PCR

Question 1

What is the PCR used for?

Answer 1

Amplifying DNA in vitro

Question 2

What are 2 features of the PCR?

Answer 2

-specific - only get amplification of selected sequence
-selective - amplify a specific sequence from a mixture of DNA sequences

Question 3

How do you calculate the number of molecules produced in PCR?

Answer 3

-2 to the power of the number of cycles
-exponential increase

Question 4

Describe one cycle of the PCR

Answer 4

1. denaturation - double stranded DNA dissociates into single stranded DNA
2. primer annealing - primers bind to complementary sequence on ssDNA (antiparallel binding)
3. primer extension - DNA polymerase synthesises new strands of DNA from the 3’ end of the primers

Question 5

What temperatures do each of the steps of PCR occur at?

Answer 5

1. denaturation - 95 degrees
2. primer annealing - 55-65 degrees
3. primer extension - 68-72 degrees

Question 6

What is needed for PCR?

Answer 6

-template - DNA that’s amplified
-DNA polymerase - copies DNA
-primers - DNA polymerase needs free 3’ OH to start
-deoxyribonucleoside triphosphates (dNTPs) - DNA bases to make new DNA strand
-buffer - correct pH + ions
-thermocycler - maintains temp

Question 7

What are the DNA polymerases that can be used in PCR?

Answer 7

-taq
-pfu

Question 8

What are the advantages and disadvantages of Taq?

Answer 8

-good thermostability, efficient, quick
-low accuracy rate, doesn’t proof-read, creates overhang

Question 9

What are the advantages and disadvantages of Pfu?

Answer 9

-superior thermostability, high accuracy, produces blunt ends
-slower, less efficient

Question 10

What are important features of primers?

Answer 10

-minimum size for specificity = 17bp
-specific to template - primer sequence is complementary + binds antiparallel
-come in pairs
-have appropriate melting temperature

Question 11

Why is it important for primers to come in pairs?

Answer 11

-bind opposite strands in opposite directions
-one binds to top strand + one binds to bottom strand
-if only 1 primer, only 1 strand is replicated

Question 12

What is Tm and how do you calculate it?

Answer 12

-the temperature at which the primer dissociates from the DNA template
-+4 degrees for every G/C and +2 degrees for every A/T

Question 13

Why is it important for primers to have the appropriate melting temperature?

Answer 13

-if Tm too high, it self anneals + if too low, it’s not specific
-around 60-64 degrees

Question 14

What does Tm determine?

Answer 14

What annealing temp (Ta) to use in PCR - around 5 degrees lower than Tm

Question 15

How can you ligate a PCR product directly into a vector?

Answer 15

-PCR products have no 5’ phosphate, so add a 5’ phosphate with T4 PNK
-taq adds a 3’ A overhang to PCR products, so remove the overhang or use a different enzyme

Question 16

What are 2 variants of the PCR?

Answer 16

-reverse transcription (RT) PCR
-quantitative PCR (qPCR)

Question 17

Describe RT PCR

Answer 17

-RNA is transcribed into DNA (cDNA)
-PCR amplifies specific cDNA sequence

Question 18

How is cDNA synthesised?

Answer 18

First strand:
-reverse transcriptase synthesises the 1st cDNA strand
-add primer
-RNA removed
Second strand:
-synthesised by Klenow fragment of DNA polymerase I
-hairpin formed by RT acts as primer
-ssDNA loop can be digested by nuclease

Question 19

How do you measure qPCR product?

Answer 19

-fluorescent dye fluoresces when it binds to dsDNA + is proportional to amount of dsDNA - not sequence specific
-fluorescent probes are sequence specific + bind to ssDNA - fluoresces when it’s displaced from template

Question 20

What is the cycle threshold?

Answer 20

Ct - the point at which fluorescence exceeds background levels

Question 21

What does a lower Ct value mean?

Answer 21

Fewer PCR cycles are needed to exceed the threshold, so more template at start

Question 22

What can you use the difference in Ct values between 2 samples to calculate and how do you calculate fold difference?

Answer 22

-calculate relative amounts of each sample
-fold difference = 2^-deltaCt