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Molecular Genetics > Week 3 > Flashcards

Week 3 Flashcards

Enzymes and PCR

1
Q

Polymerases

A

Make new DNA

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2
Q

Ligases

A

Join pieces of DNA

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3
Q

Nucleases

A

Involved in DNA repair and cell protection by cleaving phosphodiester bonds between base pairs.

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4
Q

How do nucleases work ?

A

Nucleases work through two mechanisms: - Through hydrolysis nucleases remove H+ from H2O, and use the OH- to break the phosphodiester bond. - The other uncommon way is using lyase, this breaks a C-O bond, then the P-O bond. Both methods leave 5’ Phosphate and a 3’ OH

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5
Q

What would be at the 5’ and 3’ end of a DNA molecule that has been exposed to a nuclease?

A

5’ would have a phosphate group 3’ would have a hydroxyl group

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6
Q

What are the 3 class specific nucleases? briefly explain how each works

A

Damage specific nucleases: Recognizes the presence of any DNA then binds and scans for damage. Structure specific nucleases: Recognizes the intermediates generated by DNA repair mechanisms Sequence specific nucleases: Recognizes a particular sequence of DNA

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7
Q

What are the 2 “approach” specific nucleases? briefly explain how each works

A
  • Exonuclease: hydrolyses from either the 5’ end or the 3’ end of either ssDNA or dsDNA. - Endonuclease: hydrolyses an internal Phophodiester bonds
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8
Q

What cation is required for nucleases to function?

A

Mg2+. If there is no Mg2+ then there will not be any nuclease activity. We can make Mg2+ unavailable by adding chelators such as EDTA.

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9
Q

What is the most commonly used nuclease and where is it traditionally sourced from?

A

Most commonly used nuclease is DNase I. It is traditionally sourced from bovine pancreas.

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10
Q

What are the characteristics of DNase I?

A
  • Cleaves ssDNA or dsDNA - Endoculease (cleaves near C or T bases) - Cleavage results in di, tri and tetranucleotides. - Acts differently depending on which cofactor is present.
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11
Q

Explain how the presence of Mg2+ and Mn2+ differ the effect of DNase I.

A
  • If Mg2+ is present Cuts each strand independently leaves random fragments -If Mn2+ is present Cleaves each strand close to each other Leaves dsDNA with 1 to 2 nucleotide overhangs
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12
Q

What is DNase I used for?

A

To remove DNA from a solution by hydrolysing phosphodiester bonds between bases causing degredation.

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13
Q

What does Exonuclease III do? and where is it from?

A
  • from E. coli , It is a exonuclease that removes mononucleotides from the 3’ termini of dsDNA. It prefers to work on blunt or 5’ overhangs but will act on 3’ overhang or nicks. Will not work on ssDNA. Used to preferentially remove dsDNA from a solution.
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14
Q

What does Nucease S1 do? and where is it from?

A

From aspergillus (a fungus), Is an exonuclease specific to concentration. It is used to remove ss overhangs (on dsDNA) to leave blunt ends.

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15
Q

what are Restriction endonucleases (RE)?

A
  • sequence specific
  • cuts DNA in a repeatable way into pieces of varying length
  • used in cloning to enable DNA from two different sources to be prepared for joining (using ligase)
  • Involved in host restriction-modification systems
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16
Q

What is ligase used for?

A
  • Used to join okazaki fragments during DNA replication
  • catalyses the formation P-O bonds between the 5’ P and 3’ OH of two strands
  • joins strands with compatible overhangs, or blunt end strands
  • In the lab they are used to create recombinant molecules
  • Sealing nicks in dsDNA
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17
Q

What is the most common type of Ligase?

A

T4 DNA ligase (taken from T4 bacteriphage)

it requires ATP as a cofactor.

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18
Q

What do polymerases do?

A

Catalyse the addition of a dNTP to the 3’ end of a DNA or RNA primer, the indentity of the dNTP is dependent of the template.

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19
Q

What is the main requirement for DNA polymerase to work?

A

-Must have a free 3’ end on the primer to work

-

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20
Q

What are the 3 different catalytic activities for DNA polymerase I?

A
  1. 5’ to 3’ polymerase - adds nuceleotides.
  2. 5’ to 3’ exonuclease - usually problematic but useful as it removes primer, then we fill the primer absence back in and ligase connects it back together.
  3. 3’ to 5’ exonuclease - If 5’ to 3’ polymerase puts in the wrong base, 3’ to 5’ exonuclease can remove the wrong base.
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21
Q

What is Klenow fragments used for?

A

Klenow Fragment = 5’ to 3’ polymerase and 3’ to 5’ exonuclease only.

  • Add bases to the 3’ end of the DNA fragments to fill in overhangs
  • Remove 3’ overhangs to create blunt ends
22
Q

explain processivity in relation to the T4 and T7 DNA polymerases

A

T4: More active but has less proccessivity than T7

T7: Higher processivit than T4 but less active

23
Q

What does the term processivity mean?

A

Processivity is the capacity to stay on the molecule. For example, a high processivity makes more DNA per binding event.

24
Q

What is Terminal Transferase and what are some characterists for it?

A

Used to create overhangs for cloning.

  • Doesnt require a template
  • Does require a primer
  • Adds bases to the 3’ end of DNA (e.g. Poly A tail)
  • Prefers 3’ overhangs
  • Blunt or 5’ overhangs are OK
  • Cofactor is cobalt
25
Q

What are Thermostable DNA polymerases?

A
  • Resistant to degradation by heat
  • Same range of properties as normal polymerases
  • Sourced from thermophilic bacteria

- Work best as 60 to 75 degrees celcius

26
Q

If we wanted to remove the primer, which polymerase would we use?

A

Pol I, as we need to have 5’ to 3’ exonuclease

27
Q

What are bacterial (host) restriction modification systems?

A
  • A primitive bacterial “immune system”
  • occur only in bacteria, a few bacteriophages and the unicellular algae Chorella
  • Uses Restriction endonuclease and Methylase to stop invading DNA
28
Q

In more detail, how do restriction endonuclease and methylase work together in relation to the bacterial RMS?

A

The restriction endonuclease acts as a frontline defence for the cell by digesting any invading DNA. It wont cut/digest its own DNA because methylase will methylate the recognition site protecting it against the RE.

29
Q

Why must must methylase be expressed before restriction endonuclease? (in relation to RMS)

A

If the RE was to be expressed before Methylase, then the cell would kill itself. Therefor Methylase must be expressed first so that it can methylate the DNA so that the RE will not digest the “good” DNA.

30
Q

What are the different types of restriction enzymes and what is the most popular one that we use?

A

Type I and Type III:

  • Recognition sequences are asymmetrical
  • Cleave away from the recognition site

Type II:

- Recognition sequences are symetrical (palindromic)

- Cleave within recognition site that is about 4 to 8 NT long

31
Q

What is PCR used for? What does it require? and what are the steps involved?

A

PCR is used to make multiple copies of a specific sequence of DNA. It requires a thermocycler machine that will cylcle a solution containing target DNA, Pimers, Nucleotides, DNA polymerase, A buffer, and Magnesium Chloride. It will repeat the steps; Denaturation, Annealing, and Extension.

32
Q

What happens during PCR Denaturation?

A

At 95 degrees celcius hydrogen bonds break between antiparrelel dsDNA giving us 2 ssDNA strands

33
Q

What happens during PCR Annealing?

A

at 50 to 75 degrees celcius primers (short strands of DNA complementary to the template strand) specific to the target gene are added. Primers will anneal 5’ to 3’ leaving a 3’ end availible for extention.

34
Q

What happens during PCR extension?

A

At 72 degrees celcius polymerase will attach free dNTP’s to the 3’ end of the primer extending the new strand in a 5’ to 3’ direction.

35
Q

What is unique about the 3rd cycle of PCR in terms of defined length?

A

In the first 2 cycles of PCR we will not have any target length molecules, however in the third cycle we will now have double stranded molecules with both strands having defined lengths giving us 2 official target length molecules. (this is due to the primer)

36
Q

In practice, what are the time frames involved with PCR steps?

A

Initital denaturation at 94 degrees celcius for 5 minutes.

then 30 rounds of:

94 degress celcius for 30 seconds

55 degrees celcius for 30 seconds

72 degrees celcius for 45 seconds

37
Q

What are the main characteristics of primers?

A
  • Required for PCR
  • each primer anneals to DNA template with hydrogen bonding
  • DNA synthesised from 5’ to 3’ direction
  • primers are extended from the 3’ end
  • the 3’ ends of primer pairs must “face” each other
38
Q

Identify where the following forward and reverse primers would attach to:

Forward P: GGTAGAGAAGCCATCCTGGCCACC

Reverse P: TGCTGAAAAACCAGAGGCTGAGGG

A
39
Q

Why is it important to have a GC content between 40 and 60% ?

A

So that the hydrogen bonds are easy enough to break (during denaturation)

40
Q

Why should primers be atleast about 15 to 18 nucleotides long?

A

So that they are specific enough, as any shorter they will be nonspecific.

41
Q

How is the melting temperature determined for primers?

A

Determined by the sequence of G+C bonds

42
Q

What are the relationships regarding specificity/yield and annealing temperatures?

A

If we increase the temperature, we increase the specificity.

43
Q

What are the main characteristics of templates in PCR?

A
  • dsDNA or ssDNA can act as a template
  • any length of DNA is OK as long as the area between the primers is intact
  • Purity can vary
  • 10^5 to 10^6 copies of template is ideal
44
Q

What are the main characteristics of dNTPs in PCR?

A

dNTP = Deoxynucleotide triphosphosphate

  • Building blocks incorperated in new DNA strand
  • Ideal concentrations is 50 to 250 microlitres
  • Slight excess is required as some get degraded by heat during rxn
  • too much dNTP can reduce availible Mg2+
  • Equal ammount of each is critical to prevent incorporation errors
45
Q

Explain the main characteristics of Mg2+ in PCR?

A
  • Essential co-factor for polymerase
  • Provides electrostatic shielding of the phosphodiester backbone of the DNA
  • Concentration between 1 to 5 millimoles
  • Delivered as either MgCl2 or MgSO4
  • To increase specificity we degrease Mg2+
46
Q

What are the main characteristics of a Buffer for PCR?

A
  • KCl, NaCl, NH4SO4 allused either alone or in combination
  • pH important (6.8 to 7.8 is standard)
  • buffer is specific to enzyme being used (usually enzymes come with own buffer)
  • will sometimes contain Mg2+ if enzyme optimum range for Mg2+ is limited

-

47
Q

Why are additives sometimes added to PCR solutions?

A

To decrease inhibitation or problems during PCR. Essentially used to increase efficiency

48
Q

How do we examine PCR products?

A

PCR products are detected using electrophoresis.

  • Use DNA stain to visualise the DNA (will bind to major groove of DNA double helix so that once we use UV light we will be able to see the bands)
49
Q

What are the difference between agarose and acrylamide mediums for electrophoresis?

A

Agarose:

  • 100bp to 60Kbp
  • Would only be able to differenciate up to 20 bp in difference

Acryamide:

  • 1bp to 1 Kbp
  • Can distinguish between individual base pair differences
50
Q

What non-specific amplification problems can occur with PCR?

A
  • Self-priming: template and/or primers
  • Too much template
  • Too much Mg2+
  • Annealing temp too low
  • Too much primer
  • Contamination
51
Q

What “no amplification” problems can we have with PCR?

A
  • Primers incorrect
  • Mg2+ too low
  • Annealing temp too high
  • extension time too short
  • nuclease contamination
  • too high dNTP concentration
52
Q
A

Molecular Genetics (4 decks)

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