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Genomics > DNA Hybridisation > Flashcards

DNA Hybridisation Flashcards

1
Q

What makes up DNA and RNA?

A
  • Phosphate
  • NItrogenous Base
  • Pentose Sugar
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2
Q

What is the structure of a Nitrogenous Base?

A

A ring structure (single or double ring) composed of caron and nitrogen (occasionally oxygen).

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3
Q

What is the strucutre of a Pentose Sugar?

A
  • 5 carbons

- Acyclical structure with oxygen bridge

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4
Q

Which carbon does the nitrogenous base, phosphate group and hydroxyl group join to respectively?

A

Nitrogenous base joins to carbon 1.
Phosphate group joins to carbon 5.
Hydroxyl group joins to carbon 3.

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5
Q

How is the phosphate group attached to carbon 5?

A

Via an ester bond

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6
Q

How many hydroxyl group are in RNA?

A

2

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7
Q

What are the four nucleotides in DNA?

A

Pyrimidines:

  • Cytosine
  • Thymine

Purines:

  • Guanine
  • Adenine
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8
Q

What is the difference in the structure of pyrimidines and purines?

A

The difference resides in the charged or polar groups providing the specificity of base pairing.

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9
Q

How is the RNA duplex structure formed?

A

Uracil substitues Thymine and base pairs with Adenine in RNA to form duplex structure.

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10
Q

What are the complementary bases?

A

Cytosine - Guanine

Thymine - Adenine

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11
Q

How many hydrogen bonds are between C-G and T-A?

A

3 and 2 respectively

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12
Q

What does the nucleotide chain of DNA form?

A

It forms a double helix which can take on different combinations.

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13
Q

What is the most common form of DNA?

A

B-DNA

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14
Q

What forms the backbone of DNA?

A

From a phosphodiester linkage which connects the 3’ and 5’ prime carbons of the deoxyribose sugar of DNA

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15
Q

What determines the stability of a structure?

A

The free energy of the molecule and energy minimisation.

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16
Q

What stabilises the structure of DNA?

A
  • Hydrogen bonding of the bases

- Internal arrangement

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17
Q

What other than hydrogen bonding contributes to the stability of DNA?

A
  • Sugar phosphates
  • Base stacking
  • Van der Waals forces
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18
Q

What is base stacking?

A

The hydrophobic interactions between the arrangement of bases set above each other internalised to the structure and excludes water from the internal environment of the structure

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19
Q

What gives DNA its overall negative charge?

A

The bases on the inside forming stacked bases and the negatively charged phosphates are on the outside giving DNA an overall negative charge.

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20
Q

What is the overall negative charge of DNA useful for?

A

It is used for electrophoresis - in a highly negative environment will migrate to the positive electrode.

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21
Q

What happens when DNA is denatured?

A

When DNA is denatured, there is conversion of a double stranded molecule to single stranded molecules.

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22
Q

What happens when there is a disruption of the hydrogen bonds?

A

The hydrogen bonds can be denatured or broken down into its constituent strands.

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23
Q

What structure is formed when DNA is denatured?

A
  • A randomly structured coli
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24
Q

What conditions does DNA need to be under to denature?

A
  • DNA solution is heated

- Induced by strong alkali or urea

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25
Q

How is denaturation of DNA measured?

A
  • It can be measured optically by absorbance at 260nm.

- Take the solution, heat it up and measure the optical density

26
Q

What is hyperchromicity?

A

Single stranded DNA absorbs UV light to a greater extent than double stranded DNA

27
Q

What does the denaturation of a specific DNA depend on?

A
  • The stability of the specific structure
28
Q

What is the melting temperature Tm?

A
  • The temperature at which 50% of the molecules (all strands separate) have melted
29
Q

What does the Tm depend on?

A

Hydrogen bonds

30
Q

What are the 5 factors that the Tm of a DNA molecule is determined by?

A
  • GC content
  • Length of DNA molecule
  • Salt concentration
  • pH
  • No. of mismatches
31
Q

The higher GC content….

A

…the more hydrogen bonds, the higher Tm

32
Q

The longer the contiguous duplex….

A

…. the higher the Tm

33
Q

Why is the Tm higher when there is a longer contiguous duplex?

A

There is more hydrogen bonds within the molecule greater stability.

34
Q

When does the length have a dimishing return?

A

There is a diminishing return on this and a length beyond about 300 base pairs contributes little or no more to the stability.

35
Q

What does salt do to the DNA duplexes?

A

It stabilises DNA duplexes.

36
Q

The higher [Na+]…

A

… the higher Tm.

37
Q

What overcomes the destabilising effect of mismatched base pairing?

A
  • Increasing the salt concentration stabilises the structure increases the Tm
38
Q

When is a duplex unstable and/or stable?

A

A duplex is stable at a given temperature in the presence of high salt concentration whilst the same duplex would be unstable and dissociate at the same temperature in low salt.

39
Q

What do chemical denaturants do to DNA?

A

They disrupt hydrogen bonds and are easy to add to a solution and can denature the DNA present in that solution.

40
Q

Give examples of chemical denaturants

A

Alkali, formamide, urea

41
Q

What is adding alkali pH the same as?

A

It is the same as adding OH groups.

42
Q

When OH is added to DNA strands?

A

They are small enough to penetrate into the molecule and break the hydrogen bonds - resulting in dissociate of the two strands.

43
Q

What is mismatch?

A

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

44
Q

What do mismatch base pairs do to the DNA duplex?

A

This reduces number of H bonds, fewer means lower Tm. The shorter contiguous stretches of double stranded sequence, meaning lower Tm.

45
Q

What is renaturation?

A
  • The reverse of denaturation.

- The formation of structure favours energy minimisation driven by change in free energy.

46
Q

What facilitates renaturation?

A

Slow cooling

Neutralisation

47
Q

Define hybridisation

A

The formation of duplex structure of two DNA molecuels that have been introduced to one another.

48
Q

What is the similarity between hybridisation and renaturation?

A

Both involve two DNA molecules taht have been introduced to each other. The molecule has not previously been part of the duplex of that molecule.

49
Q

Why are perfect matches formed?

A
  • Have a higher Tm
  • Are thermodynamically favoured over mismatches
  • Can use this property to form a complementary molecule with no mismatches
50
Q

What is stringency?

A

The concept of manipulating the conditions to select duplexes with a perfect match only.

51
Q

How are conditions manipulated?

A

Temperature is near Tm or there is a low salt concentration whih mean only complementary sequences are formed stabely.

52
Q

Define northern blotting

A

used for the identification of RNA species within a dense population of RNA

53
Q

define southern blotting

A

used for the identification of DNA species within a dense population of DNA

54
Q

Microarrays

A

measure the absence or presence of particular species within a population of molecules.

55
Q

What are nucleic acid hybridisation techniques used for?

A
  • Identify the presence of nucleic acid containing specific sequence of bases
  • Allows the absolute or relative quantitation of these sequences in a mixture
56
Q

What is a probe?

A
  • A ssDNA (or RNA) molecule
  • Typically 20-1000 bases in length
  • Labelled with fluorescent or luminescent molecule
  • In some techniques, thousands or millions of probes are used simultaneously.
57
Q

Disadvantages of northern blotting

A
  • Analysis of mRNA or DNA
  • Limited technique only detects one gene at a time and small numbers of samples
  • The gel-based techniques are time-consuming and messy.
  • Largely superseded by quantitative PCR
58
Q

How are microarrays carried out?

A
  • An ordered assembly of thousands of nucleic acid probes
  • Probes are fixed to a solid surface, then sample of interest is hybridised to the probes.
    Simultaneously measuring 50,000 different transcripts in a Cell, Tissue or Organ.
59
Q

How many SNPs can microarrays detect simultaneously?

A

2.5 million

60
Q

When are microarrays used?

A

In Genome wide association studies

Genomics (19 decks)

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