Lec 3

Practice vocabulary flashcards covering key terms and concepts in plasmid cloning, restriction endonucleases, PCR primer design, and site-directed mutagenesis.

Plasmid Cloning Vector

A circular, double-stranded DNA molecule containing an origin of replication, a selection marker, and a multiple cloning site, used to introduce foreign DNA into host organisms.

Origin of Replication (ori)

A specific sequence on a plasmid vector (e.g., $pMB1\,ori$ in pUC18) where DNA replication begins.

Selection Marker

A gene on a plasmid, typically conferring antibiotic resistance (such as $amp$ for ampicillin), that serves as a differentiating factor to identify cells that have successfully taken up the vector.

Multiple Cloning Site (MCS)

A defined region within a cloning vector engineered with unique recognition sites for multiple restriction endonucleases to allow insertion of target DNA fragments.

Restriction Endonuclease

An enzyme that recognizes a specific short DNA sequence (commonly $6\,bp$, but ranging from $4\,bp$ to $8\,bp$) and cleaves both strands of the DNA duplex.

Palindromic Sequence

A DNA sequence characterized as a short inverted repeat that reads identical in the $5' \rightarrow 3'$ direction on both the forward and complementary strands.

Sticky Ends

Single-stranded overhangs (typically $4\text{ bases}$ long) generated by a staggered cleavage mechanism of restriction endonucleases (such as BamHI or Sau3AI) that can base-pair with matching complementary ends.

Blunt Ends

Fully base-paired double-stranded DNA termini lacking single-stranded overhangs, produced when a restriction endonuclease (such as SmaI) cleaves both strands straight across the center of its recognition site.

Blunt-End Cloning

A cloning method in which a blunt-ended PCR product or insert is ligated into a vector linearized with blunt ends; it is straightforward and universally compatible among blunt ends, but provides no control over insert orientation.

Directional Cloning

A cloning approach where the insert and vector are cut with two distinct restriction enzymes (e.g., EcoRI and HindIII), producing non-compatible sticky ends that ensure the insert ligates into the plasmid in a single, predictable orientation.

Primer 5' Flexibility

The feature of PCR primers allowing arbitrary non-complementary sequences (such as $4\text{ to }6\text{ nucleotides}$ followed by a restriction endonuclease site) to be attached at the $5'$ end without impairing polymerase function.

Primer 3' Inflexibility

The requirement that the $3'$ end of a primer must have exact complementarity ($18\text{ to }24\,nt$) to the template DNA, ensuring the free $3'\text{-OH}$ group is positioned correctly for DNA polymerase extension.

PCR Annealing Temperature ($T_{\text{anneal}}$) Rule

The reaction parameter predicted as $T_{\text{anneal}} = T_{\text{melting}} - 5^\circ\text{C}$, calculated exclusively using the $18\text{ to }24\,nt$ template-complementary sequence and excluding added $5'$ non-complementary sequence.

Site-Directed Mutagenesis

A targeted in vitro method that utilizes mutagenic PCR primers to introduce specific, predetermined nucleotide alterations into a gene sequence to study the function of specific amino acids or protein domains.

Overlap-Extension PCR

A two-stage mutagenesis technique in which internal mutant primers generate two fragments sharing complementary sequence across the mutation, which then anneal together and are amplified as a full-length mutant product by flanking primers.

Transformation

The process of introducing recombinant plasmid DNA directly into bacterial cells, typically achieved via chemical competence or electroporation.

Ligation

The enzymatic step in cloning wherein DNA ligase seals compatible vector and insert DNA termini together through the formation of covalent phosphodiester bonds.