HMG - Week 8: Recombinant DNA Technology: Cloning and Expression Vectors LO

Critical vocabulary terms and definitions regarding recombinant DNA technology, including vector types, enzymatic tools, library construction, and detection systems based on the lecture notes.

Cloning vector

A DNA molecule used to carry foreign DNA into a host cell where it can be replicated and copied many times.

Origin of replication (ori)

A DNA sequence that serves as the starting point for DNA replication, allowing for the duplication of a plasmid within a host cell.

Multiple cloning site (MCS)

A region in a cloning vector containing many unique restriction sites that allows for the insertion of foreign DNA.

Plasmids

Small circular double-stranded DNA molecules found naturally in bacteria that replicate independently of the bacterial chromosome.

Bacteriophage Vectors

Viruses that infect bacteria used as vectors to carry larger DNA inserts than plasmids and eventually lyse the host cell.

Yeast Artificial Chromosomes (YACs)

Artificial chromosomes maintained in yeast cells that can accommodate very large DNA fragments ranging from hundreds of kilobases to megabases.

Expression vectors

Specialised cloning vectors designed to replicate DNA and produce the protein encoded by the inserted gene.

Inducible Promoters

Promoters that can be switched ON or OFF to control protein expression, preventing toxic protein production and regulating cell metabolism.

lac Promoter

A specific promoter that is normally OFF but turns ON transcription when IPTG is added.

ampR Gene

A gene commonly found in plasmids like pUC19 that provides ampicillin resistance, used as a selectable marker.

lacZ Gene

A gene that encodes $\beta$-galactosidase and is used in blue-white screening; its disruption indicates successful DNA insertion.

Blue-White Screening (Blue Result)

The result when no insert is present, meaning the lacZ gene remains functional and breaks down X-gal to produce blue colonies.

Blue-White Screening (White Result)

The result when an insert is present, disrupting the lacZ gene so no $\beta$-galactosidase is produced and X-gal is not broken down.

Restriction enzymes

Also known as restriction endonucleases, these are molecular scissors that cut DNA at specific 4–8 base pair recognition sequences.

Palindromic sequences

DNA sequences that read the same in the $5'\rightarrow3'$ direction on both strands; these are typically recognized by restriction enzymes.

Sticky Ends

DNA ends with single-stranded overhangs produced by enzymes like EcoRI, BamHI, or PstI that can hydrogen bond with complementary sequences.

Blunt Ends

DNA ends with no overhangs produced by enzymes like SmaI, which are generally harder to ligate than sticky ends.

DNA Ligase

An enzyme that seals phosphodiester bonds to join DNA fragments together into a recombinant DNA molecule.

Restriction Site Probability Formula

$\text{Probability} = (1/4)^n$, where $n$ is the number of bases in the recognition sequence.

Genomic Library

A collection of cloned DNA fragments representing the entire genome of an organism, including exons, introns, and intergenic DNA.

cDNA Library

A collection containing DNA copies of mRNA molecules expressed in a cell, produced using reverse transcriptase and containing only exons.

Southern Blotting

A technique used to detect a specific DNA sequence within a complex sample through gel electrophoresis, denaturation, and labeled probe hybridization.

Probe

A labelled single-stranded DNA fragment complementary to a target sequence used in Southern blotting for detection.

Yeast Two-Hybrid System

An experimental design used to detect protein-protein interactions by bringing together a DNA-binding domain and an activation domain to activate a reporter gene.

Bait Protein

In the yeast two-hybrid system, the protein that is attached to the DNA-binding domain (DBD).

Prey Protein

In the yeast two-hybrid system, the protein that is attached to the activation domain (AD).