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Lab Skills > PCR > Flashcards

PCR Flashcards

1
Q

What are the three steps of PCR?

A

Denaturation, annealing and extension

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2
Q

What happens in denaturation?

A

The double strand DNA templates are heated to separate the strands.

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3
Q

What happens in annealing?

A

Primers bind to flanking regions on the target DNA.

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4
Q

What happens in extension?

A

DNA polymerase extends from the 3’ end of each primer along the template strand.

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5
Q

How many cycles of PCR are usually done?

A

25-35

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6
Q

In what direction is the DNA amplified?

A

5’-3’

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7
Q

Why are Klenow fragments of DNA polymerase I from E coli no longer used in PCR?

A

As they are heat sensitive and so are destroyed at the high temperatures required for PCR

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8
Q

Which DNA polymerase is used now?

A

Taq DNA polymerase from Thermus aquaticus.

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9
Q

What are the drawbacks of Taq Polymerase?

A

It is unstable above 90 degrees and doesn’t proofread

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10
Q

What is the function of thermocyclers?

A

It automates temperature cycling and incubation times.

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11
Q

When were thermocyclers first introduced?

A

1985

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12
Q

From what sources can the template DNA be taken?

A

Any source i.e. genomic DNA, complementary DNA or plasmid DNA

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13
Q

What is the equation for copy number?

A

Avogadro’s constant x no. moles

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14
Q

What are the 7 components to a PCR reaction?

A
  • PCR buffer
  • dNTPs
  • MgCl2
  • Primers
  • Taq DNA
  • Sterile distilled water
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15
Q

In what order should the components be added to the PCR solution?

A

Start with the material that has the highest volume and work through to the lowest.

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16
Q

How long are primers?

A

Around 15-30 bases

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17
Q

What is the usual Tm range?

A

55-70 degrees and the two primers must be within 5 degrees of each other.

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18
Q

What should be avoided in PCR?

A
  • Hairpin DNA
  • Self dimers
  • Repeats of more than four nucleotides
  • GC-rich ends
19
Q

What are the optimal conditions for PCR?

A
  • 15-30 nucleotides long
  • A Tm of 55-70
  • 40-60% GC
  • One G or C at the 3’ end
20
Q

In what proportions are the four nucleotides added to the PCR solution?

A

In equimolar quantities

21
Q

When may an unbalanced concentration of dNTPs be used?

A

In order to promote a higher degree of mis incorporation by non-proofreading DNA polymerase.

22
Q

What is the recommended concentration of each dNTP?

A

0.2 mM

23
Q

Why is a higher concentration of dNTP used in the presence of Mg2+?

A

As Mg2+ binds to the dNTPs and reduces their availability for incorporation.

24
Q

With what are dNTPs sometimes replaced?

A

dUTP

25
Q

What is the function of magnesium in PCR?

A

It has cofactor activity with DNA polymerase enabling incorporation of dNTPs during polymerisation.

26
Q

What does the Mg2+ active site catalyse?

A

Phosphodiester bond formation between the 3’ OH primer and the phosphate groups of the dNTPs.

27
Q

What is the function of buffers in PCR?

A

To provide a suitable chemical environment for the activity of DNA polymerase.

28
Q

What pH is buffered in PCR?

A

pH 8-9.5

29
Q

What is pH stabilised by in PCR?

A

Tris-HCl

30
Q

What is reverse transcription?

A

When RNA is isolated from a sample and mRNA is converted back to cDNA

31
Q

Why is endpoint PCR performed?

A

To quantitate RNA expression.

32
Q

What can PCR be used to detect in genotyping?

A

Sequence variations in alleles of specific cells.

33
Q

How is the primer altered for use in genotyping?

A

It is designed to flank regions of interest and assess genetic variations based on the presence or absence of amplicons and/or their length.

34
Q

What is sequencing of PCR amplicons used to study?

A

Single nucleotide variations and single-nucleotide polymorphisms.

35
Q

What can PCR be used to clone?

A

DNA fragments of interest.

36
Q

What happens in direct PCR cloning?

A

Desired regions of the DNA source are amplified and inserted into specifically designed vectors. Or primers with extra nucleotides at 5’ end.

37
Q

Why is it recommended to purify the DNA sequence?

A

To remove excess reagents and non-full length oligonucleotides.

38
Q

What is PCR cloning used for?

A

To screen whether colonies carry a desired insert.

39
Q

What is mutagenesis?

A

Cloning to introduce desired mutations.

40
Q

What is site directed mutagenesis?

A

Primers that incorporate base substitutes, deletions or insertions.

41
Q

What important considerations are needed in site directed mutagenesis?

A

Primer design, DNA polymerase choice and RNA parameters.

42
Q

Why are high-fidelity PCR runs recommended when preparing sequencing templates?

A

To maintain DNA accuracy

43
Q

What is Sanger sequencing?

A

When PCR amplifies fragment of purified DNA and subjects them to sequencing reactions.

44
Q

How is the DNA altered in sequencing?

A

Primers are tagged at the 5’ end with binding sites for sequencing primers.

Lab Skills (1 decks)

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