Restriction Endonucleases Flashcards
(24 cards)
What are restriction endonucleases?
Enzymes that recognize specific DNA sequences (restriction sites) and cut the DNA at or near those sites.
How are restriction endonucleases named?
Named after the bacterium of origin:
1st letter = genus
Next 2 letters = species
Next = strain (if applicable)
Roman numeral = order of discovery
(E.g., EcoRI = E. coli strain R, 1st discovered)
What is unique about restriction enzyme recognition sites?
They are typically palindromic sequences, reading the same 5’ to 3’ on both DNA strands.
What are the two types of ends formed by restriction enzymes?
Sticky ends: Overhanging sequences (5’ or 3’)
Blunt ends: Straight cuts with no overhang
How does the length of the recognition site affect cutting frequency?
4 bp → every ~256 bp
6 bp → every ~4,096 bp
8 bp → every ~65,536 bp
What is degeneracy in restriction sites?
Some enzymes recognize variable sequences (e.g., Y = C or T, R = A or G)
What is methylation sensitivity?
Some enzymes only cut unmethylated DNA or are blocked by methylation.
What is the natural role of restriction enzymes in bacteria?
Defense against bacteriophages by cutting foreign DNA; own DNA protected by methylation.
Why are restriction enzymes important in molecular biology?
They enable DNA fragmentation, mapping, and recombination—core to cloning, vector design, and gene editing.
What is a restriction map?
A diagram showing enzyme cut sites on a known DNA sequence (e.g., plasmids like pUC19).
Besides restriction enzymes, how else can DNA be fragmented?
Mechanical shearing, which is random and less precise (e.g., sonication).
What is a polylinker (MCS) in a plasmid vector?
A short DNA region with multiple restriction enzyme recognition sites allowing insertion of DNA fragments.
What happens when DNA is inserted into the polylinker?
It disrupts the adjacent gene (e.g., lacZ), enabling selection like blue-white screening.
How does blue-white screening work?
Insertional inactivation of lacZ prevents blue pigment production; white colonies likely contain the insert.
What are the three main components of a restriction digest?
Template DNA, restriction enzyme, buffer (plus water to final volume).
What is the role of the buffer in a restriction digest?
It provides optimal pH, salt, and cofactors for enzyme activity.
Why is buffer selection important for restriction enzymes?
Different enzymes require specific conditions—wrong buffers can reduce activity.
What is star activity in restriction enzymes?
Non-specific DNA cleavage due to incorrect conditions (e.g., wrong buffer, too long incubation).
How are restriction enzymes inactivated?
Most are heat-inactivated at 65–80°C depending on the enzyme.
Why does uncut plasmid DNA appear smaller on a gel?
It’s supercoiled and migrates faster than linear DNA of the same size.
What do multiple bands from restriction digests indicate?
Successful cuts at specific sites; band size reflects fragment length.
How are PCR and restriction digests linked in cloning?
PCR can amplify DNA for digestion and insertion into vectors.
How are sticky ends used in cloning?
They allow directional ligation with complementary overhangs for precise insert integration.
