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Cell Biology > Polymerase Chain Reaction - PCR > Flashcards

Polymerase Chain Reaction - PCR Flashcards

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1
Q

DNA strands are…

A

…antiparallel

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2
Q

PCR is a method of…

A

amplifying small amounts of DNA molecules so that we end up with a large number of molecules and can visualise then by gel electrophoresis and used in gene cloning experiments.

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3
Q

What are the uses of PCR?

A
  • forensic DNA analysis – uses microsatellites to detect genetic variation
  • amplifying DNA from archaeological specimens – bones
  • gene cloning – amplifying genes to place into a plasmid to express a protein/GM
  • medical diagnosis
  • examining genetic variation in conservation biology
  • finding orthologous genes
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4
Q

each strand of the double helix actually is…

A

…antiparallel to its complementary strand

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5
Q

DNA is polymerised only in…

A

…one direction

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6
Q

the 5 and 3 here refer to the …

A

…carbon atoms on the deoxyribose sugar that joins with phosphate groups

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7
Q

DNA polymerase needs …

A

…small primers to start it of - needs a free 3’ end

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8
Q

Define primer

A

short sequences of RNA

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9
Q

WHat are primers made by?

A

made by RNA polymerase

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10
Q

in order to synthesise new strands of DNA several things are needed:

A

1) a complementary strand of DNA (template strand)
2) short ‘oligonucleotide’ primers ( a free 3’-OH end)
3) molecules of the 4 bases dATP, dCTP, dGTP and dTTP
4) a DNA polymerase enzyme
5) magnesium (Mg2+) ions – a cofactor
6) buffer – correct pH for the enzyme to work

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11
Q

in PCR the aim is to …

A

…amplify a certain sequence of DNA

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12
Q

if we know some of the sequence of that DNA we can …

A

…synthesise DNA oligonucleotide primers

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13
Q

if we can then make the DNA strand single stranded we can make …

A

…the primers pair to the single strands

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14
Q

if we have free nucleotides and a DNA polymerase we can then …

A

…make a complementary strand

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15
Q

How many steps of PCR?

A

3

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16
Q

FIrst step of PCR?

A

Denaturation

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17
Q

Describe denaturation

A
  • By heating the DNA to 95°C we can denature the double strand because the weak hydrogen bonds are broken
  • The two strands are separated
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18
Q

Step 2 of PCR?

A

Annealing

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19
Q

Describe annealing

A
  • bring down the temperature to the annealing temperature of the primers
    if we have in excess lots of primer molecules
  • then those primers will anneal to their complementary sequence
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20
Q

Step 3 of PCR?

A

Extension

21
Q

if we now add in a polymerase and the nucleotides dATP, dCTP, dGTP, dTTP then the …

A

…complementary strand will polymerise

22
Q

After extension, we have only doubled the strands – we need more to make a useable amount of DNA, so the process …

A

…goes through another cycle…and another…till we have done ~30 cycles

  • as the process is exponential – we end up with huge numbers of strands
23
Q

most proteins do not like being heated up – they …

A

…denature

24
Q

some bacteria live at …

A

…very hot temperatures (in hot springs for example

25
Q

we use heat stable polymerases isolated from …

A

…the thermophilic bacteria Thermus aquaticus – Taq polymerase

26
Q

because the polymerase is heat stable we can …

A

… continuously cycle the PCR reaction between 3 temperatures

27
Q

What are the three temperatures PCR can be cycled between?

A

1) denaturation temperature
2) annealing temperature
3) extension temperature

28
Q

WHats the denaturation temperature?

A

95 deg c (takes 1 minutes)

29
Q

Whats the annealing temperature?

A

40-65°C usually (takes 1 minute)

30
Q

WHats the extension temperature?

A

72 deg C (time depends on the length of DNA being amplified)

31
Q

Why is extension temperature 72 deg C?

A

the optimum temperature at which Taq works

32
Q

results of PCR can be seen on …

A

…gel electrophoresis

33
Q

the first lane is a …

A

…ladder

34
Q

What are ladders in gel electrophoresis?

A

Size markers

35
Q

The second lane is…

A

…a negative control

36
Q

What is a negative control?

A

no DNA added to the PCR reaction

37
Q

Lanes 3 and 4 are from…

A

…whitefly DNA and both have the virus

38
Q

How many lanes in gel electrophoresis?

A

4 lanes

39
Q

Why do proteins denature at high temp?

A

Changes structure

40
Q

What are the reactants used in PCR?

A

target DNA, primers, polymerase and nucleotides

41
Q

WHat is done with the reactants in PCR?

A

pipetted into a PCR tube

42
Q

After the reactants are pipetted into a PCR tube, the tubes are then placed into a…

A

thermal cycler

43
Q

Function of the thermal cycler used in PCR?

A

which heats the tubes up to the correct temperature for the correct amount of time and ‘cycles’

44
Q

the design of primers is very important and several things need to be taken into consideration:

A

1) obviously the sequence has to be complementary
2) length of primer – usually around 20-25 bases (the longer it is the higher the annealing temperature and the more specific it is)
3) at least 50% of the bases should be G or C (because they have more hydrogen bonds – they increase the annealing temperature)
4) there should not be repeats within the bases
5) the primers shouldn’t be complementary to each other
6) if possible the 3’ (last) base should be a G or C

45
Q

all living organisms have …

A

…DNA genomes

46
Q

In many cases DNA genomes of different organisms have…

A

…different versions of the same genes – express the same or similar proteins which do the same job in the cell – these are called orthologues

47
Q

Orthologues are derived from….

A

…same gene but are only different as they have separated due to a speciation event

48
Q

orthologous genes are …

A

…conserved

49
Q

the closer that species are related to each other (through evolution) the more …

A

…their DNA sequences are similar

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