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Genomics > 2. DNA Hybridisation > Flashcards

2. DNA Hybridisation Flashcards

1
Q

RECAP: Where do the nitrogenous base, phosphate group and hydroxyl group join onto?

A
  • Nitrogenous base joined to carbon 1
  • Phosphate group joined to carbon 5
  • Hydroxyl group carbon 3
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2
Q

RECAP: Which nucleotides are pyrimidine and purines?

A

pYrimidines:
• cYtosine
• thYmine

pUrines:
• gUanine
• adenine

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3
Q

RECAP: What about the structure provides specificity of base pairing?

A

The charged or polar groups

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4
Q

RECAP:What type on bonding determines Watson and Crick base pairing? and how many between each base pair?

A

Hydrogen bonds between oppositely charged groups

AT = 2
GC = 3 – So this is a stronger bond
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5
Q

RECAP: How are sugar phosphates linked?

A

Backbone of DNA is formed by phosphodiester linkage. – Connects the 3 and 5 prime carbons of deoxyribose sugar

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6
Q

RECAP: What is the stability of the DNA structure determined by?

A

Free energy of the molecule and energy minimisation.

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7
Q

RECAP: What is base stacking?

A

A form of hydrophobic interactions, arrangement of bases set above each other, which excludes water from the internal structure.

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8
Q

RECAP: What provides structural stability to DNA?

A

Hydrogen bonding of the bases, and internal arrangement and additional stability by base stacking and van der Waals (individually small).

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9
Q

RECAP: What give DNA an overall negative charge?

A

The negatively charged phosphates external

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10
Q

What happens when DNA is denatured? And what causes this to happen?

A

• Conversion of ds molecule to ss molecules – which forms randomly structured coil.
• Caused by disruption of the H bonds
o Occurs when DNA in solution is heated
o Can be induced by strong alkali or urea

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11
Q

How can denaturation be optically measured?

A

Ss DNA absorbs UV light to a greater extent than ds DNA

Hyperchromicity: Increased absorption of light at 260nm on denaturation

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12
Q

What is Tm?

A
  • The melting temperature. The point at which 50% of all strands separate.
  • This characteristic is specific to an individual double helical structure and we can use this knowledge to control formation of the duplex
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13
Q

What does the stability and Tm of a DNA molecule depend on?

A
  • GC content
  • Length of molecule
  • Salt conc
  • pH (alkali is a denaturant)
  • No of mismatches (unmatched base pairs)
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14
Q

What is the relationship between GC content and Tm?

A

• Higher GC content= more hydrogen bonds = higher Tm

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15
Q

How can you measure the %GC?

A

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

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16
Q

What is the relationship between molecule length and Tm?

A

Longer contiguous duplex = more hydrogen bonds = higher Tm

17
Q

What is the relationship between Tm and salt conc [Na+]?

A

High [Na+] = High Tm
• Increasing the salt concentration overcomes the destabilising effect of mismatched base pairing – reducing specificity of base pairing at a given temperature

18
Q

What is the relationship between Tm and pH?

A

• Chemical denaturants disrupt hydrogen bonds:
Alkali, formamide, urea
- Dissociated -OH- group disrupts H bonds.

19
Q

What is the relationship between Tm and mismatches?

A
  • A mismatch is defined as a base pair combination that is unable to form hydrogen bonds
  • 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
20
Q

What is renaturation?

A

• The reversal of denaturation
• This occurs as a result of a change in free energy upon:
– Slow cooling
– Neutralisation

21
Q

What is hybridisation? and how is this used to form a stable DNA molecule?

A

Formation of duplex structure of two DNA molecules that have been introduced to one another, for example a short synthetic DNA (or primer) and genomic DNA.
• Perfect matches have a higher Tm
• Are thermodynamically favoured over Mismatches
• Preventing mismatches forming between two molecules can be achieved by performing a hybridisation at the Tm of the duplex molecule

22
Q

What is stringency? what happens under high stringency?

A

Stringency is the concept of manipulating the conditions to select duplexes with a perfect match only.
Only complementary sequences are stable determined by a
• Temperature near Tm
• Low salt concentration

23
Q

Hybridisation is important in which of the many nucleic acid based techniques? And how?

A 
• Northern blotting
• Southern blotting
• Microarrays
• Dideoxy and Next Gen Sequencing
• PCR
• Cloning
These all rely upon the avoidance of mis-matches using Tm and manipulating the conditions under which hybridisation is carried out.
involves use of probes
24
Q

What is a probe?

A

Probes that are used to detect nucleic acids are designed to be complementary to a specific region of a target gene sequence which is unique to that gene

  • 20-1000 bps
  • labelled
  • complementary
25
Q

Describe the process of northern or southern blotting.

A
  • The technique of Southern or Northern blotting uses DNA or RNA respectively that is separated by gel electrophoresis
  • which is then transferred by mass capillary flow to a nylon membrane
  • It is covalently bond to the membrane and then hybridised with a labelled probe
  • The probe can be visualised by some means
26
Q

What is a microarray used for? And how?

A
  • A microarray might be used for gene expression profiling for example a comparison of drug treated cells and untreated cells.
  • Also, assess the presence or absence of millions of individual SNPs simply through hybridisation of genomic DNA to an array. ~ Result: homozygous or heterozygous for each SNP. Used in GWAS.
  • RNA is extracted
  • Labelled
  • Hybridised to the array and the amount and location of the label measured
  • This tells us how much of each and every one of the transcripts in the human genome that are being expressed
    • The intensity of the colour in a microarray shows us the level of hybridisation.
27
Q

What is a microarray used for? And how?

A
  • A microarray might be used for gene expression profiling for example a comparison of drug treated cells and untreated cells.
  • Also, assess the presence or absence of millions of individual SNPs simply through hybridisation of genomic DNA to an array. ~ Result: homozygous or heterozygous for each SNP. Used in GWAS.
  • RNA is extracted
  • Labelled
  • Hybridised to the array and the amount and location of the label measured
  • This tells us how much of each and every one of the transcripts in the human genome that are being expressed
    • The intensity of the colour in a microarray shows us the level of hybridisation.
28
Q

What is a microarray?

A
  • An ordered assembly of thousands 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

Genomics (12 decks)

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