Lab 5.

Question 1

What is PCR?

Answer 1

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

Question 2

Who invented PCR?

Answer 2

Carey Mullis.

Question 3

What are the 3 steps involved in PCR?

Answer 3

Denaturation.

Annealing.

Extension.

Question 4

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

Answer 4

Around 30-40 times.

Question 5

What are the primers that are used in PCR?

Answer 5

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

Question 6

How many nucleotides do the primers usually consist of?

Answer 6

Between 17-25 nucleotides.

Question 7

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

Answer 7

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

Poorly designed primes could also amplify many unwanted DNA fragments.

Question 8

What 3 things does DNA amplification allow for?

Answer 8

DNA identification and manipulation.

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

The detection of mutations.

Question 9

What happens during the denaturation step of PCR?

Answer 9

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

Question 10

What happens during the annealing step of PCR?

Answer 10

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

Question 11

What happnens in the extension phase of PCR?

Answer 11

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

Question 12

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

Answer 12

Denaturation takes place at 94 degrees C.

Annealing takes place at 60 degrees C.

Extension takes place at 70 degrees C.

Question 13

What is the goal of designing good primers?

Answer 13

The specific amplification of a DNA sequence.

To obtain high yields of copied DNA.

Question 14

Why are primers of a specific length?

Answer 14

This length is long enough to be specific.

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

Question 15

What does primer design require?

Answer 15

Extensive sequence analysis through the use of computers.

Question 16

Primers should only be complimentary to what?

Answer 16

To the target sequence.

Question 17

Why should primers only be complimentary to the target region?

Answer 17

So that taq polymerase only copies the target region.

Question 18

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

Answer 18

The forward and reverse primer.

Question 19

How are the bases found 2 primers always written?

Answer 19

In the 5,3 direction.

Question 20

The forward primer is always complimentary to which DNA strand?

Answer 20

The 5 to 3 strand.

Question 21

The reverse primer is always complimentary to which DNA strand?

Answer 21

The 3 to 5 strand.

Question 22

How is the reverse primer always written?

Answer 22

In the 5 to 3 direction.

Question 23

What is the Tm (melting temperature)?

Answer 23

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

Question 24

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

Answer 24

The melting temperature (Tm) of the primers.

Question 25

At what temperatures will all the DNA | molecules will be in the single stranded form?

Answer 25

At temperatures above the Tm.

Question 26

At what temperature is annealing usually carried out?

Answer 26

At around 5 degrees below the Tm.

Question 27

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

Answer 27

Primers with melting temperatures between 52-58 degrees.

Question 28

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

Answer 28

Secondary annealing.

Question 29

What happens if the annealing temperature is too high?

Answer 29

The primer will not bind to the target DNA.

Question 30

What happens if the annealing temperature is too low?

Answer 30

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

Question 31

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

Answer 31

Mis-annealing.

Question 32

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

Answer 32

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

Question 33

The Tm of a DNA molecule is dependent on what?

Answer 33

It's DNA sequence.

Question 34

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

Answer 34

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

Question 35

What is the Wallace rule?

Answer 35

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

Question 36

What happens if the primers are complementary to each other?

Answer 36

They will anneal with themselves.

Question 37

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

Answer 37

Those that lead to hairpins. Those that lead to primer dimers.

Question 38

How are hairpin structures formed from primers?

Answer 38

The primer molecule will pair with itself.

Question 39

Can hairpin primers bind to DNA?

Answer 39

No.

Question 40

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

Answer 40

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

Question 41

How is a primer dimer formed?

Answer 41

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

Question 42

What is heterodimer formation?

Answer 42

When the forward primer binds to the reverse primer.

Question 43

What is self-dimer formation?

Answer 43

When base pairing occurs between one of the two primers.

Question 44

Primers should be of what length?

Answer 44

17-28 bases.

Question 45

Primers should be of what G/C content?

Answer 45

50-60%.

Question 46

How should primers end?

Answer 46

With a G or C, or CG or GC.

Question 47

Why should primers end with a G or a C?

Answer 47

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

Question 48

What Tm's are preferred?

Answer 48

Tms between 55-80°C.

Question 49

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

Answer 49

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

Question 50

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

Answer 50

Becuase primer dimers will be synthesised over any other product.

Question 51

Primers should contain what kind of sequence?

Answer 51

A unique DNA sequence.