PCR

Question 1

Describe steps of PCR

Answer 1

1. Denature: @ 95C break H bond w/in bases so dsDNA -> ssDNA
2. Annealing: @ 50-75C. RNA/DNA primers bind to complementary strand
3. Extension: @ 72C. Pol. binds to primer & make new DNA strands

Question 2

What is the difference in the products amplified after cycles 1, 2 and 3?

Answer 2

1: 3 types: OG+ 2x strands cut short @ 1 end
2: 4 types: + strands cut short on both ends
3: target sequence (both ends cut) rise exponetially

Question 3

Which components are required for DNA replication and PCR react?

Answer 3

Primers, Pol., dNTP (deoxynucleotide triphosphate), DNA template strand

Question 4

Which two variables can a scientist alter in a PCR to increase or decrease specificity. Explain how it can be done

Answer 4

Var: Annealing temps. (Ta) and Mg2+ [ ]
Increase specificity: Incr. Ta OR decr. Mg2+
Decrease: Decr. Ta OR Incr. Mg2+

Question 5

Why is the specificity of primers important?

Answer 5

so it will match & bind to appropriate sequence on template DNA & elongation will start from 3’ end of primer (5’ to 3’ direction)

Question 6

When designing primers what factors need to be taken into consideration?

Answer 6

Sequence of template must be known. 3’ ends of FWD & RVS primers should face each other.

Question 7

What happens to primer during PCR?

Answer 7

Annealing: primer binds to DNA template on complementary sequences
Extension: dNTP added from 3’ end of primer and strand extends (5’ to 3’)

Question 8

State the diff. b/w DNA replication in vivo (cell) & in vitro (PCR)

Answer 8

Vivo: helicase unzip DNA; semi-discontinous; RNA primers; strands become shorter each replication; requires DNA ligase to join Okazaki fragments; not require Mg2+ cofactor and buffer
Vitro: denature DNA; continuous; DNA/RNA primers; strands doesn’t shorten each cycle; not require DNA ligase; require Mg2+ cofactor and buffer

Question 9

How can we visualise PCR products?

Answer 9

electrophoresis: stain DNA w/ Gel green or Midori green

Question 10

Why is thermostable polymerase used in PCR?

Answer 10

because it is resistant to heat denaturation (optimum temp: 60-75C). SUitable for extension (@ 72C)

Question 11

Why are lids of PCR heated?

Answer 11

prevent liquid (mainly H2O) vapourising and condensing on lids= remains liquid

Question 12

Primers are designed in conserved regions outside of the target DNA. Explain why?

Answer 12

Conserved regions are coding regions of genes (exons). Designing primers based on these coding regions reduces the probability of mismatches b/w primer and target

Question 13

What organism was Taq DNA Polymerase isolated from?

Answer 13

Thermophilic bacteria

Question 14

Why is so PCR so useful for a wide variety of applications in Molecular Biology?

Answer 14

Can amplify DNA copies or interested DNA = sufficient quantity to conduct tests