Restriction Enzymes Flashcards
What is a plasmid?
Extra-chromosomal DNA that replicates independent of a chromosome.
Plasmids are smaller than a chromosome and can be present in multiple copies.
What are plasmids used for?
Plasmids are used in molecular biology research to move genes of interest into an organism so the genes can be studied.
What is a recombinant plasmid?
Recombinant plasmids are the result of genes being ligated into a plasmid, combining DNA from two different sources.
What is DNA ligase?
DNA ligase is an enzyme involved in DNA replication, gluing two pieces of DNA together.
Ligase makes phosphodiester bonds connect to the BACKBONE of the two pieces of DNA. ATP (ribose, not deoxy) is provided as an energy source.
What do restricted enzyme digested pieces do?
These have base sequences on their ends that are complementary to one another. Hydrogen bonding can occur before the phosphodiester bond is made.
How is a recombinant plasmid made?
A vector/plasmid without an insert has an insert ligated into it.
What is pUC18?
Commonly used plasmid in molecular biology.
What do most plasmids contain?
What does pUC18 contain?
Most plasmids contain an antibiotic resistance gene.
pUC18 contains an ampicillin resistance gene (ampR).
Other plasmids may barry a gene that makes the bacterium resistant to another antibiotic.
What happens when ampicillin is added to media with a plasmid and Escherichia coli?
When a plasmid is moved into E. coli, the ampicillin will force the bacterium to keep the plasmid inside the cell. Now, the gene of interest can be studied in E. coli.
What is an origin of replication (ori)?
The origin of replication is where replication begins.
Plasmids have this; it is needed for the plasmid to be replicated in the bacterium.
What are polylinker or polycloning sites?
Polylinker/polycloning sites are located on plasmids. They contain several restriction enzyme sites that are very close together.
When a gene is glued into a plasmid, that is where it will be glued.
What do bacteria containing pUC18 have?
Bacteria containing pUC18 have hundreds of copies per cell.
They are also forced to maintain the plasmid by being grown with ampicillin in the media.
How can plasmids be moved into a bacterium?
Transformation.
Example of transformation?
What is a restriction endonuclease?
An enzyme that cuts the DNA at a specific DNA sequence (the restriction enzyme site) within the DNA strands, rather than at the very end. They can leave a sticky end (or cohesive end or overhang) or blunt end on a double-stranded DNA.
What is EcoR1?
A restriction enzyme that cuts at 5’GAATTC3’, between the G and the A.
This sequence is considered palindromic.
What is HindIII?
A restriction enzyme that cuts the backbone between the AA sequence between 5’AAGCTT3’.
What does it mean when EcoR1 generates a 5’ overhang?
It means that there will be a strand that is sticking out with its 5’ end.
Does EcoRV make an overhang like EcoR1?
No, EcoRV makes a blunt end. There will be no overhang.
When ligase is added along with ATP, it can make the phosphodiester bonds between the insert and the plasmid. How many phosphodiester bonds are needed?
How many pieces of insert is needed for a vector/plasmid?
How many EcoR1 sites will be in the recombinant plasmid?
Why do bacteria make restriction endonucleases?
Bacteria produce restriction endonucleases to chop up DNA from a bacteriophage that may have infected the bacterium.
“Restriction” is used because it limits bacteriophage infection.
What is the restriction modification system?
- Restriction enzyme/endonuclease: used to cut up bacteriophage DNA
- Methylase: adds methyl group (CH3) to some bases in restriction enzyme sites on bacterial chromosome
