PCR and methods for creating mutations

Question 1

What are the three stages of elongation?

Answer 1

1. Exponentatial amplification: Doubling every cycle.
2. Levelling off: [dNTP and primer] is falling.
3. Plateau: Component exhaustion.

Question 2

Why is magnesium important in PCRs?

Answer 2

Polymerases generally require Mg for conformation.

Question 3

What are PCR inhibitors?

Answer 3

Proteins or lipids which through different mechanisms inhibit the PCR reaction.

Question 4

Why is hot-start PCR a thing?

Answer 4

When warming up the PCR, the primers hybridize at T < Tm. This leads to non-specific amplicons (which are generally outcompeted by correct amplicons).

Question 5

Why can a touchdown PCR be beneficial?

Answer 5

1. You start by running x annealing cycles at T > Tm for primers.
2. Annealing T successively drops to Tm for primers.

The reason for why the aforementioned steps are good, is that only primers binding to the complementary region with very high affinity will be stable enough to withstand the temparetures. Only the correct amplicon is amplified.

Question 6

What’s an inverse PCR?

Answer 6

You amplify plasmids from a known sequence out into the unknown. You can use this to sequence transposon-elements or the like.

Question 7

Apart from there being 45-60% GC, where else is a G/C important?

Answer 7

3’ end of oligo: GC-clamp.

Question 8

What are back-to-back PCRs used for?

Answer 8

Inducing deletions, insertions and substitutions.

Question 9

Why is error-prone PCR unpredictable?

Answer 9

The error rate follows a normal distribution, you never know where or how many mutations are going to be induced.

You make the polymerase sloppy, not the primers.