Lec. 2

Comprehensive vocabulary flashcards covering the components, cycle stages, primer design rules, and gel electrophoresis troubleshooting protocols for Polymerase Chain Reaction (PCR).

Template DNA

The original DNA sample containing the target sequence to be amplified, serving as the physical site for replication to begin in a PCR reaction.

Taq Polymerase

A heat-stable DNA polymerase enzyme used in PCR to synthesize new complementary DNA strands during the extension phase.

Denaturation Step

The phase of PCR (typically performed at $95\,^\circ\text{C}$) where high temperature is used to unwind and separate double-stranded DNA into single strands without requiring helicase.

Annealing Step

The PCR stage (typically around $60\,^\circ\text{C}$ for $30\,\text{seconds}$) during which forward and reverse primers bind specifically to their complementary target sequences on the single-stranded template DNA.

Annealing Temperature Formula

The reaction setting calculated using the melting temperature of the primers, defined as $T_{\text{annealing}} = T_{\text{melting}} - 5\,^\circ\text{C}$.

Extension Step

The step in each PCR cycle (typically performed at $72\,^\circ\text{C}$) where DNA polymerase is optimally active and synthesizes new DNA strands initiating from the $3'$ ends of the annealed primers.

Exponential PCR Amplification

The cyclical process where both original template strands and newly synthesized strands serve as templates in subsequent cycles, doubling target DNA strands with each cycle.

Optimal Primer Length

A sequence typically consisting of $18\text{--}24\,\text{nucleotides}$ chosen to ensure specificity and appropriate hybridization stability.

Primer Melting Temperature ($T_{\text{m}}$) Range

The temperature at which half of the primer-duplex dissociates, ideally kept between $55\,^\circ\text{C}$ and $70\,^\circ\text{C}$ and closely matched between both paired primers.

Hairpin Structure

An undesirable secondary structure formed when a portion of a single primer binds to another complementary section within its own sequence.

3' End Primer Design Rule

A guideline recommending that the $3'$ end of a primer contains at least one G or C nucleotide (and ideally no more than 4) to ensure stable binding for polymerase initiation.

Conventional Primer Sequence Writing

The standard practice of writing all synthesized oligonucleotide sequences strictly from $5'$ to $3'$, requiring the reverse primer to be formatted as the reverse complement of the coding strand.

Non-Specific Product

An unintended PCR band appearing at an unexpected molecular weight due to primers annealing at imperfectly matched sites; often resolved by raising the annealing temperature.

Primer-Dimers

Short, low molecular weight artifacts (often visible below $500\,\text{bp}$ on an agarose gel) produced when forward and reverse primers hybridize to each other due to intermolecular complementarity.

Agarose Gel Electrophoresis Analysis in PCR

A diagnostic method used to evaluate PCR success by separating amplified DNA fragments by size against a DNA molecular weight marker to confirm the expected band size and troubleshoot artifacts.