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Genomics > DNA Hybridisation: DNA Complementarity, Hybridisation And Its Application > Flashcards

DNA Hybridisation: DNA Complementarity, Hybridisation And Its Application Flashcards

1
Q

Where does the nitrogenous base of DNA join the sugar?

A

C1

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2
Q

Where does the phosphate group of DNA join the sugar?

A

C5

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3
Q

Where does the hydroxyl group of DNA join the sugar?

A

C3

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4
Q

Which carbons in the pentose ring is the oxygen bridge between?

A

C1-C4

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5
Q

Which are the pyrimidine bases?

A

Cytosine and thymine

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6
Q

Which are the purine bases?

A

Guanine and adenine

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7
Q

What is base stacking caused by?

A

Hydrophobic interactions that change the arrangement of bases set above each other on the inside of the DNA structure

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8
Q

How many hydrogen bonds does the C-G base pairing form?

A

3

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9
Q

How many hydrogen bonds does the A-T base pairing form?

A

2

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10
Q

Under what conditions does DNA denature?

A

When the DNA in solution is heated or exposed to a strong alkali and urea

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11
Q

What does the single DNA strand look like?

A

A randomly coiled string

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12
Q

How can you measure the denaturation point of DNA?

A

Using optical density at 260nm

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13
Q

What happens to the absorbance of DNA at higher temperatures?

A

Increases

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14
Q

Why does the absorbance of DNA increase at higher temperatures?

A

Because single stranded DNA absorbs more

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15
Q

What is hyperchromicity?

A

Increased absorption of light at 260nm on denaturation

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16
Q

What is the melting temperature (Tm) of DNA?

A

The point at which half of the DNA strands have separated

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17
Q

What factors determine the melting temperature of DNA?

A

GC content, length of DNA molecule, salt concentration, pH and mismatches

18
Q

How does GC content determine the melting point of DNA?

A

Higher GC content (more guanine-cytosine bps) -> more H bonds -> higher melting point

19
Q

How does length of the DNA molecule determine the melting point of DNA?

A

Longer = higher melting point

20
Q

Why does length of the DNA molecule determine the melting point of DNA?

A

More H bonds means it’s more stable

21
Q

How does salt concentration determine the melting point of DNA?

A

Salt stabilises the DNA helix so higher sodium conc increases the melting point

22
Q

What does high salt conc reduce in DNA at a given temperature?

A

The specificity of base pairing

23
Q

How does pH affect the melting point of DNA?

A

Alkalinity disrupts the H bonds and lowers the melting point

24
Q

How do mismatches affect the melting point of DNA?

A

More mismatches mean a lower melting point

25
Q

Why do mismatches affect the melting point of DNA?

A

Less H bonds, so less stable

26
Q

What’s the opposite of denaturation?

A

Renaturation

27
Q

What is renaturation facilitated by?

A

Slow cooling and neutralisation

28
Q

What is the basis for specificity of DNA renaturation?

A

Complementarity

29
Q

What is stringency?

A

Limiting hybridisation between imperfectly matched sequences

30
Q

What does stringency allow us to manipulate?

A

Specificity

31
Q

What is high stringency determined by?

A

A temperature near the melting point or a low salt conc

32
Q

What happens to DNA at high stringency?

A

Only complementary sequences are stable

33
Q

What techniques do complementarity and hybridisation underlie?

A

Northern and southern blotting, microarrays, dideoxy (Sanger) and next gen sequencing, PCR and cloning

34
Q

What is northern and southern blotting?

A

An analysis of mRNA or DNA

35
Q

What limits the use of northern and southern blotting?

A

It can only detect one gene at a time in small numbers of samples, which makes it slow. Also gel based so time consuming and messy

36
Q

How do microarrays work (basic)?

A

Thousands of nucleic acid probes fixed to a solid surface, then a sample of interest is hybridised to the probes

37
Q

What do nucleic acid hybridisation techniques allow?

A

The absolute or relative quantitation of sequences in a mixture

38
Q

Basically, how do nucleic acid techniques work?

A

Label a probe and hybridise it in a mixed population of DNA. This captures specific DNA sequences

39
Q

How does hybridisation form specific duplexes?

A

Using the complementarity of nucleic acids

40
Q

What are the characteristics of a probe DNA molecule?

A

Single stranded, 20-1000 bases in length, labelled with a fluorescent or luminescent molecule

41
Q

What is the process of northern or southern blotting?

A

Extract DNA or RNA
Gel electrophoresis
Transfer to nylon membrane
Add labelled probe and let it hybridise to the sample
Detect hybridisation using electrophoresis

Genomics (16 decks)

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