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Nucleic Acid Biochem II > 13-14 Labelling and Restriction Analysis > Flashcards

13-14 Labelling and Restriction Analysis Flashcards

200-203 248-250 216-219

1
Q

What is the melting temperature of nucleic acids (Tm)?

A

The temperature at which half of the nucleic acid strands are denatured

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2
Q

What two things make duplex DNA want to be in a random coil?

A
  • Electrostatic repulsion of phosphate groups

- Higher entropy of random coil

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3
Q

What two things make duplex DNA want to be in a double helix formation?

A
  • Hydrogen bonding between base pairs

- Base stacking (van der waals interactions)

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4
Q

What three factors affect hybrid stability? And in what way?

A
  • Base composition (GC content)
  • Length of nucleic acid sequence (longer = more stable)
  • Sequence similarity between hybrids (mismatching = less stable)
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5
Q

What is the temperature at which DNA achieves the maximum rate of DNA-DNA reassociation? What is this temperature referred as?

A

25 degrees Celsius below Tm

This temp is also known as the annealing temperature

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6
Q

What two nucleic acid types are hybridized in southern blots?

A

DNA and DNA

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7
Q

What two nucleic acid types are hybridized in northern blots?

A

DNA and RNA

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8
Q

What type of nucleic acid analysis is most likely to be used for gene detection and mapping of complex genomes and in gene expression studies?

A

Hybridization analysis

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9
Q

What are DNA microarrays?

A

Hybridization analysis involving microscopic DNA spots attached to a solid surface

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10
Q

What are three types of hybridization analysis?

A
  • Southern blot (DNA/DNA)
  • Northern blot (DNA/RNA)
  • DNA microarray
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11
Q

What type of nucleic acid analysis involves fixing complex nucleic acid samples to solid surfaces?

A

Hybridization analysis

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12
Q

What are the two broad steps in hybridization analysis?

A
  • Complex nucleic acids are fixed to a solid surface

- They are then probed with a nucleic acid sequence of interest

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13
Q

What is the attachment of radioactive, fluorescent or other type of marker to DNA molecules?

A

DNA labelling

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14
Q

What is the DNA labelling method of incorporating radioactive nucleotides along the length of a fragment of DNA called?

A

Random primer labelling

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15
Q

How does random primer labelling work? What can strands treated with these be used for?

A
  • A duplex strand of DNA is denatured
  • A random primer is annealed
  • The primers are extended by Klenow fragments (of DNA pol I) in presence of radioactive precursors
  • DNA polymerase I finishes polymerization, result is two duplex strands of DNA with radioactive primers in two daugher strands

If these strands are melted they can be used as probes

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16
Q

What is nick translation labelling?

A

A labelling technique in molecular biology in which DNA Polymerase I is used to replace some of the nucleotides of a DNA sequence with their labeled analogues, creating a tagged DNA sequence which can be used as a probe in Fluorescent in situ hybridization or blotting techniques.

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17
Q

How is nick translation labelling done?

A
  • DNase I randomly nicks one strand of DNA
  • DNA Pol I makes new DNA in 5’ to 3’ direction with radioactively labelled nucleotides starting from nick while removing non-radioactive nucleotides ahead
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18
Q

What are four types of radioactive elements used in nucleic acid labelling?

A
  • 32P
  • 33P
  • 35S
  • 3H
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19
Q

When labelling nucleic acid with 35 sulfur (35S), where does this element go?

A

On the primary phosphate, replacing an oxygen.

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20
Q

What two techniques for detecting radioactively labelled nucleic acid molecules?

A
  • Autoradiography (x-ray film)

- Phosphorimaging (phosphorescent screen)

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21
Q

What are two techniques for detecting nucleic acids without radioactive labels?

A
  • FLuorescense (dyes)

- Chemiluminescence (reaction between label and additional chemicals makes light, detected by film)

22
Q

What are the steps of southern blotting? (5)

A
  • Gel electrophoresis
  • NaOH denature
  • Transfer to nitrocellulose membrane
  • Incubation of nitrocellulose bound DNA in 32P labeled DNA or RNA or a specific sequence
  • Autoradiography
23
Q

What components are needed for transfer of DNA from agarose gel to nitrocellulose membrane for southern blotting? (in order - 6)

A
  • Weight
  • Paper towels
  • Nitrocellulose
  • Wick
  • Buffer
  • Gel electrophoretogram
24
Q

What are the steps of northern blotting? (5)

A
  • Extract RNA from cells
  • Denature
  • Agarose gel electrophoresis
  • Blotting,
  • Northern hybridization autoradiography with DNA probe hybridizing to RNA transcript
25
Q

What is northern blotting primarily used to detect? What type of probe is used?

A

mRNA transcripts

A DNA probe is used

26
Q

What are the four steps of microarray?

A
  • mRNA from cells are isolated at two times, representing all genes expressed in the cells at these times
  • mRNA are converted to cDNAs by reverse transcriptase using fluorescent labeled DNTs
  • cDNAs are added to microarray and fluorescent cDNAs anneal to complementary sequences on the microarray
  • Each fluorescent spot represents a gene expressed in the cells
27
Q

What does each fluorescent spot of a microarray represent?

A

A gene expressed in the cell

The gene expressed will be one that is expressed at both stages of the cell’s development/lifecycle. That is what is being detected, Green spots indicate early stage, yellow both stages and red represents later stage.

28
Q

Do restriction endonucleases only catalyze duplex DNA at specific sites?

A

No, they can cleave at non-specific base sequences as well.

The name ‘restriction’ comes from ‘modification’

29
Q

How are restriction endonucleases used in bacteria?

A

Bacteria methylate their own DNA and use restriction endonucleases to cleave foreign DNA (eg. viral DNA)

30
Q

In bacteria, restriction endonucleases work in pairs with ____ to protect the cell against bacteriophages

A

Strain-specific restriction enzymes and methylases work in pairs

31
Q

How many types of restriction endonucleases are there?

A

3

32
Q

What does a type I restriction endonuclease do?

A
  • Methylase and endonuclease activity in one enzyme
33
Q

Do type I restriction endonucleases need ATP to cleave DNA?

A

Yes

34
Q

Where are cleavage sites for type I restriction endonucleases?

A

Outside of the sequences recognition site, sometimes up to 10 kbp away.

35
Q

What does type II restriction endonuclease do?

A
  • It only has endonuclease activity
36
Q

Where do type II restriction endonucleases cleave on a sequence of DNA?

A

Within the recognition site

37
Q

Do type II restriction endonucleases need ATP?

A

No

38
Q

What are two types of ends that restriction endonuclease enzymes can generate?

A
  • Sticky ends (with a 3’ or 5’ overhang)

- Blunt ends (no overhags)

39
Q

Do type III restriction endonucleases have methylation activity?

A

Yes

40
Q

Where do type III restriction endonucleases cleave on a DNA sequence?

A

Only about 25 base pairs away from recognition site

41
Q

What are palindromic DNA sequences?

A

Strand-wise symmetrical about a centre of symmetry

42
Q

Are restriction enzyme recognition or cleavage sites commonly palindromic?

A

Recognition sites, though this is the same thing for type II restriction endonucleases

43
Q

What are restriction enzymes that recognize the same DNA sequence called? Do they cleave at the same position?

A

Isoschizomers

They do not necessarily cleave at the same positions

44
Q

In gel electrophoresis, DNA moves towards what electrode?

A

The anode

45
Q

What two things allows electrophoresis to separate DNA fragments on the basis of size alone

A
  • DNA has one unit charge/residue (eg. charge is proportional to length)
  • The molecular sieving effect determines the relative mobility of DNA molecules at a given gel concentration
46
Q

Mobility of DNA fragments is inversely proportional to ________?

A

Mobility of DNA fragments is inversely proportional to the logarithm of their molecular weight

47
Q

What are two types of gel used in molecular biology for electrophoresis?

A
  • Agarose (large nucleic acids)

- Polyacrylamide (proteins and small nucleic acids)

48
Q

What is an advantage of polyacrylamide gel electrophoresis for nucleic acid analysis?

A

It can resolve fragments that differ by as little as one nucleotide in length

49
Q

How are DNA molecules restriction enzyme mapped?

A

By digesting with two different restriction endonucleases. Three samples are made.

  • One digested with the first enzyme
  • One digested with both
  • One digested with the second enzyme

This is usually only feasible for small enzymes with few cut sites

50
Q

What is an ORF map?

A

An open reading frame map. It can be used to determine potential protein-coding regions in DNA by looking at all 6 possible frame translations, indicating possible start and stop codon locations for each.

51
Q

What is a Klenow fragment and what is it used for?

A

A enzymatically cleaved portion of DNA pol I. Can polymerize in 5’ to 3’ direction and exonucleate in the 3’ to 5’ direction, but can’t exonucleate in the 5’ to 3’ direction.

It is used to make probes with random primer labelling

Nucleic Acid Biochem II (8 decks)

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