Gene Cloning Flashcards
(18 cards)
What is a vector in DNA cloning?
A DNA molecule (usually a plasmid) that carries foreign DNA into a host cell and facilitates its replication.
Name the main components required for DNA cloning.
1) Foreign DNA, 2) Host organism, 3) Vector DNA, 4) Insertion method, 5) Transformation method, 6) Selection method.
What are YACs and BACs?
YACs = Yeast Artificial Chromosomes (200–3000 kb); BACs = Bacterial Artificial Chromosomes (100–300 kb).
What are plasmids?
Small circular dsDNA molecules that replicate independently in host cells and often carry antibiotic resistance genes.
What is the function of the origin of replication (ORI) in plasmids?
It enables the plasmid to replicate independently within the host cell.
What does the multiple cloning site (MCS) do?
Provides unique restriction sites for inserting foreign DNA into the vector.
What is the purpose of the lacZ gene in cloning vectors?
It encodes β-galactosidase; disruption by foreign DNA enables blue-white screening.
What is Gateway® technology?
A cloning method using BP and LR clonase enzymes for recombination-based DNA insertion.
In blue-white screening, what does a white colony indicate?
It contains an insert and produces non-functional β-galactosidase.
Name three applications of Gateway® cloning.
Native protein expression, fusion protein studies, multiple gene expression.
What is T-A cloning?
A method using PCR products with A-overhangs and vectors with T-overhangs; relies on base pairing and ligation.
How does TOPO cloning work?
Uses topoisomerase I to cleave and ligate DNA at specific sequences for unidirectional cloning.
What are the steps in transformation by heat shock?
1) Make cells competent, 2) Mix with DNA, 3) Heat shock to allow DNA entry.
What is electroporation?
A method that uses an electric field to make cell membranes permeable for DNA uptake.
What is conjugation in DNA transfer?
Natural transmission of plasmids from one bacterium to another via cell-to-cell contact.
What are selectable markers used for?
To identify host cells that have successfully taken up foreign DNA, often via antibiotic resistance.
Give two examples of selection methods.
Antibiotic resistance and blue-white screening.
What is the purpose of large-scale propagation and storage of clones?
To produce high quantities of DNA and to preserve clones long-term (e.g., cryopreservation at –80°C).
