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Genomics SGUL Sem 3 > DNA Hybridization > Flashcards

DNA Hybridization Flashcards

1
Q

What is a pentose sugar?

A

5 carbons that form a cyclical structure with an oxygen bridge

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2
Q

What are the pyrimidines

A

Cytosine, Thymine, Uracil

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3
Q

What are the purines?

A

Adenine, Guanine

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4
Q

On what basis does hydrogen bonding form?

A

Watson and Crick base pairing

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5
Q

Between what two groups does bonding occur?

A

Purines and pyrimidines

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6
Q

How many hydrogen bonds are there between T and A?

A

2

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7
Q

How many hydrogen bonds are there between C and G?

A

3

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8
Q

Between what base pairs is hydrogen bonding the strongest and why?

A

C and G, 3 H bonds

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9
Q

What are sugar phosphates linked by?

A

Phosphodiester bonds

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10
Q

What kind of interactions does base stacking involve?

A

Hydrophobic interactions

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11
Q

Why are bases stacked?

A

To exclude water from the inside of the molecule around the bases

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12
Q

Describe what the Van der Waals forces are like in DNA?

A

Individually small but they contribute to the stability

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13
Q

What is the backbone of DNA formed by?

A

Phosphodiester linkage

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14
Q

What do phosphodiester bonds join?

A

3 and 5 prime carbons of deoxyribose of DNA

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15
Q

What does the stability of DNA depend on?

A

Free energy of the molecule
Energy minimisation

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16
Q

What do phosphate groups interact with?

A

Proteins

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17
Q

What bonds can be broken to denature DNA?

A

Hydrogen bonds

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18
Q

What disrupts hydrogen bonding?

A

Alkalis
Heat

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19
Q

What is hyperchromicity?

A

Increased absorption of light at 260nm on denaturation

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

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20
Q

What is Tm?

A

The point at which 50% of all strands separate is called the melting temperature

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21
Q

What increases as the molecule is denature

A

Absorption

22
Q

What 5 things affect Tm?

A

GC content
Length of molecule

Salt concentration
pH
Mismatches

23
Q

What does a higher Gc content mean?

A

More hydrogen bonds
Higher Tm

24
Q

What is the relationship between the length of DNA and Tm?

A

The longer the contiguous duplex the higher the Tm

More Hydrogen bonds within the molecule greater stability

25
Q

Up until what point does adding more hydrogen bonds increase stability?

A

Up until 300 bp

26
Q

What does high salt do to DNA duplexes?

A

Stabilizes them

27
Q

What is the relationship between Na+ and Tm?

A

High Na+ = High Tm

28
Q

What does increasing the salt concentration do to the stability and Tm?

A

Stabilizes the structure and increases the Tm

Overcomes the effect of mismatched base pairing reducing specificity of base pairing in high salt environments

29
Q

What does increasing salt do to base pairing?

A

Reduces the specificity of base pairing at a given temperature

30
Q

What are 3 chemicals that disrupt hydrogen bonds?

A

Urea
Formamide
Alkali

31
Q

What kind of pH destabilizes DNA duplexes?

A

High

32
Q

What is a mismatch?

A

A base pair that is unable to form hydrogen bonds

33
Q

Why do mismatches weaken DNA?

A

→ It reduces the number of hydrogen bonds

→ Fewer H bonds means a lower Tm

34
Q

How do mismatches destabilize base pairing?

A

→ They allow other molecules to penetrate within the structure of the molecule

→ Shorter contiguous stretches of double stranded sequence = lower Tm

→Distorts the structure and destabilises adjacent base pairing

35
Q

What is the reverse of denaturation called?

A

Renaturation

36
Q

What is renaturation facilitated by?

A

Slow cooling and neutralization

37
Q

What is hybridization?

A

Renaturation but with the introduction of a foreign molecule into a solution

38
Q

Why is renaturation favored?

A

Formation of the structure favors energy minimization which is driven by a change in free energy (∆G)

39
Q

Why do perfect matches have a higher Tm?

A

→ They are thermodynamically favored over mismatches
→ Mis-matches destabilise DNA and reduce the Tm

40
Q

How can you manipulate specificity?

A

Limiting hybridization between imperfectly matched sequences allows to manipulate specificity

41
Q

What is high stringency?

A

→ Temperature near the Tm
→ Low salt concentration

42
Q

How does nucleic acid hybridization work?

A

→ Identifies the presence of nucleic acid containing a specific sequence of bases

→ A labelled molecule is used to investigate a mixed population of molecules to find out how many of a specific sequence there are

43
Q

How do you find a specific strand within DNA with a probe?

A

Label a probe and hybridize it with a strand you are looking for that corresponds to a gene

44
Q

What is a probe?

A

→ A single strand of DA

→ Labelled with a fluorescent molecule

→ Used to detect nucleic acids in a diagnostic

→ Under high stringency conditions will form a perfectly matched duplex with their complement

45
Q

Why is PCR used over gel techniques?

A

→ Gel techniques are messy

→ Only detects one gene at a time and small numbers of samples

46
Q

How does a microarray work?

A

→ Ordered assembly of probes are attached to the surface of a glass matrix

→ Hybridization is used to attach the complementary molecules to the surface of the matrix

→ Label visualised and measured

→ This can measure 50,000 transcripts in a cell

47
Q

What are SNPs?

A

→ Single nucleotide polymorphisms are the most common type of genetic variation among people

→ Each SNP represent a difference in a single nucleotide

48
Q

What does the denaturation of a DNA duplex depend on?

A

Upon the stability of the structure determined by its sequence of bases

49
Q

What do nucleic acid based techniques rely on?

A

→ The specificity of complementary of base pairing

→ The avoidance of mis-matches by using stringency and the calculated Tm of the duplex

→ Manipulating the conditions under which hybridisation is carried out

50
Q

What are the characteristics of a probe?

A

→ Always single stranded

→ Can be made from either DNA or RNA depending on the technique

→ Usually between 20 and 1000 bases in length but relative short synthetic molecules

→ Made chemically

→ Labelled with a fluorescent, or luminescent molecule

51
Q

Describe the process of Southern/Northern blotting.

A

→ DNA or RNA that is separated by gel electrophoresis

→ Transferred by mass capillary flow of a buffer from a reservoir to a nylon membrane carrying the nucleic acid with it

→ Captured by and covalently bonded to the membrane

→ Then hybridised with a labelled probe

Genomics SGUL Sem 3 (21 decks)

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