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Fundamentals of Molecular Biology > DNA extraction and enzymes > Flashcards

DNA extraction and enzymes Flashcards

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1
Q

What 3 things must the DNA be separated from during extraction?

A
  1. Lipids
  2. Proteins
  3. Metal cations
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2
Q

Why must metal cations be removed?

A

They are used as cofactors by DNAses.

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3
Q

Which reagents dissolve lipids? Give 3 examples.

A
  1. Phenol
  2. Chloroform
  3. Detergents
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4
Q

Which 4 reagents remove proteins by denaturation and to what degree?

A
  1. Phenol - strongly denaturing.
  2. Chloroform - weakly denaturing.
  3. Detergents - strongly denaturing.
  4. Guanidine thiocyanate - strongly denaturing.
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5
Q

Which 2 reagents are hydrophobic?

A

Phenol and Chloroform.

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6
Q

Which reagent is hydrophilic?

A

Detergent.

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7
Q

Which reagent digests protein as oppose to denature it?

A

Proteinases.

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8
Q

Guanidine thiocyanate is said to be a chaotropic agent - what does that mean?

A

It causes DNA to bind reversibly to silica, allowing it to be separated.

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9
Q

What does EDTA do?

A

Chelates metal cations so they cannot be used by DNAses as cofactors.

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10
Q

Are pH buffers needed in DNA extraction?

A

Yes.

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11
Q

What does alcohol in combination with various salts do to DNA?

A

Causes it to form a solid ppt.

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12
Q

What do commercial resins do?

A

Selectively bind to DNA/proteins.

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13
Q

What are nucleases?

A

Enzymes that cut DNA.

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14
Q

What is an exonuclease?

A

An enzyme that removes nucleotides from the end of a molecule.

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15
Q

What does ribonuclease A do?

A

Attacks ssRNA on the 3’ end of pyramidine nucleotides. This causes unhybridised RNA to be removed.

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16
Q

Under what 2 conditions must enzymes be stored and why?

A
  1. -20 degrees to prevent denaturation

2. In glycerol to prevent oxidation

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17
Q

What are restrictions endonucleases (REs)?

A

Enzymes that cleave DNA internally.

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18
Q

What 3 major conditions are required for REs to work?

A
  1. Mg2+
  2. pH 7-8
  3. 37 degrees
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19
Q

Which RE has a different optimum temperature and what is it?

A

Taq1 requires 65 degrees.

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20
Q

Where do REs cleave DNA?

A

At palindromic recognition sequences.

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21
Q

Does each RE have a specific recognition site?

A

Yes.

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22
Q

What are the 3 major classes of RE and how do they differ?

A
  1. Type I - cleave approx. 1000bp away from the recognition site.
  2. Type II - cleave at the recognition site.
  3. Type III - cleave approx. 24-26bp away from the recognition site.
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23
Q

Where do REs come from?

A

Isolated from bacteria that use then as defence against viral infection.

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24
Q

Why is bacterial DNA unaffected by REs?

A

Bacteria produce methylases with the same recognition sites as the REs. They then methylate the DNA to prevent it from being cut.

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25
Q

How are REs named? Give the 4 main steps.

A
  1. First letter of the genus.
  2. First two letters of the species.
  3. Letter denoting the strain.
  4. Number giving the class of RE.
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26
Q

Type II REs usually cut in a recognisable way. What is this?

A

They tend to leave 5’ terminal phosphates.

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27
Q

There are 3 main types of cut from a RE. What are they and give examples of enzymes.

A
  1. Blunt ends e.g. Bal1
  2. 5’ overhang e.g. Asp718
  3. 3’ overhang e.g. Kpn1
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28
Q

What enzyme can be used to digest overhangs and create blunt ends?

A

S1 nuclease.

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29
Q

What is characteristic about RE molecules and why?

A

They are homodimers that sit anti-parallel. Each homodimer binds to a strand of DNA. As the DNA sequence is palindromic the homodimers are aligned to mimic this.

30
Q

What is a homodimer?

A

A molecule with 2 identical sub-units.

31
Q

Where does the energy for cutting come from in RE reactions?

A

The energy is ‘free energy’ - it is provided by hydrolysis of the sugar-phosphate backbone of the DNA strand.

32
Q

What are isoschizomers?

A

Different REs (often from different organisms) that have the same recognition site.

33
Q

How do you calculate how often an RE cuts? Give 4 steps.

A
  1. There are 4 possible bases
  2. Identify the number of bases in the recognition site.
  3. Multiply 4 by that number, e.g. recog. site contains 4 bases: 4x4x4x4
  4. Answer is how many bases one cut will occur in, e.g. 4x4x4x4=256, one cut occurs every 256bp.
34
Q

What is 1 unit of enzyme?

A

“The amount of enzyme required to cut 1μg of Lambda DNA to completion in 1 hour at 37 degrees.”

35
Q

When does ‘1 unit’ not apply to an enzyme? Give 2 reasons.

A
  1. If that enzyme cannot recognise Lambda.

2. If that enzyme has an optimum temperature other than 37 degrees.

36
Q

What is a DNA polymerase?

A

“Enzymes that synthesise nucleic acids on a pre-existing template.”

37
Q

Which DNA polymerase is most commonly used in vitro for replication?

A

DNA polymerase I.

38
Q

What are the requirements of DNA polymerase I?

A

A primer, nucleotides and a single strand of DNA.

39
Q

DNA polymerase I has 3 main functions. What are they?

A
  1. 5’ to 3’ polymerase for replication
  2. 5’ to 3’ exonuclease for repair
  3. 3’ to 5’ exonuclease for proof-reading
40
Q

What is the Klenow fragment?

A

When DNA polymerase I is digested with subtilisin it produces the Klenow fragment that can solely act as a 3’ to 5’ exonuclease. ALL 5’ TO 3’ ACTIVITY IS LOST.

41
Q

What do DNA ligases do?

A

Join DNA fragments in ligation.

42
Q

Sticky ends are easier to ligate than blunt ends. Which enzyme can be used to join blunt ends?

A

T4 DNA ligase.

43
Q

How does T4 DNA ligase work?

A

It forms an enzyme-AMP complex than binds to 5’ terminal phosphate.

44
Q

What does T4 DNA ligase require as a cofactor?

A

ATP.

45
Q

At what temperature can sticky ends by joined at?

A

12-16 degrees.

46
Q

What does alkaline phosphatase do?

A

Prevents self-ligation by removing the 5’ terminal phosphate group. Thus T4 DNA ligase cannot join blunt ends together.

47
Q

What does T4 polynucleotide kinase do?

A

Adds a 5’ terminal phosphate, reversing the actions of alkaline phosphatase.

48
Q

What is the function of DNA ligase in nature?

A

To seal ‘nicks’ on newly synthesised DNA, repairs breakages between bases on a single strand in the double-stranded molecule.

49
Q

At what temperature does blunt ligation occur?

A

Room temperature.

50
Q

In what process will you find DNA dependent RNA polymerases?

A

Transcription.

51
Q

In what process will you find DNA dependent DNA polymerases?

A

Replication.

52
Q

In what process will you find RNA dependent DNA polymerases?

A

Reverse transcription by retroviruses.

53
Q

From what organism does DNA polymerase I originate?

A

E. coli.

54
Q

From what organism does Taq polymerase originate?

A

Thermus aquaticus.

55
Q

What is Amplitaq?

A

A GMed version of taq polymerase that is thermostable between 37-94 degrees.

56
Q

What is amplitaq used in?

A

PCR.

57
Q

What is SEQUENASE?

A

The brand name for bacteriophage T7 polymerase.

58
Q

What is bacteriophage T7 polymerase composed of?

A

It is a hybrid polymerase made from 2 sub-units: thioredoxin from the E.coli host and T7 polymerase from the bacteriophage.

59
Q

What is SEQUENASE used for and why?

A

DNA sequencing as can synthesise very long chains.

60
Q

What is SEQUENASE 2.0?

A

SEQUENASE lacking the 3’ to 5’ exonuclease activity, this is to make it faster.

61
Q

Where does reverse transcriptase come from?

A

Retroviruses.

62
Q

What is the problem with reverse transcriptase?

A

It is v. error prone.

63
Q

What is reverse transcriptase used for?

A

Creating cDNA from RNA to build cDNA libraries.

64
Q

Where does S1 nuclease originate from?

A

The fungus A. oryzae

65
Q

What enzyme can be used to open hairpin loops in newly synthesised cDNA?

A

S1 nuclease.

66
Q

Where does ribonuclease A come from?

A

Bovine pancreases.

67
Q

What does deoxyribonuclease do?

A

Randomly cleaves ss and ds DNA.

68
Q

What does deoxyribonuclease require?

A

Mg2+.

69
Q

What are the 3 uses of deoxyribonuclease?

A
  1. Introduces random nicks in DNA for radio labelling.
  2. Generates random sequences for cloning.
  3. Analyses DNA-protein complexes in footprinting.
70
Q

What is DNA footprinting?

A

A study of the specificity of DNA-binding proteins.

Fundamentals of Molecular Biology (15 decks)

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