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Genomics > DNA hybridisation: DNA Complementarity and its application > Flashcards

DNA hybridisation: DNA Complementarity and its application Flashcards

1
Q

What do nucleotides make up?

A

Make up DNA and RNA

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2
Q

Components of a nucleotide?

A

Nitrogenous base
Pentose sugar
Phosphate group

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3
Q

List the purines

A

Adenine

Guanine

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4
Q

List the pyrimadines

A

Cytosine
Thymine
Uracil

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5
Q

What are the 3 bondings in a double helix?

A
  • Sugar phosphates which are linked by phosphodiester bonds
  • Base stacking by hydrophobic interactions which excludes water from internal structure
  • Van der waal forces are small but contribute to stability
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6
Q

What is double stranded DNA formed from?

A

From 2 antiparallel strands

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7
Q

In a double stranded DNA, where are the negatively charged phosphate groups located?

A

Negatively charged phosphates on the outside of the double stranded DNA

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8
Q

When DNA is denatured, what happens to the double stranded molecule?

A

Conversion of double stranded molecule into a single stranded molecule

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9
Q

What is disrupted due to denaturation?

A

Disruption of Hydrogen bonds within double helix

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10
Q

What can also induce the disruption of hydrogen bonds?

A

Induced by strong alkali or urea

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11
Q

How can denaturation be measured?

A

Can be measured optically by absorbance at 260nm

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12
Q

What increases due to denaturation and what is it called?

A

Absorption increases and this is called hyperchromicity

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13
Q

What is Tm?

A

Point at which 50% of all strands separate

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14
Q

What is Tm largely dependent on?

A

Largely dependent on hydrogen bonds:

  • GC content
  • Length of DNA molecule
  • Salt concentration
  • pH
  • Mismatches
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15
Q

What does a higher GC content mean?

A

Means more H bonds therefore higher Tm

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16
Q

Equation for %GC

A

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

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17
Q

What happens to Tm value due to a larger duplex and why?

A

The larger the duplex, the higher the Tm

-There’re more H bonds within the molecule therefore greater stability

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18
Q

When is there no contribution to Tm due to length of DNA molecule?

A

Beyond 300bp, little or no contribution to Tm

19
Q

What does salt stabilise?

A

Salt stabilises DNA duplexes

20
Q

How does an increase in [Na+] contribute to Tm?

A

Results in a high Tm

-As it overcomes destabilising effect of mismatched base pairing

21
Q

What does an alkali pH cause to DNA?

A

Alkali resorts in destablisation

22
Q

How does an alkali pH cause denaturation?

A

The OH- ions disrupt hydrogen bond pairing

23
Q

The effect of alkali pH on Tm

A

Fewer H bonds therefore lower Tm

24
Q

What is mismatch defined as?

A

Defined as base pair which is unable to form hydrogen bonds

25
Q

What is renaturation?

A

Reversal of denaturation

26
Q

What does the formation of structures in renaturation favour?

A

Formation of structures favours energy minimisation driven by change in free energy

27
Q

What is renaturation facilitated by?

A
  • Slow cooling

- Neutralisation

28
Q

What is hybridization?

A

Formation of duplex structure of 2 DNA molecules that have been introduced to one another

29
Q

What is thermodynamically favoured over mismatches?

A

Perfect matches have a higher Tm therefore favoured more than mismatches

30
Q

What allows us to manipulate specificity?

A

Limiting hybridisation between imperfectly matched sequence

31
Q

What is stable under high stringency and what is it determined by?

A

Only complementary sequences are stable and is determined by a:

  • Temperature near Tm
  • Low salt concentration
32
Q

What does nucleic acid hybridisation technique identify?

A

Identifies presence of nucleic acid containing specific sequence of bases

33
Q

What does nucleic acid hybridisation allow?

A

Allows the absolute or relative quantitation of these sequence in a mixture

34
Q

What does hybridisation use to form specific duplexes?

A

Hybridisation uses the ability of nucleic acids to form specific duplexes

35
Q

What do probes detect?

A

Probes detect the presence of complementary nucleic acid by hybridisation

36
Q

What molecules are probes?

A

ssDNA or RNA molecules

37
Q

Length of probes

A

20-1000 bases in length

38
Q

What are probes labelled with?

A

Labelled with a fluorescent or luminescent molecules

39
Q

What does NA blotting technique analyse?

A

Analyses mRNA or DNA

40
Q

Why is nucleic acid blotting technique limited?

A

Limited as they only detect one gene at a time

41
Q

What is nucleic acid blotting technique superseded by?

A

Superseded by PCR

42
Q

What are micro-arrays?

A

Ordered assembly of thousands of nucleic acid probes

43
Q

What are micro-arrays fixed to and what do we do to the sample of interest?

A

Fixed to solid surface then sample of interest is hybridised to the probes

44
Q

What do micro-arrays detect and what are they used in?

A

Detect SNP’s and used in genome wide association studies

Genomics (11 decks)

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