L13 PCR

Question 1

What is PCR?

Answer 1

• Amplify DNA in vitro (in a tube).
• Specific - will only get amplification of your selected sequence.
• Selective - can amplify a specific sequence from a mixture of DNA sequences

Question 2

What features are needed when making anew starnd?

Answer 2

It must be:
1. Complementary nucleotide base pairing (A-T and G-C)
2. Double-stranded
3. Antiparallel
4. Semi-conservative (template and newly synthesised daughter)

Question 3

What do we need for PCR replication?

Answer 3

1. Need to unwind DNA
2. Need a primer
3. Polymerase enzyme
4. New nucleotides

Question 4

What is the basic principle of PCR?

Answer 4

PCR is a cyclic process that amplifies a specific DNA sequence. Each cycle involves three steps:

• Denaturation: The double-stranded DNA is heated to separate the two strands.
• Annealing: Primers, short single-stranded DNA sequences complementary to the target sequence, bind to the template DNA.
• Extension: DNA polymerase extends the primers, synthesizing new DNA strands complementary to the templates.

Question 5

How does PCR amplify DNA exponentially?

Answer 5

In each PCR cycle, the number of DNA molecules doubles. This exponential amplification is achieved by repeating the following steps:
* Denaturation
* Annealing
* Extension
After 30 cycles, you can theoretically generate over one billion copies of the target DNA sequence from a single starting molecule.

Question 6

What do we need for PCR?

Answer 6

1. Template - desired DNA
2. DNA polymerase - copies DNA
3. Primers - DNA polymerase need free 3’-OH provided by a primer.
4. dNTPs - DNA bases to make new strands
5. Buffer - correct pH and ions
6. Thermocycler - maintains appropriate temperature.

Question 7

What is the challenge of using DNA polymerase in PCR?

Answer 7

The high temperatures required for denaturation (95°C) can inactivate most DNA polymerases. To overcome this challenge, thermostable DNA polymerases, such as Taq polymerase, are used in PCR. These enzymes are derived from thermophilic bacteria and can withstand high temperatures without denaturing.

Question 8

What are the DNA polymerases used in PCR and why?

Answer 8

Taq polymerase is used because of its thermostability, extension rate( how fast it can replicate), processivity and fidelity.

Question 9

Which polymerase is more effective than Taq and why?

Answer 9

Pfu with superior thermostability and higher fidelity.