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Genomics Semester 3 > DNA Complementarity Hybridisation and it's Application > Flashcards

DNA Complementarity Hybridisation and it's Application Flashcards

1
Q

What are DNA and RNA made up of?

A

Nucleotides

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2
Q

What are nucleotides comprised of ?

A

Nitrogenous Base
Phosphate
Pentose Sugar

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3
Q

What is a nitrogenous base?

A

A ring structure composed of carbon and nitrogen. Comprising of either a single or double ring

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4
Q

What is a Pentose sugar?

A

A Pentose sugar is a 5 carbon cyclical structure with an oxygen bridge

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5
Q

At which Carbon is the Nitrogenous base attached to?

A

C1

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6
Q

At which Carbon is the phosphate group attached to?

A

C5

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7
Q

Which carbon is the hydroxyl group attached on?

A

C3

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8
Q

Between which carbons is there a oxygen bridge?

A

C1 and C4

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9
Q

Through what bond does the phosphate and the next nitrogenous base attach by?

A

Phosphodiester bond

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10
Q

How many nucleotides is DNA made up of and what are they?

A

DNA is made up of 4 nucleotides

  • cytosine
  • thymine
  • adenine
  • guanine
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11
Q

Which of the nucleotides are Pyrimidines ?

A

Cytosine and thymine

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12
Q

Which of the nucleotides are Purine?

A

Adenine and Guanine

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13
Q

What provides the specificity of base pairing?

A

The difference in the structures due to the charged and polar groups

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14
Q

In RNA what substitutes Thymine?

A

Uracil

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15
Q

DNA double helix is formed due to what?

A
  • Hyrdogen bonding of Watson’s and Cricks base pairing
  • Bonding between amine and carboxyl gorups between A-T or C-G
  • Each pair there is a single purine and pyrimidine
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16
Q

What is the difference between A-T and C-G pairing

A

The number of hydrogen bonds formed

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17
Q

Why is C-G pairing stronger?

A

C-G forms 3 hydrogen bonds whereas A-T forms 2

More hydrogen bonds

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18
Q

What does base pairing contribute to in terms of the DNA structure?

A

It explains the stability of the double helix structure

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19
Q

What are the 3 structures of the Nucleotide Chain of DNA?

A
  1. Sugar Phosphates
  2. Base Stacking
  3. Van der Waals froces
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20
Q

What are the Sugar phosphates linked by?

A

Phosphodiester bonds

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21
Q

Through what interactions does base stacking occur? What is exculded thorugh this?

A

Hydrophobic interactions

Arrangement of bases set above eachother internalised to the strucure and exlcudes water

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22
Q

How big are the Van der Waal forces?

A

Individually small but still contributes to the stability

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23
Q

What is the backbone structure formed from?

A

Phosphodiester Linkage

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24
Q

How is the stability of the structure determined?

A

It is determined by the free energy of the molecule and energy minimisation

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25
Q

How is Stability of the helix derived?

A

Stability is derived from:

  • hydrogen bonding of the bases
  • internal arrangement and addition stability of base stacking
  • alongside vdW
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26
Q

Where does the stability come from in a single stranded DNA molecule?

A

From the sugar phosphate backbone

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27
Q

From what strands is the double helix formed from?

A

Two antiparallel strands

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28
Q

What does having antiparallel strand mean?

A

They have an opposite orinetation

  • bases are on the inside forming stacked bases
  • the negatively charged phopshates external givign dNA an overall negative charge
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29
Q

Why is it important tohave charged phosphate groups on the otuside?

A

Importantn for the interation with proteins

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30
Q

What is Denautirng

A

Denatriing is the breaking of hydrogen bonds inot its consitiuent starnds

conversion of double strnaded molecule into a single stranded molecule

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31
Q

During what condidiotns are the hydrogne bonds disrupted?

A
  • DNA is heated

- Induced by strong alkali or urea

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32
Q

On denaturaion what type of strand does it form?

A

Randomly structured coil

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33
Q

How can denaturation be measured?

A

It can be measured optically by abd=sorbace at 260nm

34
Q

What is Hyperchromacity?

A

Increased absorption of light at 260nm on denatutration

Single stranded DNA absorbs UV lgiht to a greater extent than double standed DNA

35
Q

What is the Melting Temperature or Tm?

A

Point at which 50% of all strands have sperarated

36
Q

The Tm is specific to what?

A

The Tm is specific to an individual double helix

Use this charactersitics to control formation of the duplex

37
Q

What does the denautartion of a specififc DNA depend on?

A

Depedns upon the stability of the specific structure

38
Q

Name 6 conditions that Tm is dependent on.

A
  1. Hydrogen Bonds
  2. G-C base pairing content
  3. Length of DNA molecule
  4. Slat concerntrarion
  5. pH
  6. Mismatches
39
Q

What is the relationship between G-C content and Tm?

A

The higher G-C content = more hydrogen bonds = higher Tm

40
Q

Why does G-C content cause a higher Tm?

A

G-C is stronger than A-T
There are 3 H bonds rather than 2
therefore more H bonds to break which requires a higher temperature

41
Q

How do you calculate the %GC

A

(G+C) / (G+C+A+T) X 100

42
Q

What is the relationship between Tm and Molecule length?

A

The longer the contiguous duplex the higher the Tm

43
Q

How does having a longer duplex cause a higher Tm?

A

There are more hydrogen bonds within the molecule therefore a greater stability

44
Q

At which point in the molecule does hydrogen bonds stop contributing to the stability?

A

Beyond 300 base pairs

45
Q

What is the relationship between Tm and Salt Concentration?

A

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

46
Q

What does salt do to DNA?

A

Salt stabilises DNA duplexes

47
Q

Compare the scenarios of having High salt concentrations/ low concentration and mismatched base pairing

A

High Salt Concentration:
- a duplex containing mismatches can form and be stable at a given temperature in the presence of this condition

Low Salt Concentration:
- the same duplex would be unstable and dissociate at the same temperature in low salt

48
Q

Mismatch base pairings also affect what?

A

They affect adjacent base pairings as a consequence of distortion of the helix
This encourages the unzipping of the molecule

49
Q

What is the relationship between Tm and pH?

A

The higher the pH, the lower the Tm

50
Q

Why does a high pH cause a lower Tm?

A

OH- ions are released which disrupts H bond pairinfs

Fewer H bonds = Lower Tm = lower stability of the structure

51
Q

What do chemical denaturants do?

A

Disrupts hydrogen bonds

52
Q

What does a high pH do to a DNA duplex?

A

It destabilises the DNA duplex

53
Q

What is a mismatch?

A

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

54
Q

What are the three effects that mismatches have?

A
  • reduces the number of hydrogen bonds formed within a duplex
  • distorts the duplex and destabilises adjacent base pairs
  • leads to zipping and unzipping
55
Q

What is the relationship between Tm and Mismatches?

A

More mismatches = less hydrogen bonds = lower Tm

Shorter contiguous stretches of double stranded sequence = lower Tm

56
Q

What is the effect of reducing the change in free energy on duplex formation?

A

Formation of a duplex less energetically favourable

57
Q

What is the process name for the reverse of denaturation?

A

Renaturation

58
Q

What facilitates Renautration?

A

Facilitated by reversal of the factors that influence denaturation

59
Q

What does renaturation depend on?

A

It depends upon the energy of the system and corresponding molecules

Occur as a result of a change in free energy

60
Q

What are the two conditions of renaturation?

A
  • Slow cooling ( cool down the heating)

- Neutralisation (neutralise the Alkali)

61
Q

What is the difference between renaturation and hybridization?

A
  • Renaturation : formation of a duplex molecule originally from a duplex molecule before
  • Hybridization: the formation of a duplex molecule by introducing a 2nd molecule ( involves 2 DNA molecules being introduced to each other)
62
Q

What is Stringency?

A

Stringency is manipulating conditions by limiting hybridization between imperfectly matched sequences allowing us to manipulate specificity.

63
Q

Explain the Stringency concept.

A

Stringency is manipulating the conditions to select duplexes with a perfect match only

64
Q

What are the results of a Low Stringency?

A

Conditions: High Salt , Low Temp

- multiple duplex with different mismatch pairing

65
Q

What are the results of a High Stringency?

A

Conditions: Temperature near Tm , Low Salt Concentration

Promote formation of non-mismatch pairing

66
Q

Name some Nucleic Acid Based Technology that uses hybridisation.

A
  • Northern Blotting
  • Southern Blotting
  • Microarrays
  • Dideoxy and Next Gen Sequencing
  • PCR
  • Cloning
67
Q

Explain how Nucleic Acid Hybridisation technique works.

A
  • identifies the presence of nucleic acids containing specific sequences of bases
  • allows the relative quantitation of these sequences in a mixture
  • uses labelled molecules which are complementary to molecules under investigation
68
Q

What is the term used for a labelled nucleic acid?

A

Probe

69
Q

How are Probes used ?

A

Probes that are used are complementary to a specific region of a target gene sequence

70
Q

How many bases are probes?

A

Probes are between 20 - 1000 bases in length dependent on the technique used

71
Q

What is a Probe?

A

A single stranded DNA molecule or RNA

Labelled with fluorescent molecules

72
Q

What is the technique where thousands of probes are used simultaneously?

A

Microarrays

73
Q

Of what technique is Northern Blotting an adaptation from?

A

Southern Blotting

74
Q

What is Northern Blotting?

A

Analysis of mRNA or DNA within an population

75
Q

What is a limitation of Northern Blotting?

A

Only detects one gene at a time and small number of samples

Time consuming and messy

76
Q

What techniques superseded Northern Blotting?

A

Quantitative PCR or Microarrays

77
Q

How does Northern Blotting work?

A
  • Separates DNA or RNA via gel electrophoresis
  • transferred to a nylon membrane via mass capillary flow
  • covalently bond to the membrane and then hybridised with a labelled probe
  • Probe can be visualised
78
Q

What are microarrays?

A

An ordered assembly of thousands nucleic acid probes

79
Q

How do microarrays work?

A

Probes are fixed to a solid surface

Then sample if interest is hybridised ti the probes

80
Q

What can microarrays be used for?

A

Microarrays can be used for gene expressions profiling

81
Q

Explain the process of gene expression profiling.

A
  • RNA is extracted
  • Labelled
  • Hybridised to the array and the amount and location of the label is measured
  • Results: Quantity of how much and who in the human genome are being expressed

Genomics Semester 3 (4 decks)

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