PCR

Question 1

What are the three steps of PCR?

Answer 1

Denaturation, annealing and extension

Question 2

What happens in denaturation?

Answer 2

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

Question 3

What happens in annealing?

Answer 3

Primers bind to flanking regions on the target DNA.

Question 4

What happens in extension?

Answer 4

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

Question 5

How many cycles of PCR are usually done?

Answer 5

25-35

Question 6

In what direction is the DNA amplified?

Answer 6

5’-3’

Question 7

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

Answer 7

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

Question 8

Which DNA polymerase is used now?

Answer 8

Taq DNA polymerase from Thermus aquaticus.

Question 9

What are the drawbacks of Taq Polymerase?

Answer 9

It is unstable above 90 degrees and doesn’t proofread

Question 10

What is the function of thermocyclers?

Answer 10

It automates temperature cycling and incubation times.

Question 11

When were thermocyclers first introduced?

Answer 11

1985

Question 12

From what sources can the template DNA be taken?

Answer 12

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

Question 13

What is the equation for copy number?

Answer 13

Avogadro’s constant x no. moles

Question 14

What are the 7 components to a PCR reaction?

Answer 14

• PCR buffer
• dNTPs
• MgCl2
• Primers
• Taq DNA
• Sterile distilled water

Question 15

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

Answer 15

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

Question 16

How long are primers?

Answer 16

Around 15-30 bases

Question 17

What is the usual Tm range?

Answer 17

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

Question 18

What should be avoided in PCR?

Answer 18

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

Question 19

What are the optimal conditions for PCR?

Answer 19

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

Question 20

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

Answer 20

In equimolar quantities

Question 21

When may an unbalanced concentration of dNTPs be used?

Answer 21

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

Question 22

What is the recommended concentration of each dNTP?

Answer 22

0.2 mM

Question 23

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

Answer 23

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

Question 24

With what are dNTPs sometimes replaced?

Answer 24

dUTP

Question 25

What is the function of magnesium in PCR?

Answer 25

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

Question 26

What does the Mg2+ active site catalyse?

Answer 26

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

Question 27

What is the function of buffers in PCR?

Answer 27

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

Question 28

What pH is buffered in PCR?

Answer 28

pH 8-9.5

Question 29

What is pH stabilised by in PCR?

Answer 29

Tris-HCl

Question 30

What is reverse transcription?

Answer 30

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

Question 31

Why is endpoint PCR performed?

Answer 31

To quantitate RNA expression.

Question 32

What can PCR be used to detect in genotyping?

Answer 32

Sequence variations in alleles of specific cells.

Question 33

How is the primer altered for use in genotyping?

Answer 33

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

Question 34

What is sequencing of PCR amplicons used to study?

Answer 34

Single nucleotide variations and single-nucleotide polymorphisms.

Question 35

What can PCR be used to clone?

Answer 35

DNA fragments of interest.

Question 36

What happens in direct PCR cloning?

Answer 36

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

Question 37

Why is it recommended to purify the DNA sequence?

Answer 37

To remove excess reagents and non-full length oligonucleotides.

Question 38

What is PCR cloning used for?

Answer 38

To screen whether colonies carry a desired insert.

Question 39

What is mutagenesis?

Answer 39

Cloning to introduce desired mutations.

Question 40

What is site directed mutagenesis?

Answer 40

Primers that incorporate base substitutes, deletions or insertions.

Question 41

What important considerations are needed in site directed mutagenesis?

Answer 41

Primer design, DNA polymerase choice and RNA parameters.

Question 42

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

Answer 42

To maintain DNA accuracy

Question 43

What is Sanger sequencing?

Answer 43

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

Question 44

How is the DNA altered in sequencing?

Answer 44

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