facilitated by?

A
  • Slow Cooling
  • Neutralisation
21
Q

What energy does Renaturation favour?

A

Favours energy minimisation driven by change in Free energy DG

22
Q

What is Hybridisation?

A

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

23
Q

How is complimentary base paring and Tm the basis of specificity?

A
  • Perfect matches have a higher Tm
  • Perfect matches are thermodynamically favoured over mismatches
24
Q

How can we form a complimentary duplex with no mis-matches?

A

We can use the property of perfect matches have a higher Tm by hybridising at the higher Tm of both duplex’s.

25
Q

What is Stringency?

A

Manipulating conditions: Limiting hybridisation between imperfectly matched sequences allows us to manipulate specificity

High Stringency = the better the match.

Low Stringency = mismatching

26
Q

What occurs under High Stringency?

A
  • only complementary sequences are stable
  • Temperature near Tm
  • Low salt concentration
27
Q

What is the function of Nucleic acid hybridisation techniques?

A

Identifies the presence of NA containing a specific sequence of bases allowing for the absolute or relative quantitation of these sequences in a mixture

28
Q

How does hybridisation work?

A

It Uses the complementarity and hybridisation of labelled nucleic acids (by attaching probes)

29
Q

What is a Probe?

A
  • A short stranded DNA/RNA molecule
  • Labelled with a fluorescent or luminescent molecule
30
Q

What are two examples of High Throughput techniques?

A
  • Microarrays
  • Next generation sequencing
31
Q

What occurs during the process of Microarrays?

A
  • An ordered assembly of thousands nucleic acid probes are fixed to a solid surface.
  • Then sample of interest is hybridised
32
Q

What occurs during the process of Next Generation Sequencing?

A

The Parallel sequencing of millions of molecules captured in a surface by hybridisation

Genomics (7 decks)

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