DNA restriction Flashcards

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1
Q

What are bacterial restriction enzymes (what is their function in nature)?

A

Bacteria have a specific set of enzymes/proteins capable of destroying the DNA of foreign organisms such as viruses (breaking up the DNA as required). These are restriction enzymes and they are capable of recognising specific DNA sequences

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2
Q

How are restriction enzymes named?

A

Using the name of the bacteria species, using the first letter of the genus name and the first two letters of the species name

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3
Q

What does enzyme activity depend on?

A
  1. Temperature
  2. The presence of cofactors
  3. Correct pH and salt conditions

e.g., Restriction enzyme EcoR1 (from Ecoli.) functions at 37 degrees Celsius (like the human body)

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4
Q

How do restriction enzymes work?

A

They recognise specific restriction sites. These are then chopped in such a way that “sticky ends” (overhangs) are produced to enable easier rejoining.
No covalent bonds (this is the phosphodiester bond in DNA) are required as a result.

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5
Q

What are key features of restriction sites?

A

They are typically palindromic sequences (the same when read backwards).
For example:
DNACCGGDNA
DNAGGCCDNA
(Ignore the “DNA” (this is just signifying the rest of the bases), but the CCGG and GGCC are palindromes of each other)

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6
Q

What makes it more likely that the right gene is chopped?

A

The longer the recognition site, the less likely a random gene will be chopped.

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7
Q

What increases with longer restriction sites?

A
  • The likelihood that other different genes are chopped
  • The cleaved fragments produced
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8
Q

What does RFLP stand for?

A

Restriction Fragment Length Polymorphism

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9
Q

How different is DNA between individuals?

A
  • > 90% of DNA sequences are the same in individuals of species
  • Enough of the DNA is different enough to distinguish one individual from another (in the remaining 10% are known alleles of polymorphisms)
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10
Q

How can RFLP be used to distinguish between different individuals?

A

Individual differences within the ~10% of DNA that contains polymorphisms can include differences in restriction enzyme cleaving sites, even if this change doesn’t result in different phenotypes.

One different nucleotide can mean that DNA can be cut with a restriction enzyme in one individual but not another.

This works because the genome is so long - meaning the probability of differences in restriction sites are high

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11
Q

What about the DNA length means that RFLP works?

A

The genome is so long that the probability of differences in restriction sites are high

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12
Q

What is the experimental procedure of RFLP?

A
  1. Extraction of DNA (which contains interesting restriction sites)
  2. PCR (amplifying the extracted DNA)
  3. Restriction digest (this either successfully restricts the DNA strands or cannot if the restriction sites are not present)
  4. Size-separation of DNA fragments by gel electrophoresis
  5. Visualisation of fragments by ethidium bromide staining (in gel) or other techniques (the number of bands indicates if restriction has occurred or not)
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13
Q

What is are two examples of industrial uses of RFLP analysis?

A
  1. Food authentication (for example can confirm that there is no dolphin bycatch etc.)
  2. Could test the origin of a species (e.g., is it invasive (from an aquarium) or did it migrate from a more local area [compare the individual with samples from both the aquarium and possible migration source]. E.g., the invasive Mediterranean green algae which was found to spread from aquaria
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14
Q

How does using PCR make RFLP a more useful procedure?

A

The amplification means that identification between different individuals can be achieved from very small amounts of starting DNA

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15
Q

What does RFLP analysis do in simple terms?

A

It distinguishes between individuals or species using differences in their restriction sites within the ~10% of DNA where polymorphisms of known alleles is present.

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16
Q

How can you find out the results of RFLP analysis?

A

Using gel electrophoresis.

The presence of more than one band indicates that restriction sites are present.