Polymerase Chain Reaction

Question 1

What is purpose of Polymerase Chain Reaction (PCR)?

Answer 1

To amplify minute amounts of DNA

Question 2

Which enzyme carries out PCR?

Answer 2

DNA Polymerase

Question 3

Which specific DNA Polymerase enzyme is used for PCR and why?

Answer 3

• Taq Polymerase found from bacteria living near hot springs.
• This enzyme can withstand high temperatures and still function.

Question 4

Where is PCR carried out?

Answer 4

In labs in a DNA Thermocycler that has test tubes. PCR is carried out in cycles.

Question 5

Describe the rate of DNA amplification

Answer 5

Exponential where each cycle doubles the amount of DNA.

Question 6

What are the 4 ingridients required for PCR?

Answer 6

• Samply of DNA
• Source of all 4 DNA nucleotides
• DNA Polymerase (specifically Taq Polyemrase)
• Two single stranded DNA primers

Question 7

What is the purpose of Sample DNA in PCR?

Answer 7

Each strand of sample DNA acts as template to produce more DNA.

Question 8

What is purpose of having a source of all 4 DNA nucleotides (A,T,C and G) in PCR?

Answer 8

So that DNA Polymerase can use them to build the DNA strands.

Question 9

What is purpose of having two single stranded DNA primers for PCR?

Answer 9

• These are synthetically produced.
• Are attached to 5’ end of both DNA strands.
• DNA polyemarse can only build only existing strands, so primer acts as existing strand.
• Are up to 30 bases long.

Question 10

What are the names given to each of the 2 single stranded DNA primers in PCR?

Answer 10

• Forward primer
• Reverse primer

Question 11

Describe the forward primer PCR.

Where it binds, and it is called forward primer

Answer 11

• Binds at start codon on the template strand
• This is the 3’ end
• This means Taq Polymerase synthesises DNA in same direction as RNA Polymerase would fuction.
• Thus, named forward primer

Not really start codon as it is on template strand.

Question 12

Describe the reverse primer in PCR.

Where it binds, and it is called reverse primer

Answer 12

• Binds at stop codon on the coding strand
• This is the 3’ end
• This means Taq Polymerase sythesises DNA in the reverse direction that RNA Polymerase would function.
• Thus named reverse primer

Question 13

What are the 3 steps in a PCR cycle?

Answer 13

1. Denature
2. Annealing
3. Extension

Question 14

What are the 3 temperatures in order of PCR do you need to remember?

Answer 14

1. 94 degrees Celsius
2. 55 degrees Celsius
3. 72 degrees Celsius

Question 15

Describe process of Denaturing in PCR

Answer 15

• DNA is exposed to 94 degrees celsius for 2 minutes.
• This breaks hydrogen bonds.
• This separates the strands of DNA.

Question 16

Describe the process of Annealing in PCR

Answer 16

• DNA is placed in environment of 55 degrees celsius for about 2 minutes.
• The two DNA Primers bind to 3’ end of both DNA strands of interest.

Question 17

Describe the process of Extension in PCR

Answer 17

• Placed in 72 degrees Celsius for 1 minute
• Taq Polymerase attaches to primers. (acts as starting point).
• Begins building and extending strand by adding free floating DNA nucleotides.
• Creates 2 double stranded DNA molecules that are identical for every DNA strand.

Question 18

How long does 1 cycle of PCR approzimately take?

Answer 18

5 minutes