Principles & Techniques Flashcards

How does digital PCR (dPCR) quantify nucleic acids without a standard curve?

Answer: By partitioning the sample into many reactions and counting positive partitions with Poisson statistics

The fraction of positive partitions, corrected with Poisson statistics, gives absolute copy number.

What enzyme is required in the first step of RT-PCR?

Answer: Reverse transcriptase

Reverse transcriptase synthesizes cDNA from an RNA template, which is then amplified by PCR.

Inverse PCR is mainly used to:

Answer: Amplify unknown sequences flanking a known region

The DNA is digested and circularized, then primers pointing outward from the known region amplify the unknown flanks.

Why does PCR eventually reach a plateau phase?

Answer: Reagents run out, the polymerase loses activity, and products reanneal to each other

Late cycles slow down as dNTPs and primers deplete, enzyme activity drops, and product strands compete with primers.

Degenerate primers are most useful when:

Answer: Only the protein sequence or related gene sequences are known

Degenerate primers are mixtures covering possible codon variants, so they can amplify related or partly unknown sequences.

Allele-specific PCR (ARMS) distinguishes alleles primarily by:

Answer: Placing the discriminating nucleotide at the primer's 3' end

A 3'-terminal mismatch prevents efficient extension, so only the matching allele amplifies.

What is the typical denaturation temperature range in standard PCR?

Answer: 94-98°C

Temperatures of 94-98°C separate dsDNA strands without destroying a thermostable polymerase over normal cycling.