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Molecular Biology Lab. > Lab 5. > Flashcards

Lab 5. Flashcards

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1
Q

What is PCR?

A

A method of amplifying a specific gene from template DNA without using a living organism.

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2
Q

Who invented PCR?

A

Carey Mullis.

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3
Q

What are the 3 steps involved in PCR?

A

Denaturation.

Annealing.

Extension.

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4
Q

How many times are the 3 steps usually repeated during a typical PCR experiment?

A

Around 30-40 times.

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5
Q

What are the primers that are used in PCR?

A

Single-stranded DNA molecules that bind to DNA strands and help RNA polymerase attach to the same strand.

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6
Q

How many nucleotides do the primers usually consist of?

A

Between 17-25 nucleotides.

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7
Q

What happens during a PCR experiment is the primer design is bad?

A

If primer design is bad then there may be no PCR product produced.

Poorly designed primes could also amplify many unwanted DNA fragments.

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8
Q

What 3 things does DNA amplification allow for?

A

DNA identification and manipulation.

The detection of infectious organisms within the body e.g. viruses.

The detection of mutations.

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9
Q

What happens during the denaturation step of PCR?

A

The DNA is denatured from double-stranded DNA to single-stranded DNA.

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10
Q

What happens during the annealing step of PCR?

A

The primers are annealed to their complementary regions on the single-stranded DNA molecules.

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11
Q

What happnens in the extension phase of PCR?

A

The primers are extended into a complementary DNA strand by a polymerase enzyme.

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12
Q

What are the 3 temperature that each step of PCR takes place at?

A

Denaturation takes place at 94 degrees C.

Annealing takes place at 60 degrees C.

Extension takes place at 70 degrees C.

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13
Q

What is the goal of designing good primers?

A

The specific amplification of a DNA sequence.

To obtain high yields of copied DNA.

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14
Q

Why are primers of a specific length?

A

This length is long enough to be specific.

It is also short enough for primers to easily bind to the template.

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15
Q

What does primer design require?

A

Extensive sequence analysis through the use of computers.

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16
Q

Primers should only be complimentary to what?

A

To the target sequence.

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17
Q

Why should primers only be complimentary to the target region?

A

So that taq polymerase only copies the target region.

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18
Q

What are the names of the 2 primers that bind to a DNA strand?

A

The forward and reverse primer.

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19
Q

How are the bases found 2 primers always written?

A

In the 5,3 direction.

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20
Q

The forward primer is always complimentary to which DNA strand?

A

The 5 to 3 strand.

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21
Q

The reverse primer is always complimentary to which DNA strand?

A

The 3 to 5 strand.

22
Q

How is the reverse primer always written?

A

In the 5 to 3 direction.

23
Q

What is the Tm (melting temperature)?

A

The temperature at which 1/2 of the DNA molecules are single stranded state and 1/2 are double stranded.

24
Q

What is the annealing temperature for a PCR reaction based on?

A

The melting temperature (Tm) of the primers.

25
Q

At what temperatures will all the DNA

molecules will be in the single stranded form?

A

At temperatures above the Tm.

26
Q

At what temperature is annealing usually carried out?

A

At around 5 degrees below the Tm.

27
Q

Primers with what melting temperatures will usually produce the best results?

A

Primers with melting temperatures between 52-58 degrees.

28
Q

What property do primers with melting temperatures above 65 degrees tend to exhibit?

A

Secondary annealing.

29
Q

What happens if the annealing temperature is too high?

A

The primer will not bind to the target DNA.

30
Q

What happens if the annealing temperature is too low?

A

The primer will bind to sequences which aren’t perfectly complementary.

31
Q

What is the process of primers binding to non-complimentary sequences known as?

A

Mis-annealing.

32
Q

What is the most important factor to consider when designing DNA primers for PCR?

A

They should have Tm’s that are within 5 degrees of each other (the closer the better).

33
Q

The Tm of a DNA molecule is dependent on what?

A

It’s DNA sequence.

34
Q

What feature about the DNA sequence will lead to a higher Tm?

A

The higher the amount of G’s and C’s within the DNA, the higher the Tm.

35
Q

What is the Wallace rule?

A

Tm (in ºC) = 2(A+T) + 4(C+G).

(A+T) = sum of A and T residues in the primer.

(C+G) = sum of C and G residues in the primer

36
Q

What happens if the primers are complementary to each other?

A

They will anneal with themselves.

37
Q

What are the 2 types of potential self-complementary sequences that can be found within primers?

A

Those that lead to hairpins.

Those that lead to primer dimers.

38
Q

How are hairpin structures formed from primers?

A

The primer molecule will pair with itself.

39
Q

Can hairpin primers bind to DNA?

A

No.

40
Q

What primers should be avoided in an effort to avoid the formation of hairpin structures?

A

Any primers that contain more than a string of 3 intramolecular base pairs.

41
Q

How is a primer dimer formed?

A

When 2 primers pair with each other rather than with the DNA molecule.

42
Q

What is heterodimer formation?

A

When the forward primer binds to the reverse primer.

43
Q

What is self-dimer formation?

A

When base pairing occurs between one of the two primers.

44
Q

Primers should be of what length?

A

17-28 bases.

45
Q

Primers should be of what G/C content?

A

50-60%.

46
Q

How should primers end?

A

With a G or C, or CG or GC.

47
Q

Why should primers end with a G or a C?

A

It prevents breathing of the ends and increases the efficiency of priming.

48
Q

What Tm’s are preferred?

A

Tms between 55-80°C.

49
Q

Why should runs of three or more Cs or Gs at the 3’-ends of primers be avoided?

A

As they may lead to mispriming at G or C-rich sequences.

50
Q

Why should the 3’-ends of primers should not be complementary?

A

Becuase primer dimers will be synthesised over any other product.

51
Q

Primers should contain what kind of sequence?

A

A unique DNA sequence.

Molecular Biology Lab. (7 decks)

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