Principles & Techniques Flashcards

Theoretically, how many copies of a single double-stranded target are produced after 30 cycles of 100% efficient PCR?

Answer: About 1 billion (2^30)

Each cycle doubles the target, so 30 cycles yield 2^30, roughly 1.07 billion copies.

What is the main purpose of hot-start PCR?

Answer: To prevent polymerase activity at low temperatures that causes mispriming and primer-dimers

Hot-start methods block polymerase activity until the first high-temperature step, reducing nonspecific products formed during setup.

In touchdown PCR, how is the annealing temperature handled over successive cycles?

Answer: It starts above the primer Tm and is lowered stepwise in later cycles

Starting with a stringent high annealing temperature favors specific targets early, which then outcompete nonspecific products.

Which polymerase property allows Pfu polymerase to have higher fidelity than Taq polymerase?

Answer: 3'→5' exonuclease (proofreading) activity

The 3'→5' exonuclease removes misincorporated nucleotides, lowering the error rate.

Why do Taq-generated PCR products suit TA cloning?

Answer: Taq adds a single non-templated 3' adenine overhang

Taq's terminal transferase activity leaves a 3' A that pairs with the 3' T overhang of TA vectors.

Using a standard Taq polymerase, approximately what extension time is recommended for a 3 kb amplicon?

Answer: About 3 minutes

Taq extends at roughly 1 kb per minute, so a 3 kb product needs about 3 minutes.

Nested PCR improves specificity mainly because:

Answer: A second primer pair binds within the first amplicon, so only correct first-round products are re-amplified

Nonspecific first-round products are unlikely to contain binding sites for the inner primers.